Isolation and Th17 Differentiation of Na?ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-β. After incubation for at least 72 hours and for up to five days at 37 °C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.     

ISOLATION AND EXPRESSION OF THE INTERLEUKIN-2 GENE FROM THE KILLER WHALE, ORCINUS ORCA

Complementary DNA encoding interleukin-2 (IL-2) was isolated, cloned, and sequenced from the killer whale, Orcinus orca . The sequence of the killer whale IL-2 coding region consists of 455 nucleotides which translate into a polypeptide containing 151 amino acids. Killer whale IL-2 displays 88%, 88%, 87%, 87%, 85%, 84%, 80%, and 71% nucleotide sequence homology and 76%, 76%, 76%, 73%, 68%, 73%, 64%, and 56% amino acid homology with the cow, sheep, pig, red deer, horse, human, manatee, and mouse, respectively. High levels of killer whale recombinant IL-2 were generated by transiently transfecting killer whale IL-2/pCMV Blue plasmid DNA into cultured monkey kidney cells (Cos-1). Generation of this recombinant IL-2 will allow the development of assays useful for assessing IL-2 levels in the serum and from isolated lymphocytes of killer whales and possibly other species of cetaceans. A major contribution and significance of the in vitro expression of killer whale IL-2 exists in its potential to be used as a therapeutic agent.     

The Target of the NSD Family of Histone Lysine Methyltransferases Depends on the Nature of the Substrate

The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.     

The Lumpy Breast

“Topics in Primary Care Medicine” presents articles on common diagnostic or therapeutic problems encountered in primary care practice. Physicians interested in contributing to the series are encouraged to contact the series'' editors.     

Lack of induced chemical defense in juvenile Alaskan woody plants in response to simulated browsing

Summary Juvenile individuals of five species of Alaskan trees and shrubs were clipped at six levels of intensity and sampled for resin, tannin, nutrient, and carbohydrate content. Clipping caused no induction of putative defensive compounds (resins or tannins), indicating that the high level of resin found in many Alaskan woody plants in response to browsing is best explained as a reversion to a juvenile growth stage rather than an induction of a specific chemical defense.     

Equilibrium binding constants and facile dissociation of novel serum albumin-dicyanoaurate(I) complexes

 Dicyanoaurate(I), Au(CN)₂ ^–, an important metabolite of chrysotherapy agents (anti-arthritic gold drugs), contains two tightly bound cyanide ligands which render it relatively unreactive toward ligand exchange reactions with potential gold-binding ligands. The extent and nature of its binding to bovine serum albumin (BSA), which may modulare the in vivo activity of Au(CN)₂ ^–, were investigated to determine whether Au(CN)₂ ^– might be more bioavailable than other gold complexes. ¹³C NMR spectroscopy, radioisotope tracers, chromatography, ultrafiltration, and atomic spectroscopy, employing Au(¹³CN)₂ ^– or Au(¹⁴CN)₂ ^– as appropriate, revealed two distinct binding mechanisms. The dominant reaction is reversible association (non-specific binding) of intact Au(CN)₂ ^– ions to form BSA·[Au(CN)₂ ^–] _n adducts. Approximately one equivalent binds with an equilibrium binding constant (pH 7.4, 25  °C) of K ₁=5.5 (±1.1)×10⁴, and three additional equivalents bind with a constant of 7.0 (±0.1)×10³. Au(¹³CN)₂ ^– associated with albumin is characterized by a broad ¹³C NMR resonance at δ_C=154.7 ppm compared to the sharp resonance of the free complex at 156.4 ppm. The BSA·[Au(CN)₂ ^–] _n adducts readily dissociate during gel exclusion chromatography and are therefore underestimated, but are retained and accurately quantitated by ultrafiltration methods. The second binding mechanism is a ligand exchange reaction at Cys-34, to form AlbSAuCN, which accounts for only a small fraction (≤11%) of the bound gold. The small extent of the latter interaction differentiates Au(CN)₂ ^– from the gold drugs such as auranofin, aurothiomalate (Myochrysin) and aurothioglucose (Solganol), which undergo ligand exchange at Cys-34 of albumin to form tightly bound gold-protein complexes. The weak interaction at Cys-34 and the facile dissociation of bound, intact Au(CN)₂ ^– are consistent with its putative role as a gold metabolite that can be accumulated intracellularly. Received: 2 July 1997 / Accepted: 24 September 1997