Class II MHC antigen-positive macrophages from human placentae suppress strong MLR and CML reactions

Placental adherent cells (PAC), derived by mild enzymic digestion of human placentae and adherence to plastic, were tested for their ability to suppress MLR and CML reactions. Stimulation of allogeneic T cells by cord blood mononuclear cells or by a strongly stimulatory B lymphoblastoid cell line were consistently inhibited by all PAC preparations tested. PAC were fractionated by Fc rosetting and Percoll gradients to produce a number of bands. Suppression correlated with the content of macrophages in each band and was strongest in band 5 which generally contained 94-100% macrophages. The suppressive effect was not inhibited by indomethacin. The possible role of macrophages in suppressing immune reactivity in the placenta is discussed.     

Modeling-Enabled Characterization of Novel NLRX1 Ligands

Nucleotide-binding domain and leucine-rich repeat containing (NLR) family are intracellular sentinels of cytosolic homeostasis that orchestrate immune and inflammatory responses in infectious and immune-mediated diseases. NLRX1 is a mitochondrial-associated NOD-like receptor involved in the modulation of immune and metabolic responses. This study utilizes molecular docking approaches to investigate the structure of NLRX1 and experimentally assesses binding to naturally occurring compounds from several natural product and lipid databases. Screening of compound libraries predicts targeting of NLRX1 by conjugated trienes, polyketides, prenol lipids, sterol lipids, and coenzyme A-containing fatty acids for activating the NLRX1 pathway. The ligands of NLRX1 were identified by docking punicic acid (PUA), eleostearic acid (ESA), and docosahexaenoic acid (DHA) to the C-terminal fragment of the human NLRX1 (cNLRX1). Their binding and that of positive control RNA to cNLRX1 were experimentally determined by surface plasmon resonance (SPR) spectroscopy. In addition, the ligand binding sites of cNLRX1 were predicted in silico and validated experimentally. Target mutagenesis studies demonstrate that mutation of 4 critical residues ASP677, PHE680, PHE681, and GLU684 to alanine resulted in diminished affinity of PUA, ESA, and DHA to NLRX1. Consistent with the regulatory actions of NLRX1 on the NF-κB pathway, treatment of bone marrow derived macrophages (BMDM)s with PUA and DHA suppressed NF-κB activity in a NLRX1 dependent mechanism. In addition, a series of pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the regulatory function of PUA on colitis is NLRX1 dependent. Thus, we identified novel small molecules that bind to NLRX1 and exert anti-inflammatory actions.     

Targeted tandem affinity purification of PSD‐95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K⁺ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K⁺ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.     

Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks

p53 and p19(ARF) are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19(ARF) deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19(ARF)-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.     

Production and purification of human indoleamine 2,3-dioxygenase (HuIDO) protein in a baculovirus expression system and production and characterization of egg yolk antibody against the purified HuIDO

The human indoleamine 2,3-dioxygenase (HuIDO) baculoviral construct, for expression of HuIDO protein with a hexa-histidine and FLAG (DYKDDDDK) tag, was produced using the BacPAK Baculovirus Expression System. HuIDO baculovirus was used to infect Sf21 insect cells to produce functionally active protein in large amounts. Conditions for protein purification by metal affinity chromatography were determined and optimized. Addition of haemin ensured optimal activity of the purified heme-containing oxygenase. The soluble purified protein was used to immunize a chicken to produce large quantities of polyclonal IgY against HuIDO. The anti-HuIDO IgY antibody specifically detected HuIDO produced by a range of cell types including transfectants and native HuIDO expression induced in IFN-gamma-stimulated cells. The antibody detected HuIDO in cell lysates by western blotting and in the cytoplasm of cells by microscopy. The antibody was unable to block the function of the enzyme, indicating that this antibody binds outside the active site of HuIDO.     

Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes

A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50-80 mm KCl, NaCl, or LiCl. In addition, mitochondria isolated from apoptotic cells using low ionic strength buffer bound a greater proportion of endogenous cytochrome c. In contrast to cytochrome c, Smac/DIABLO and Omi/HtrA2 were released independent of ionic strength, and AIF and EndoG behaved as if they are exposed to the intermembrane space but tethered to or within the inner membrane. AIF and EndoG were also not released by active caspases, which suggests their involvement in apoptosis may be limited. In summary, whereas tBid permeabilizes the outer membrane to cytochrome c, Smac/DIABLO, and Omi/HtrA2, the release of cytochrome c during apoptosis will be underestimated unless sufficient ionic strength is maintained to overcome the electrostatic association of cytochrome c with membranes.     

The effects of cropping corn on the extractability of zinc added to a calcareous soil

The effects of cropping corn on the decrease in the extractability of Zn added to a calcareous soil were studied by a pot experiment and chemical extractions. The results show that the concentrations of Zn exchangeable with MgCl₂ (EXC-Zn) and extractable with DTPA (DTPA-Zn) in the soils with added Zn decreased with time. The processes associated with the decrease in extractability in DTPA of Zn added to soil can be described aptly by a diffusion equation which gives the proportion of added Zn in the non-DTPA fraction as a function of the square root of incubation time. This result suggests that the diffusion of Zn cations into microporous solids is a rate-limiting reaction. The relative diffusion rate coefficients (D/r²) were found to be 1.95×10^-10 and 3.34×10^-10 sec^-1 in the soils with added Zn of 20 and 60 mg kg^-1, respectively. Compared with uncropped soil, the concentrations of DTPA-Zn in the soils with added Zn were decreased by cropping. The decrease of DTPA-Zn in the soils in the presence of corn can be attributed to both its acquisition by corn and other processes associated with the growing of corn. The activity of plant roots would appear to enhance the process of decrease in the extractability in DTPA of Zn added to the soil. The source of Zn uptake by corn was affected by the loading or activity of Zn in soil. In the soil with low available Zn, the DTPA non-extractable Zn (non-DTPA-Zn) was mobilized and taken up by corn. In the soils with high available Zn, e.g. the recently added Zn, only EXC-Zn and a part of the DTPA-Zn were taken up by corn.