Cellular localization of D-lactate dehydrogenase and NADH oxidase from Archaeoglobus fulgidus

Members of the genus Archaeoglobus are hyperthermophilic sulfate reducers with an optimal growth temperature of 83 degrees C. Archaeoglobus fulgidus can utilize simple compounds including D-lactate, L-lactate and pyruvate as the sole substrate for carbon and electrons for dissimilatory sulfate reduction. Previously we showed that this organism makes a D-lactate dehydrogenase (Dld) that requires FAD and Zn2+ for activity. To determine the cellular location and topology of Dld and to identify proteins that interact with Dld, an antibody directed against Dld was prepared. Immunocytochemical studies using gold particle-coated secondary antibodies show that more than 85% of Dld is associated with the membrane. A truncated form of Dld was detected in immunoblots of whole cells treated with protease, showing that Dld is an integral membrane protein and that a significant portion of Dld, including part of the FAD-binding pocket, is outside the membrane facing the S-layer. The gene encoding Dld is part of an operon that includes noxA2, which encodes one of several NADH oxidases in A. fulgidus. Previous studies have shown that NoxA2 remains bound to Dld during purification. Thin sections of A. fulgidus probed simultaneously with antibodies against Dld and NoxA2 show that both proteins co-localized to the same sites in the membrane. Although these data show a tight interaction between NoxA2 and Dld, the role of NoxA2 in electron transport reactions is unknown. Rather, NoxA2 may protect proteins involved in electron transfer by reducing O2 to H2O2 or H2O.     

Proteomic studies of the intrinsically unstructured mammalian proteome

Intrinsically unstructured proteins (IUPs) represent an important class of proteins primarily involved in cellular signaling and regulation. The aim of this study was to develop methodology for the enrichment and identification of IUPs. We show that heat treatment of NIH3T3 mouse fibroblast cell extracts at 98 degrees C selects for IUPs. The majority of these IUPs were cytosolic or nuclear proteins involved in cell signaling or regulation. These studies represent the first large-scale experimental investigation of the intrinsically unstructured mammalian proteome.     

Deuterium oxide effects on excitation-contraction coupling of skeletal muscle

²H₂O (99.8%) Ringer's solution greatly reduces the twitch and tetanus of frog sartorius muscle and, as specially shown here, slows the onset features of the mechanical output of the twitch by: (a) increasing the time (L_R) from stimulus to start of latency relaxation; (b) slowing the developmet of the latency relaxation, and (c) greatly decreasing the rate of onset of tension development. These changes reflect effects of ²H₂O on excitation-contraction coupling and they represent the critical direct effects of ²H₂O on muscle since it does not depress either the action potential or the intrinsic myofibrillar contractility. The increase in L_R is attributed to slowed inward electrical propagation in the T-tubule. But the critical effect of ²H₂O on frog muscle is to greatly depress mobilization of activator Ca²⁺. The depression of the Ca²⁺ mobilization and of its effects on the activation of contraction evidently result from (a) a lowered rate of release of Ca²⁺ from the sarcoplasmic reticulum, as indicated by the slowed development of the latency relaxation, (b) a decreased amount of Ca²⁺ released in a twitch, and (c) a reduced speed of diffusion of the Ca²⁺ to the contractile filaments. The depressed mobilization of Ca²⁺ is apparently the essential cause of ²H₂O's general depression of twitch and tetanus output.     

Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation

Ubiquitin-like proteins (ub-lps) are conjugated by a conserved enzymatic pathway, involving ATP-dependent activation at the C terminus by an activating enzyme (E1) and formation of a thiolester intermediate with a conjugating enzyme (E2) prior to ligation to the target. Ubc9, the E2 for SUMO, synthesizes polymeric chains in the presence of its E1 and MgATP. To better understand conjugation of ub-lps, we have performed mutational analysis of Saccharomyces cerevisiae Ubc9p, which conjugates the SUMO family member Smt3p. We have identified Ubc9p surfaces involved in thiolester bond and Smt3p-Smt3p chain formation. The residues involved in thiolester bond formation map to a surface we show is the E1 binding site, and E2s for other ub-lps are likely to bind to their E1s at a homologous site. We also find that this same surface binds Smt3p. A mutation that impairs binding to E1 but not Smt3p impairs thiolester bond formation, suggesting that it is the E1 interaction at this site that is crucial. Interestingly, other E2s and their relatives also use this same surface for binding to ubiquitin, E3s, and other proteins, revealing this to be a multipurpose binding site and suggesting that the entire E1-E2-E3 pathway has coevolved for a given ub-lp.     

