Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site

The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.     

Five-color flow cytometric analysis of swine lymphocytes for detection of proliferation,apoptosis, viability,and phenotype

BACKGROUND: The objective of this study was to develop a method to simultaneously examine phenotype, proliferation, apoptosis, and death of antigen-stimulated porcine lymphocytes. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from pigs vaccinated with a Brachyspira hyodysenteriae bacterin. RESULTS: Once isolated, PBMCs were stained with the fluorescent membrane intercalating dye, PKH67, and cultured with or without B. hyodysenteriae whole-cell sonicate antigen. Serial samples of nonstimulated and B. hyodysenteriae-stimulated PBMCs were harvested for flow cytometric analysis. Fluorochrome excitation was performed with spatially separated air-cooled argon and red helium neon laser beams. Five-color analysis included signal detection of PKH67 (proliferation), phycoerythrin (cell surface antigen), Texas Red phycoerythrin tandem (cell surface antigen), allophycocyanin (annexin V), and 7-amino-actinomysin D (7AAD; viability). For analysis, gates were set on live (annexin V(-), 7AAD(-)), intact apoptotic (annexin V(+), 7AAD(dim)), and live plus intact apoptotic (annexin V(+/-), 7AAD(dim/-)) cells, and the phenotypes of PBMCs within these populations were determined during the course of the in vitro response. Dead cells (i.e., 7AAD(bright)) were excluded from the analysis. CONCLUSION: Application of this method for the determination of porcine lymphocyte subset proliferation is presented.     

Correlation between Photoinhibition Sensitivity and the Rates and Relative Extents of Xanthophyll Cycle De-epoxidation in Chlorina Mutants of Barley (Hordeum vulgare L.)

We compared photoinhibition sensitivity to high irradiance (HI) in wild-type barley (wt) and both its chlorina f ₁₀₄-nuclear gene mutant, that restricts chlorophyll (Chl) a and Chl b synthesis, and its f ₂-nuclear gene mutant, that inhibits all Chl b synthesis. Both F_v/F_m and _PS2 decreased more significantly in f ₂ than f ₁₀₄ and wt with duration of HI exposure. Chl degraded more rapidly in the f ₂ than in either f ₁₀₄ or wt. Most sensitivity to photoinhibition was exhibited for f ₂, whereas there was little difference in response to HI between the f ₁₀₄ and wt. The highest de-epoxidation (DES) value at every time point of exposure to HI was measured for f ₂, whereas the wt had the lowest value among the three strains. There were two lifetime components resolved for the conversion of violaxanthin (V) to zeaxanthin plus antheraxanthin (Z + A). The most rapid lifetime was around 6 min and the slower lifetime was >140 min, in both the mutants and wt. However, the wt and f ₁₀₄ both displayed larger amplitudes of both de-epoxidation lifetimes than f ₂. The difference between the final de-epoxidation state (DES = [Z + A]/[V + A + Z]) in the light compared to the dark expressed as DES for wt, f ₁₀₄, and f ₂ was 0.630, 0.623, and 0.420, respectively. The slow lifetime component and overall larger DES in the wt and f ₁₀₄ correlated with more photoprotection, as indicated by relatively higher F_v/F_m and _PS2, compared to the f ₂. Hence the photoprotection against photoinhibition has no relationship with the absolute DES value, but there is a strong relationship with de-epoxidation rate and relative extent or DES.     

Osmotic characteristics of mouse spermatozoa in the presence of extenders and sugars

