Regulation of Chemokines, CCL3 and CCL4, by interferon gamma and Nitric oxide synthase 2 in mouse macrophages and during Salmonella enterica Serovar Typhimurium infection

Background. Interferon gamma (IFN-gamma) increases the expression of multiple genes and responses; however, the mechanisms by which IFN-gamma downmodulates cellular responses is not well understood. In this study, the repression of CCL3 and CCL4 by IFN-gamma and nitric oxide synthase 2 (NOS2) in macrophages and upon Salmonella typhimurium infection of mice was investigated. Methods. Small molecule regulators and adherent peritoneal exudates cells (A-PECs) from Nos2(-/-)mice were used to identify the contribution of signaling molecules during IFN-gamma-mediated in vitro regulation of CCL3, CCL4, and CXCL10. In addition, infection of bone marrow-derived macrophages (BMDMs) and mice (C57BL/6, Ifn-gamma(-/), and Nos2(-/-)) with S. typhimurium were used to gain an understanding of the in vivo regulation of these chemokines. Results. IFN-gamma repressed CCL3 and CCL4 in a signal transducer and activator of transcription 1 (STAT1)-NOS2-p38 mitogen activated protein kinase (p38MAPK)-activating transcription factor 3 (ATF3) dependent pathway in A-PECs. Also, during intracellular replication of S. typhimurium in BMDMs, IFN-gamma and NOS2 repressed CCL3 and CCL4 production. The physiological roles of these observations were revealed during oral infection of mice with S. typhimurium, wherein endogenous IFN-gamma and NOS2 enhanced serum amounts of tumor necrosis factor alpha and CXCL10 but repressed CCL3 and CCL4. Conclusions. This study sheds novel mechanistic insight on the regulation of CCL3 and CCL4 in mouse macrophages and during S. typhimurium oral infection.

Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

Toll-Like Receptors 7, 8, and 9 Expression and Function in Primary Human Cervical Cancer Langerhans Cells Evidence of Anergy

Objective: Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Methodology: Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a(+)CD207(+) cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1 beta, IL-6, IL-10, IL-12p70, interferon (IFN) alpha, interferon gamma, and tumor necrosis factor (TNF) alpha by Luminex multiplex bead array. Human papillomavirus was genotyped. Results: We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs. expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1 beta, and tumor necrosis factor alpha to TLR8 ligand and interferon alpha in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Conclusions: Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

Intracellular Pathogen Sensor NOD2 Programs Macrophages to Trigger Notch1 Activation

Intracellular pathogen sensor, NOD2, has been implicated in regulation of wide range of anti-inflammatory responses critical during development of a diverse array of inflammatory diseases; however, underlying molecular details are still imprecisely understood. In this study, we demonstrate that NOD2 programs macrophages to trigger Notch1 signaling. Signaling perturbations or genetic approaches suggest signaling integration through cross-talk between Notch1-PI3K during the NOD2-triggered expression of a multitude of immunological parameters including COX-2/PGE(2) and IL-10. NOD2 stimulation enhanced active recruitment of CSL/RBP-Jk on the COX-2 promoter in vivo. Intriguingly, nitric oxide assumes critical importance in NOD2-mediated activation of Notch1 signaling as iNOS(-/-) macrophages exhibited compromised ability to execute NOD2-triggered Notch1 signaling responses. Correlative evidence demonstrates that this mechanism operates in vivo in brain and splenocytes derived from wild type, but not from iNOS(-/-) mice. Importantly, NOD2-driven activation of the Notch1-PI3K signaling axis contributes to its capacity to impart survival of macrophages against TNF-alpha or IFN-gamma-mediated apoptosis and resolution of inflammation. Current investigation identifies Notch1-PI3K as signaling cohorts involved in the NOD2-triggered expression of a battery of genes associated with anti-inflammatory functions. These findings serve as a paradigm to understand the pathogenesis of NOD2-associated inflammatory diseases and clearly pave a way toward development of novel therapeutics.