Immunohistochemical expression of rabphilin-3A-like (Noc2) in normal and tumor tissues of human endocrine pancreas

Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans.     

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular basis of recognition by Gal/GalNAc specific legume lectins: influence of Glu 129 on the specificity of peanut agglutinin (PNA) towards C2-substituents of galactose

The ability to discriminate between galactose and N- acetylgalactosamine, observed in some lectins, is crucial for their biological activity as well as their usefulness as tools in biology and medicine. However, the molecular basis of differential binding of lectins to these two sugars is poorly understood. Peanut agglutinin (PNA) is one of the few galactose-specific legume lectins which does not bind N- acetylgalactosamine at all and is, therefore, ideal for the study of the basis of specificity towards C-2 substituted derivatives of galactopyranosides. Examination of the three-dimensional structure of PNA in complex with lactose revealed the presence of both a longer loop and bulkier residues in the region surrounding the C-2 hydroxyl of the galactopyranoside ring, which can sterically prevent the accommodation of a bulky substituent in this position. One such residue, is a glutamic acid at position 129 which protrudes into the binding site and perhaps directly obstructs any substitution at the C-2 position. Two mutants in bacterially expressed PNA were therefore constructed. These were E129D and E129A, in which Glu129 was replaced by Asp and Ala, respectively. The specificity of the mutants for galactose, galactosamine, and N- acetylgalactosamine was examined through observing the inhibition of hemagglutination and binding of the lectin to immobilized asialofetuin. The results showed that the affinity of E129A and E129D for C-2-substituted derivatives of the galactose varies. The mutant E129D showed significant binding towards N- acetylgalactosamine, suggesting that the residue Glu 129 is crucial in imparting exclusive galactose-specificity upon PNA. This study not only attempts to provide an explanation for the inability of PNA to accommodate C-2-substituted derivatives at its primary subsite, but also seeks to present a basis for engineering lectins with altered specificities.      

Effect of cocaine on responses of mouse phrenic nerve-diaphragm preparation

Effects of 5 to 40 microM cocaine on the compound action potential (AP) and tension responses of the mouse phrenic nerve-diaphragm preparation were monitored following nerve and muscle stimulation at 37 degrees C. Cocaine caused concentration dependent reduction in amplitude of the nerve AP, muscle AP, and tension response to a single nerve stimulus, and greater reduction in amplitude of these responses to repetitive nerve stimuli at 100 Hz for 0.5 sec. Cocaine caused similar reduction in the muscle AP and tension responses to direct muscle stimulation in the presence or absence of curare, and markedly reduced the overshoot, total potential, and maximum rate of rise and fall of intracellularly recorded muscle AP, without affecting the resting potential, or the contracture responses evoked by caffeine. These results indicate that cocaine reduces skeletal muscle function by reducing the excitability of muscle and nerve membranes, without significantly affecting neuromuscular transmission, excitation-contraction coupling or contractility.     

Detubulation effects on the action of zinc on frog skeletal muscle action potential

Summary Detubulation of the untreated fiber decreases the time constant of the action potential's foot ( _f ) and increases the maximal rate of rise of the spike ( ). Zinc at all concentrations, and regardless of whether the fiber is intact or detubulated, increases _f and decreases , and thus seems to decrease Na activation of the fiber. Detubulation was used principally to elucidate the localization and mechanism of the Zn²⁺-induced retardation of the falling phase of the frog sartorius fiber action potential, which evidently results from a general depression of delayed rectification. At 50 to 1000 m, Zn²⁺ not only prolongs repolarization of intact fibers (measured by increase int _0.5, the half-time of the spike's fall), but also produces a marked hump early in this phase. Detubulation of zinc-free fibers reducest _0.5 to about 80% of its intact value, and under Zn²⁺ treatmentt _0.5 is increased but only to about 80% of that produced in the intact fiber, and the falling-phase hump is completely obliterated. Thus, detubulation decreasest _0.5 in Zn²⁺-treated fibers not only by generally eliminating T-tubular participation in action potential generation, but also by subtracting a Zn²⁺-induced retardation of tubular delayed rectification. Tubular delayed rectification must therefore be an intrinsic feature of normal excitation. These results are further analyzed by means of (i) Stanfield's findings about retardation of delayed rectification by Zn²⁺ and (ii) Adrian-Peachey's theory of T-tubule participation in action potential generation, which suggests that the Zn²⁺-evoked repolarization hump signals onset of Zn²⁺-altered active participation of T-tubules in determining the spike's shape.Died April 17, 1978. Previously known as: P.M.K. Dass, and so listed in the reference Dass and Sandow (1972).