Defining nicotinic agonist binding surfaces through photoaffinity labeling

Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.     

Inactivation of Alicyclobacillus acidoterrestris vegetative cells in model system,apple, orange and tomato juices by high hydrostatic pressure

The objective of this study is to determine the effect of high hydrostatic pressure (HHP) on inactivation of Alicyclobacillus acidoterrestris vegetative cells in a model system (BAM broth) and in orange, apple and tomato juices. The shelf-life stability of pressurized juices is also studied. In general the viability loss was enhanced significantly as the level of pressure and temperature were increased (P < 0.05). 4.70 log cycle reduction was obtained after pressurization at 350 MPa at 50 °C for 20 min in BAM broth whereas thermal treatment at 50 °C for 20 min caused only 1.13 log cycle inactivation showing the effectiveness of HHP treatment on inactivation. The D values for pressure (350 MPa at 50 °C) and temperature (50 °C) treatments were 4.37 and 18.86 min in BAM broth, respectively. All juices were inoculated with A. acidoterrestris cells to 10⁶ c.f.u./ml and were pressurized at 350 MPa at 50 °C for 20 min. More than 4 log cycle reduction was achieved in all juices studied immediately after pressurization. The pressurized juices were also stored up to 3 weeks at 30 °C and the viable cell numbers of A. acidoterrestris in orange, apple and tomato juices were 3.79, 2.59 and 2.27 log cycles, respectively after 3 weeks. This study has indicated that A. acidoterrestris vegetative cells can be killed by HHP at a predictable rate even at temperatures at which the microorganism would normally grow.     

Murine interferon-gamma inducible protein-10 (IP-10) secreted by Lactococcus lactis chemo-attracts human CD3+ lymphocytes

Chemokines are members of the super family of cytokines necessary for leukocyte recruitment in tissues and lymphoid organs. The interferon-gamma inducible protein-10 (IP-10) chemo-attracts CXCR3-expressing cells, such as activated T lymphocytes and monocytes. We have genetically engineered a strain of Lactococcus lactis to secrete a biologically active murine IP-10 that interacts with human CXCR3, its homolog receptor, and chemo-attracts human CD3+ T lymphocytes.     

Improvement of yield of Pleurotus eryngii var. eryngii by substrate supplementation and use of a casing overlay

Improved yield and biological efficiency (BE) of Pleurotus eryngii var. eryngii were achieved by supplementation of substrate with a commercial delayed-release nutrient and use of a casing overlay. Yield increases of 14% were achieved from cased substrates that were supplemented at time of casing with delayed-release nutrient (Remo’s). Use of a casing layer enhanced yield by 141% over non-cased substrates. When casing and substrate supplementation were combined, yield increased 179% over non-cased/non-supplemented substrates. Mushrooms harvested from cased substrates were darker in color and solids contents were lower compared to non-cased substrates. An additional break of mushrooms was harvested from non-cased “spent” substrate by fragmenting and re-supplementing the substrate prior to the application of a casing overlay. Three production methods were compared for their effect on mushroom yield: “standard”, “casing” and “casing after first break”. Casing of the substrate before first break (“casing” production method) resulted in the highest yield and biological efficiency.     

Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94

The structural basis for the coupling of ATP binding and hydrolysis to chaperone activity remains a central question in Hsp90 biology. By analogy to MutL, ATP binding to Hsp90 is thought to promote intramolecular N-terminal dimerization, yielding a molecular clamp functioning in substrate protein activation. Though observed in studies with recombinant domains, whether such quaternary states are present in native Hsp90s is unknown. In this study, native subunit interactions in GRP94, the endoplasmic reticulum Hsp90, were analyzed using chemical cross-linking in conjunction with tandem mass spectrometry. We report the identification of two distinct intermolecular interaction sites. Consistent with previous studies, one site comprises the C-terminal dimerization domain. The remaining site represents a novel intermolecular contact between the N-terminal and middle (M) domains of opposing subunits. This N+M domain interaction was present in the nucleotide-empty, ADP-, ATP-, or geldanamycin-bound states and could be selectively disrupted upon addition of synthetic geldanamycin dimers. These results identify a compact, intertwined quaternary conformation of native GRP94 and suggest that intersubunit N+M interactions are integral to the structural biology of Hsp90.     

Axoplasm isolation from peripheral nerve

Localized changes in the composition of axonal cytoplasm (axoplasm) are critical for many biological processes, including axon guidance, responses to injury, neurite outgrowth, and axon‐glia interactions. Biochemical and molecular studies of these mechanisms have been heavily focused on in vitro systems because of the difficulty of obtaining subcellular extracts from mammalian tissues in vivo. As in vitro systems might not replicate the in vivo situation, reliable methods of axoplasm extraction from whole nerve would be helpful for mechanistic studies on axons. Here we develop and evaluate a new procedure for preparation of axoplasm from rat peripheral nerve, based on incubation of separated short segements of nerve fascicles in hypotonic medium to separate myelin and lyse nonaxonal structures, followed by extraction of the remaining axon‐enriched material. We show that this new procedure reduces serum and glial cell contamination and facilitates proteomic analyses of axonal contents. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010     

Single ryanodine receptor channel basis of caffeine's action on Ca2+ sparks

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ～75% and single RyR2 opening frequency ～150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.     

Cyclohexane-1,2-dione hydrolase from denitrifying Azoarcus sp. strain 22Lin, a novel member of the thiamine diphosphate enzyme family

Alicyclic compounds with hydroxyl groups represent common structures in numerous natural compounds, such as terpenes and steroids. Their degradation by microorganisms in the absence of dioxygen may involve a C-C bond ring cleavage to form an aliphatic intermediate that can be further oxidized. The cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) from denitrifying Azoarcus sp. strain 22Lin, grown on cyclohexane-1,2-diol as a sole electron donor and carbon source, is the first thiamine diphosphate (ThDP)-dependent enzyme characterized to date that cleaves a cyclic aliphatic compound. The degradation of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate comprises the cleavage of a C-C bond adjacent to a carbonyl group, a typical feature of reactions catalyzed by ThDP-dependent enzymes. In the subsequent NAD(+)-dependent reaction, 6-oxohexanoate is oxidized to adipate. CDH has been purified to homogeneity by the criteria of gel electrophoresis (a single band at ～59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (～105 kDa; gel filtration). As isolated, CDH contains 0.8 ± 0.05 ThDP, 1.0 ± 0.02 Mg(2+), and 1.0 ± 0.015 flavin adenine dinucleotide (FAD) per monomer as a second organic cofactor, the role of which remains unclear. Strong reductants, Ti(III)-citrate, Na(+)-dithionite, and the photochemical 5-deazaflavin/oxalate system, led to a partial reduction of the FAD chromophore. The cleavage product of CDO, 6-oxohexanoate, was also a substrate; the corresponding cyclic 1,3- and 1,4-diones did not react with CDH, nor did the cis- and trans-cyclohexane diols. The enzymes acetohydroxyacid synthase (AHAS) from Saccharomyces cerevisiae, pyruvate oxidase (POX) from Lactobacillus plantarum, benzoylformate decarboxylase from Pseudomonas putida, and pyruvate decarboxylase from Zymomonas mobilis were identified as the closest relatives of CDH by comparative amino acid sequence analysis, and a ThDP binding motif and a 2-fold Rossmann fold for FAD binding could be localized at the C-terminal end and central region of CDH, respectively. A first mechanism for the ring cleavage of CDO is presented, and it is suggested that the FAD cofactor in CDH is an evolutionary relict.