Successful cryopreservation requires cells to tolerate volume excursions experienced during permeating cryoprotectant equilibration and during cooling and warming. However, prior studies have demonstrated that mouse spermatozoa are extremely sensitive to osmotically induced volume changes. A series of three experiments were conducted 1) to test the efficacy of two commonly used extender media components, egg yolk (EY) and skim milk (SM), in broadening the osmotic tolerance limits (OTL) of ICR and B6C3F1 murine spermatozoa; 2) to determine if the extender components affected sperm plasma membrane permeability coefficients for water and cryoprotective agent (CPA) characteristics; and 3) to test the effects of permeating and nonpermeating CPA on mouse sperm morphology. In experiment 1, sperm samples were added to 150, 225, 300, 450, or 600 mOsm NaCl, EY, SM, sucrose, or choline chloride at 22 degrees C and then returned to isosmotic conditions. In experiment 2, epididymal sperm were preequilibrated in 1 M glycerol (Gly) or 2 M ethylene glycol (EG) prepared in SM extender, abruptly exposed to isosmotic conditions at 22, 15, or 2 degrees C, and the corresponding volume excursions were measured and analyzed. In experiment 3, the effects of permeating CPA (0.3 M EG or dimethyl sulfoxide) or nonpermeating CPA (12% sucrose or 18% raffinose) on sperm morphology (i.e., principle midpiece folding and putative membrane fusion) were evaluated. Experiment 1 showed that spermatozoa from ICR and B6C3F1 mice have effectively broader OTL when exposed to EY or SM extenders. The results of experiment 2 indicated that, for ICR sperm, the activation energy (E(a)) for the hydraulic conductivity (L(p)) was unchanged in SM extender. However, for B6C3F1 sperm, there were significant differences in E(a) of L(p) in the presence of Gly and EG. The result of experiment 3 indicated that permeating CPAs damage sperm membrane integrity, causing a high frequency of head-to-tail or tail-to-tail membrane fusion, whereas this occurrence in the presence of nonpermeating CPA was less than 3%. Finally, the results of experiments 1 and 2 were combined in a mathematical model to predict Gly and EG addition and removal in the presence of SM extender, which would prevent mouse sperm membrane damage. These predictions indicated that, for ICR sperm, both Gly and EG may be added and removed in a single step. However, for B6C3F1 spermatozoa, Gly required a two-step addition while EG only required a single step. For removal from B6C3F1 sperm, Gly required a three-step removal process while EG required a two-step removal.     

Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties

Oligosaccharyltransferase (OST) is an integral membrane protein that catalyzes N-linked glycosylation of nascent proteins in the lumen of the endoplasmic reticulum. Although the yeast OST is an octamer assembled from nonhomologous subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Wbp1p, Swp1p, and Stt3p), the composition of the vertebrate OST was less well defined. The roles of specific OST subunits remained enigmatic. Here we show that genomes of most multicellular eukaryotes encode two homologs of Stt3p and mammals express two homologs of Ost3p. The Stt3p and Ost3p homologs are assembled together with the previously described mammalian OST subunits (ribophorins I and II, OST48, and DAD1) into complexes that differ significantly in enzymatic activity. Tissue and cell type-specific differences in expression of the Stt3p homologs suggest that the enzymatic properties of oligosaccharyltransferase are regulated in eukaryotes to respond to alterations in glycoprotein flux through the secretory pathway and may contribute to tissue-specific glycan heterogeneity.     

Separation methods applicable to the evaluation of enzyme-inhibitor and enzyme-substrate interactions

Enzymes catalyze a rich variety of metabolic transformations, and do so with very high catalytic rates under mild conditions, and with high reaction regioselectivity and stereospecificity. These characteristics make biocatalysis highly attractive from the perspectives of biotechnology, analytical chemistry, and organic synthesis. This review, containing 128 references, focuses on the use of separation techniques in the elucidation of enzyme-inhibitor and enzyme-substrate interactions. While coverage of the literature is selective, a broad perspective is maintained. Topics considered include chromatographic methods with soluble or immobilized enzymes, capillary electrophoresis, biomolecular interaction analysis tandem mass spectrometry (BIA-MS), phage and ribosomal display, and immobilized enzyme reactors (IMERs). Examples were selected to demonstrate the relevance and application of these methods for determining enzyme kinetic parameters, ranking of enzyme inhibitors, and stereoselective synthesis and separation of chiral entities.     

Avidin- or streptavidin-biotin as a highly sensitive method to stain total protein on membranes

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate α-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5ng of transferred protein in a single band and is thus 5–10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.