MicroRNA-155 Is Required for Mycobacterium bovis BCG-Mediated Apoptosis of Macrophages

Pathogenic rnycobacteria, including Mycobacterium tuberculosis and Mycobacterium bovis, cause significant morbidity and mortality worldwide. However, the vaccine strain Mycobacterium bovis BCG, unlike virulent strains, triggers extensive apoptosis of infected macrophages, a step necessary for the elicitation of robust protective immunity. We here demonstrate that M. bovis BCG triggers Toll-like receptor 2 (TLR2)-dependent microRNA-155 (miR-155) expression, which involves signaling cross talk among phosphatidylinositol 3-kinase (PI3K), protein kinase C delta (PKC delta), and mitogen-activated protein kinases (MAPKs) and recruitment of NF-kappa B and c-ETS to miR-155 promoter. Genetic and signaling perturbations presented the evidence that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-alpha). Enhanced activation of PKA signaling resulted in the generation of PKA C-alpha; phosphorylation of MSK1, cyclic AMP response element binding protein (CREB), and histone H3; and recruitment of phospho-CREB to the apoptotic gene promoters. The miR-155-triggered activation of caspase-3, BAK1, and cytochrome c translocation involved signaling integration of MAPKs and epigenetic or posttranslational modification of histones or CREB. Importantly, M. bovis BCG infection-induced apoptosis was severely compromised in macrophages derived from miR-155 knockout mice. Gain-of-function and loss-of-function studies validated the requirement of miR-155 for M. bovis BCG's ability to trigger apoptosis. Overall, M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, strongly implicating a novel role for miR-155 in orchestrating cellular reprogramming during immune responses to mycobacterial infection.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

Helix-sheet interconversion in a synthetic pore lining peptide derived from a designed ion channel

We have designed a four-helix protein that is expected to tetramerize in the membrane to form an ion channel with a structurally well defined pore. A synthetic peptide corresponding to the channel lining helix facilitates ion transport across liposomal membranes and largely helical in membranes. Detailed circular dichroism studies of the peptide in methanol, water and methanal-water mixtures reveal that it is helical in methanol, beta-structured in 97.5% water and a combination of these two structures at intermediate compositions of methanol and water. A fluorescence resonance energy transfer study of the peptide shows that the peptide is monomeric in methanol but undergoes extensive anti-parallel aggregation in aqueous solution.

Satellite-derived direct radiative effect of aerosols dependent on cloud cover

Aerosols from biomass burning can alter the radiative balance of the Earth by reflecting and absorbing solar radiation(1). Whether aerosols exert a net cooling or a net warming effect will depend on the aerosol type and the albedo of the underlying surface(2). Here, we use a satellite-based approach to quantify the direct, top-of-atmosphere radiative effect of aerosol layers advected over the partly cloudy boundary layer of the southeastern Atlantic Ocean during July-October of 2006 and 2007. We show that the warming effect of aerosols increases with underlying cloud coverage. This relationship is nearly linear, making it possible to define a critical cloud fraction at which the aerosols switch from exerting a net cooling to a net warming effect. For this region and time period, the critical cloud fraction is about 0.4, and is strongly sensitive to the amount of solar radiation the aerosols absorb and the albedo of the underlying clouds. We estimate that the regional-mean warming effect of aerosols is three times higher when large-scale spatial covariation between cloud cover and aerosols is taken into account. These results demonstrate the importance of cloud prediction for the accurate quantification of aerosol direct effects.

Requirement Of A Diperoxovanadate-Derived Intermediate for the Interdependent Oxidation Of Vanadyl And Nadh

