Allosterism and Na++-d-glucose cotransport kinetics in rabbit jejunal vesicles: Compatibility with mixed positive and negative cooperativities in a homo- dimeric or tetrameric structure and experimental evidence for only one transport protein involved

Summary We first present two simple dimeric models of cotransport that may account for all of the kinetics of Na⁺⁺-d-glucose cotransport published so far in the small intestine. Both the sigmoidicity in the Na⁺⁺ activation of transport (positive cooperativity) and the upward deviations from linearity in the Eadie-Hofstee plots relative to glucose concentrations (negative cooperativity) can be rationalized within the concept of allosteric kinetic mechanisms corresponding to either of two models involving sequential or mixed concerted and sequential conformational changes. Such models also allow for 2 Na⁺⁺ 1 S and 1 Na⁺⁺ 1 S stoichiometries of cotransport at low and high substrate concentrations, respectively, and for partial inhibition by inhibitors or substrate analogues. Moreover, it is shown that the dimeric models may present physiological advantages over the seemingly admitted hypothesis of two different cotransporters in that tissue. We next address the reevaluation of Na⁺⁺-d-glucose cotransport kinetics in rabbit intestinal brush border membrane vesicles using stable membrane preparations, a dynamic approach with the Fast Sampling Rapid Filtration Apparatus (FSRFA), and both nonlinear regression and statistical analyses. Under different conditions of temperatures, Na⁺⁺ concentrations, and membrane potentials clamped using two different techniques, we demonstrate that our data can be fully accounted for by the presence of only one carrier in rabbit jejunal brush border membranes since transport kinetics relative to glucose concentrations satisfy simple Michaelis-Menten kinetics. Although supporting a monomeric structure of the cotransporter, such a conclusion would conflict with previous kinetic data and more recent studies implying a polymeric structure of the carrier protein. We thus consider a number of alternatives trying to reconcile the observation of Michaelis-Menten kinetics with allosteric mechanisms of cotransport associated with both positive and negative cooperativities for Na⁺⁺ and glucose binding, respectively. Such models, implying energy storage and release steps through conformational changes associated with ligand binding to an allosteric protein, provide a rational hypothesis to understand the long-time debated question of energy transduction from the Na⁺⁺ electrochemical gradient to the transporter.This research was supported by grant MT-7607 from the Medical Research Council of Canada. One of the authors (A.B.) was supported by a scholarship from the Fonds de la Recherche en Santé du Québec and C. C. was supported by a fellowship from the GRTM. The technical assistance of Mrs. C. Leroy has been greatly appreciated. The authors also thank D.D. Maenz and C. Malo for insightful discussions and C. Gauthier for the art work.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The K_m values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a K_i of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.     

S-adenosyl-l-methionine inhibits phosphoinositide metabolism in the rat brain synaptosomal suspensions

S-adenosyl-l-methionine (AdoMet) has been reported to affect events linked to noradrenergic neurotransmission. In the present work, we studied the effect of AdoMet on norepinephrine (NE)-stimulated inositol phosphate production in³H-inositol-labelled crude synaptosomal suspensions of rat brain. AdoMet (50–1000 M) decreased both the synthesis of labelled polyphosphoinositide (30–50%) and the release of inositol mono- and bisphosphate (40–50%). The AdoMet effect was not dependent on NE concentration (10–1000 M), suggesting that the inhibition of inositol phosphate release was not the result of a modification of the norepinephrine binding to its receptor sites. S-adenosyl-L-homocysteine (AdoHcy) (1 mM) an inhibitor of methyltransferase activities, partially inhibited (70%) the AdoMet (0.1 mM) effect, indicating that the methylation processes cannot explain all the effects observed. We conclude that, in addition to previously reported effects of AdoMet on NE transport, AdoMet may reduce NE-linked intracellular signalling.     

