Structural Characterization of the Recombinant P40 Heavy Chain Subunit Monomer and Homodimer of Murine IL-12

Interleukin-12 (IL-12) is a heterodimeric cytokine that consists of two structurally unrelated subunits, P35 and P40. However, when expressed alone in Chinese hamster ovary (CHO) cells, murine P40 showed two species of different molecular weights under nonreducing conditions, a monomeric form of 45 kDa and a homodimer of >97 kDa. Under reducing conditions the two forms migrated as an identical array of species of 40-45 kDa. The monomer was separated from the homodimer under nonreducing conditions by heparin affinity chromatography and the disulfide bond structures of both species were determined by peptide mapping, Edman sequencing, and mass spectrometry. The peptide maps of the two species were identical except for a single peak that changed retention time. Sequencing showed that this peak contained two peptides of identical sequences in both maps. Mass spectrometric analysis of the peak from the >97-kDa species revealed an ion of double the expected mass, thus indicating that the peptide pair had dimerized. Mass analysis of the peak from the 40- to 45-kDa species showed that the peptide pair contained a mass difference that corresponded to that of an extra cysteine and which disappeared upon reduction. Amino acid analysis confirmed that the monomeric form of rmP40 is modified by a reducible cysteine. Structural analysis of the remainder of the cysteine-containing peaks showed that both species of rmP40 contained the same set of intramolecular disulfide bonds. The murine P40 homodimer arises from formation of a single intermolecular disulfide bond at Cys¹⁷⁵. In the monomeric P40, however, this cysteine is capped by an additional cysteine. Purified rmP40 monomer and homodimer inhibited the IL-12-dependent induction of interferon-γ, but neither appeared capable of inducing IL-12-like biological activity.     

Regulation of the selective uptake of high density lipoprotein-associated cholesteryl esters by human fibroblasts and Hep G2 hepatoma cells

We have previously shown that the liver and steroidogenic tissues of rats in vivo and a wider range of cells in vitro, including human cells, selectively take up high density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles. This process is regulated in tissues of rats and in cultured rat cells according to their cholesterol status. In the present study, we examined regulation of HDL selective uptake in cultured human fibroblasts and Hep G2 hepatoma cells. The cholesterol content of these cells was modified by a 20-hr incubation with either low density lipoprotein (LDL) or free cholesterol. Uptake of HDL components was examined in a subsequent 4-6-hr assay using intracellularly trapped tracers: 125I-labeled N-methyl-tyramine-cellobiose-apoA-I (125I-NMTC-apoA-I) to trace apoA-I, and [3H]cholesteryl oleyl ether to trace cholesteryl esters. In the case of fibroblasts, pretreatment with either LDL or free cholesterol resulted in decreased selective uptake (total [3H]cholesteryl ether uptake minus that due to particle uptake as measured by 125I-NMTC-apoA-I). In contrast, HDL particle uptake increased with either form of cholesterol loading. The amount of HDL that was reversibly cell-associated (bound) was increased by prior exposure to free cholesterol, but was decreased by prior exposure to LDL. In the case of Hep G2 cells, exposure to free cholesterol only slightly increased HDL particle uptake; selective uptake decreased after both forms of cholesterol loading, and reversibly bound HDL increased after exposure to free cholesterol, but either did not change or decreased after exposure to LDL. It was excluded that either LDL carried over into the HDL uptake assay or that products secreted by the cultured cells influenced these results. Thus, selective uptake by cells of both hepatic and extrahepatic origin was down-regulated by cholesterol loading, under which conditions HDL particle uptake increased. Total HDL binding was not directly correlated with either the rate of selective uptake or the rate of HDL particle uptake or the cholesterol status of the cells, suggesting more than one type of HDL binding site.     