Oxygen release accompanying oxidation of vanadyl by diperoxovanadate was suppressed on addition of NADH. The added NADH was rapidly oxidized, oxygen in the medium was consumed, and the reaction terminated on exhaustion of either NADH or vanadyl. The consumption of oxygen and disappearance of NADH needed small concentrations of diperoxovanadate to initiate and increased with increase in the concentration of vanadyl and NADH or decrease of pH. The products of the reaction were found to be NAD(+) from NADH and vanadate oligomers from vanadyl and oxygen. The reaction was insensitive to catalase and was not dependent on H2O2. The reaction was inhibited by superoxide dismutase, cytochrome c, EDTA, Mn2+, histidine, and DMPO, but not by hydroxyl radical scavengers such as ethanol and benzoate, The ESR spectrum of the reaction mixture showed the presence of the 1:2:2:1 quartet signal typical of a DMPO-OH adduct, but this was not modified by ethanol, This oxygen radical species, possibly of (OV)-O-. type derived from diperoxovanadate, is proposed to have a role in the reactions of oxygen release and NADH oxidation

Microbial transformation of a fungicide-derived amine, dimethyl-p-phenylene diamine by Alcaligenes DM4

Abstract Microbial transformation of N, N-dimethyl-p-phenylene diamine (DMPDA), a microbial product formed from the fungicide fenaminosulf (p-dimethylaminobenzenediazo sodium sulfonate) was studied by enriching microbes in soils treated with the amine. Microorganisms isolated from DMPDA-treated soil belonged to the genera of Micrococcus, Alcaligenes, and Corynebacterium. Of the various isolates, Alcaligenes DM4 showed maximal growth on DMPDA utilizing it as sources of carbon and nitrogen. When grown in mineral salts basal medium containing 0.05% DMPDA to serve as carbon and nitrogen sources, Alcaligenes DM4 grew exponentially up to 18 h. Even though the characterization of the complete pathway of microbial degradation of DMPDA could not be carried out due to the auto-oxidation of the compound, the initial transformation product of DMPDA by Alcaligenes DM4 has been identified as a dimer. The dimer is generated into the culture medium presumably by the extra-cellular oxidase of Alcaligenes DM4. It is suggested that the risk-benefit evaluation on the use of fenaminosulf is to be made taking into consideration the microbial transformations of the fungicide.

Bile Acid Derived PET-Based Cation Sensors: Molecular Structure Dependence of their Sensitivity

A number of bile acid derived photoinduced electron transfer (PET) based sensors for metal ions are prepared. A general strategy for designing the sensor with a modular nature allows for making different molecules capable of sensing different metal ions by a change in the fluorophore and receptor unit. Keeping the basic molecular structure the same, different bile acid base fluoroionophores were prepared inorder to achieve the highest sensitivity toward the metal ions. Thesensors showed similar binding constants for the same metal ion, but the degree Of fluorescence enhancement upon addition of the metal salts were different. The sensitivities of the sensors towards a certain metal were determined from the observed fluorescence enhancement upon addition of the metal salt.

Complexes of lanthanide perchlorates with two new O,N,O ligands derived from antipyraldehyde and acetic and benzoic acid hydrazides

Antipyrine is a well known ligand for lanthanides (I). A forage through the organic literature of pyrazolones reveals that the 4-position of antipyrine is amenable to a wide variety of organic reactions. It should thus be possible to introduce suitable functional groups at this position and design new multidentate ligands for metal ions. It is also found that the coordination chemistry of lanthanides is much less well developed and far fewer ligands have been used for complexation with lanthanide ions compared to that of the d-transition metal ions. Keeping these points in view we have reported earlier, complexes of lanthanides with a bidentate ligand N,N-diethyl-antipyrine-4-carboxamide (2). In this communication we report the synthesis of two new ligands from Schiff base condensation of antipyraldehyde and the hydrazides of acetic and benzoic acids and the complexes formed by these hydrazones with lanthanide perchlorates.

Complexes of lanthanide perchlorates with two new " O,N,O " ligands derived from antipyraldehyde and acetic and benzoic acid hydrazides

Antipyrlne is a well known llgand for lanthanldes (i). A forage through the organic literature of pyrazolones reveals that the 4-position of antipyrlne is amenable to a wide variety of organic reactions. It should thus be possible to introduce suitable functional groups at this position and design new multidentate ligands for metal ions. It is also found that the coordination chemistry of lanthanides is much less well developed and far fewer ligands have been used for complexation with lanthanide ions compared to that of the d-transition metal ions.