Writing failure: knowledge production,temporalities, ethics,and traces

This volume follows failures out into the world, exploring how they unfold ethnographically. Taking a longer view shows how objects, narratives, and diagnoses of failures may be crafted, acted on, suffered, resisted – unmade or recomposed. Thus while tropes and diagnoses of failure can temporarily (re)organize, narrate, and stabilize the world, the kinds of failures explored here also indicate a mode of uncontainable excess that refuses the boundedness of knowledge objects, temporalities, and spaces. This volume offers three main interventions. The first concerns knowledge production: how objects of failure are crafted through selective ways of knowing that occlude both other modes of apprehension at different scales and failure's many affective valences. The second thinks through the knotted temporalities – whether pasts, futures, suspended presents, or repetition and sedimentation – that make and are made by failure. Finally, writing about unfurling failures requires careful attention to non-linear reverberations and traces as well as to open-ended and mobile narratives that produce different social and material effects.     

Growth and respiratory oxidation of reduced sulfur compounds by intact cells ofThiobacillus novellus (type strain) grown on thiosulfate

Thiobacillus novellus (type strain) was grown chemolithoautrophically on thiosulfate in batch cultures under microaerophilic conditions. Under these conditions,T. novellus grew exponentially (=0.05–0.06 h^–1). The respiratory oxidation rates of tetrathionate, thiosulfate, elemental sulfur (S^o), and sulfite were measured respirometrically with an oxygen electrode, with exponentially growing cells. Cells growing on thiosulfate as the unique energy source retain thiosulfate-oxidizing activity, S^o-oxidizing activity (SOA), and very high sulfite-oxidizing activity, but lack respiratory tetrathionate-oxidizing activity. HQNO (50 m), an inhibitor of the quinone-cytochrome b region, strongly inhibited the SOA (70%), moderately the sulfite-oxidizing activity (45%), and poorly the thiosulfate-oxidizing activity (15%), 1mm KCN totally inhibited (>89%) all respiratory activities. This study confirms that inThiobacillus novellus, as well as in otherThiobacilli, SOA is present in cells grown with thiosulfate as sole electron donor. SOA appears not to be an oxygenase; it is linked to the respiratory chain, and the electrons are probably released in the quinone-cytochrome b region.     

Cobalamin producers and prokaryotic consumers in the Northwest Atlantic

Cobalamin availability can influence primary productivity and ecological interactions in marine microbial communities. The characterization of cobalamin sources and sinks is a first step in investigating cobalamin dynamics and its impact on productivity. Here, we identify potential cobalamin sources and sinks on the Scotian Shelf and Slope in the Northwest Atlantic Ocean. Functional and taxonomic annotation of bulk metagenomic reads, combined with analysis of genome bins, were used to identify potential cobalamin sources and sinks. Cobalamin synthesis potential was mainly attributed to Rhodobacteraceae, Thaumarchaeota, and cyanobacteria (Synechococcus and Prochlorococcus). Cobalamin remodelling potential was mainly attributed to Alteromonadales, Pseudomonadales, Rhizobiales, Oceanospirilalles, Rhodobacteraceae, and Verrucomicrobia, while potential cobalamin consumers include Flavobacteriaceae, Actinobacteria, Porticoccaceae, Methylophiliaceae, and Thermoplasmatota. These complementary approaches identified taxa with the potential to be involved in cobalamin cycling on the Scotian Shelf and revealed genomic information required for further characterization. The Cob operon of Rhodobacterales bacterium HTCC2255, a strain with known importance in cobalamin cycling, was similar to a major cobalamin producer bin, suggesting that a related strain may represent a critical cobalamin source in this region. These results enable future inquiries that will enhance our understanding of how cobalamin shapes microbial interdependencies and productivity in this region.     

Wax as a Mechanism for Protection against Photoinhibition — A Study of Cotyledon orbiculata

The leaves of the Crassulacean acid metabolism plant Cotyledon orbiculata have a waxy coating which is highly reflective but can be easily removed by brushing. This provided an ideal system in which to investigate the role of epidermal wax as a possible photoprotectant. Removal of the wax, prior to exposure to natural sunlight, resulted in substantial decreases in F_v/F_m and in severe cases evidence of photoinhibitory damage, as indicated by a rise in F_o. Leaves from which wax had been removed also showed higher conversion of violaxanthin to zeaxanthin than waxed leaves. Recovery of brushed leaves over a 12 day period was correlated with an increase in the total pool of xanthophyll cycle components. This study suggests that the presence of highly reflective wax on the epidermis may confer significant photoprotection to plants exposed to high solar radiation environments.