Population structure and marker�Ctrait association analysis of the US peanut (Arachis hypogaea L.) mini-core collection

Peanut (Arachis hypogaea L.) is one of the most important oilseed and nutritional crops in the world. To efficiently utilize the germplasm collection, a peanut mini-core containing 112 accessions was established in the United States. To determine the population structure and its impact on marker-trait association, this mini-core collection was assessed by genotyping 94 accessions with 81 SSR markers and two functional SNP markers from fatty acid desaturase 2 (FAD2). Seed quality traits (including oil content, fatty acid composition, flavonoids, and resveratrol) were obtained through nuclear magnetic resonance (NMR), gas chromatography (GC), and high-performance liquid chromatography (HPLC) analysis. Genetic diversity and population structure analysis identified four major subpopulations that are related to four botanical varieties. Model comparison with different levels of population structure and kinship control was conducted for each trait and association analyses with the selected models verified that the functional SNP from the FAD2A gene is significantly associated with oleic acid (C18:1), linoleic acid (C18:2), and oleic-to-linoleic (O/L) ratio across this diverse collection. Even though the allele distribution of FAD2A was structured among the four subpopulations, the effect of FAD2A gene remained significant after controlling population structure and had a likelihood-ratio-based R ( 2 ) (R ( LR ) ( 2 ) ) value of 0.05 (oleic acid), 0.09 (linoleic acid), and 0.07 (O/L ratio) because the FAD2A alleles were not completely fixed within subpopulations. Our genetic analysis demonstrated that this peanut mini-core panel is suitable for association mapping. Phenotypic characterization for seed quality traits and association testing of the functional SNP from FAD2A gene provided information for further breeding and genetic research.     

MicroRNA pathways modulate polyglutamine-induced neurodegeneration

Nine human neurodegenerative diseases are due to expansion of a CAG repeat- encoding glutamine within the open reading frame of the respective genes. Polyglutamine (polyQ) expansion confers dominant toxicity, resulting in neuronal degeneration. MicroRNAs (miRNAs) have been shown to modulate programmed cell death during development. To address whether miRNA pathways play a role in neurodegeneration, we tested whether genes critical for miRNA processing modulated toxicity induced by the spinocerebellar ataxia type 3 (SCA3) protein. These studies revealed a striking enhancement of polyQ toxicity upon reduction of miRNA processing in Drosophila and human cells. In parallel genetic screens, we identified the miRNA bantam (ban) as a potent modulator of both polyQ and tau toxicity in flies. Our studies suggest that ban functions downstream of toxicity of the SCA3 protein, to prevent degeneration. These findings indicate that miRNA pathways dramatically modulate polyQ- and tau-induced neurodegeneration, providing the foundation for new insight into therapeutics.     

Oxyresveratrol dampens neuroimmune responses in vivo: a selective effect on TNF-alpha

Consumption of nutrients rich in hydroxystilbenes has been promoted because of their health benefits, including dampening of inflammatory responses. However, few studies have examined their effects in vivo. Here, we show that the hydroxystilbene oxyresveratrol (trans-2,3',4,5'-tetrahydroxystilbene: o-RES) blocked hypothermia but caused no significant effect on the febrile response to the immune stimulus, bacterial LPS in rats. This was associated with a reduction in the LPS-induced plasma cytokine, tumor necrosis factor (TNF)-alpha, but not IL-6. Both IL-6-stimulated STAT-3 and LPS-induced cycoloxygenase-2 expression in the hypothalamus were not affected by o-RES. These data strongly suggest that the o-RES-induced dampening of neuroimmune responses is largely due to its inhibitory effect on TNF-alpha production. In contrast to in vitro experiments, o-RES has no direct effect on NF-kappaB signaling pathway in vivo. The specific inhibitory effect of o-RES on TNF-alpha opens new avenues for the clinical use of o-RES in pathological conditions where excessive production of TNF-alpha is deleterious.     

The interaction profile of homologous recombination repair proteins RAD51C,RAD51D and XRCC2 as determined by proteomic analysis

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.