Cloning and characterization of mouse Lmp3 cDNA, encoding a proteasome β subunit

We isolated and sequenced a cDNA encoding mouse proteasome subunit LMP3 from a macrophage cDNA library. The gene encodes a 264-amino-acid protein with a calculated molecular mass of 29.11 kDa and an isoelectric point (pl) of 5.44. Comparison of the predicted protein sequence with that of the human and rat homologues, N3, revealed 11 and eight changes, respectively, in the cleaved NH₂-terminal presequence of the precursor protein (pre-LMP3), and six and 10 changes, respectively, in the processed product. To corroborate the predicted molecular mass and pI, we analyzed LMP3 by immunoprecipitation with a mAb to human N3 that crossreacts with mouse LMP3. Precursor and processed forms of LMP3 were identified by 2D NEPHGE-PAGE, and their mobilities suggest the Lmp3 clone encodes the entire protein sequence.     

Spatio-Temporal Dynamics of Field Cricket Calling Behaviour: Implications for Female Mate Search and Mate Choice

Amount of calling activity (calling effort) is a strong determinant of male mating success in species such as orthopterans and anurans that use acoustic communication in the context of mating behaviour. While many studies in crickets have investigated the determinants of calling effort, patterns of variability in male calling effort in natural choruses remain largely unexplored. Within-individual variability in calling activity across multiple nights of calling can influence female mate search and mate choice strategies. Moreover, calling site fidelity across multiple nights of calling can also affect the female mate sampling strategy. We therefore investigated the spatio-temporal dynamics of acoustic signaling behaviour in a wild population of the field cricket species Plebeiogryllus guttiventris. We first studied the consistency of calling activity by quantifying variation in male calling effort across multiple nights of calling using repeatability analysis. Callers were inconsistent in their calling effort across nights and did not optimize nightly calling effort to increase their total number of nights spent calling. We also estimated calling site fidelity of males across multiple nights by quantifying movement of callers. Callers frequently changed their calling sites across calling nights with substantial displacement but without any significant directionality. Finally, we investigated trade-offs between within-night calling effort and energetically expensive calling song features such as call intensity and chirp rate. Calling effort was not correlated with any of the calling song features, suggesting that energetically expensive song features do not constrain male calling effort. The two key features of signaling behaviour, calling effort and call intensity, which determine the duration and spatial coverage of the sexual signal, are therefore uncorrelated and function independently.     

Low Band Gap Benzimidazole COF Supported Ni3N as Highly Active OER Catalyst

Covalent organic frameworks (COFs) have structures and morphologies closely resembling graphenes, whose modular construction permits atomic‐level manipulations. This, combined with their porous structure, makes them excellent catalyst supports. Here, the high electrocatalytic activity of a composite, formed by supporting Ni₃N nanoparticles on a benzimidazole COF, for oxygen evolution reaction is shown. The composite oxidizes alkaline water with a near‐record low overpotential of 230 mV @ 10 mA cm^?2 (η ₁₀). This high activity is attributed to the ability of the COF to confine the Ni₃N nanoparticles to size regimes otherwise difficult to obtain and to its low band gap character (1.49 eV) arising from the synergy between the conducting Ni₃N nanoparticles and the π‐conjugated COF. The COF itself, as a metal‐free self‐standing framework, has an oxygen evolution reaction activity with η ₁₀ of 400 mV. The periodic structure of the COF makes it serve as a matrix to disperse the catalytically active Ni₃N nanoparticles favoring their high accessibility and thereby good charge‐transport within the composite. This is evident from the amount of O₂ evolved (230 mmol h^?1 g^?1), which, to the best of our knowledge, is the highest reported. The work reveals the emergence of COF as supports for electrocatalysts.     

Biologically active, recombinant DNA in clathrin-coated vesicles isolated from rat livers after in vivo injection of liposome-encapsulated DNA

DNA entrapped in liposomes containing lactosylceramide in the bilayers is found to be associated with clathrin-coated vesicles isolated from the rat livers after intravenous injection of these liposomes. The presence of the exogenous DNA in the coated vesicles was detected by Southern blotting. The amount of DNA present in the coated vesicles does not appear to vary up to 4 h after injection of the liposomes into the animals. The recognition of the lactosyl group present in the liposome by the galactose receptor present on the surface of the different liver cells may lead to their internalization in a way analogous to receptor-mediated endocytosis of various macromolecules. DNA present in the lumen of the coated vesicles is found to be biologically active as evidenced by its replication in bacterial cells and mouse fibroblasts.     

Synthesis and lysis of formate by immobilized cells of Escherichia coli

Formate hydrogenlyase (FHL) activity was induced in a strain of Escherichia coli S13 during anaerobic growth in yeast extract-tryptone medium containing 100 mM formate. The cells obtained at the optimum growth phase were immobilized in 2.5% (w/v) agar gel when 50-60% of the whole cell FHL activity was retained. The immobilized FHL system had good storage stability and recycling efficiency. In the lysis of formate, an increase of formate concentration to 1.18M increased QH(2) (initial) value of the immobilized cell, and subsequently cells, hydrogen evolution, in general, ceased after 6 to 8 of incubation, resulting in incomplete lysis of formate. Presence of small amount of glucose (28 mM) was more or less quantitatively lysed with concomitant disappearence of glucose from the medium. Synthesis of formate from hydrogen and bicarbonate solution by the immobilized cells was also characterized. Presence of glucose (10 mM) in 50 mM bicarbonate solution stimulated formate synthesis by immobilized cells. The pH optimum range, K(m), and specific activity of the immobilized cells for the lysis of formate were 6.8-7.2 0.4M, and 66 mL/g cell-h, respectively. The cells could fix hydrogen to the extent of 24.4% (w/w) of its own wet cell mass in a 72-h reaction cycle. Potentiality of the immobilized FHL system for biotechnological exploitation was discussed.     

Effect of three organophosphates on respiration in rat brain and liver tissue

The effects of three organophosphate pesticides, i.e. monocrotophos, dichlorvos, and phosphamidon on respiration in rat brain and liver tissue slices have been studied. Among these pesticides dichlorvos causes significant inhibition of respiration both in brain and liver.     

Antidepressants and brain respiration

Imipramine and clorgyline, at concentrations of 0.002 M, inhibit the respiration of brain tissue by 82 and 71 per cent respectively, while chloropromazine and tranylcypromine, at concentrations of 0.01 M, inhibit it about 25 per cent. Deprenyl and amphetamine at a concentration of 0.002 M inhibit brain tissue respiration by 12 and 18 per cent respectively. Respiration in brain is least affected by lithium chloride (only 5 per cent inhibition).     

Fusion of negatively charged liposomes with clathrin-uncoated vesicles

The interaction of lipid vesicles with uncoated vesicles from bovine brain has been studied by fluorescence energy transfer between fluorescent lipid analogs (NBD-PE, Rh-DOPE), by loss of fluorescence self-quenching (NBD-PE, carboxyfluorescein) and by freeze-fracture electron microscopy. The fluorescence techniques monitor the mixing of membranous lipids and the induced release of encapsulated material. The results demonstrate a mixing of the negatively charged lipid (PA, PS) vesicles with the uncoated vesicles. In parallel with the lipid mixing a release of intravesicularly encapsulated material takes place. Lipid vesicles composed of zwitterionic lipids (PC, DOPC, PC:PE) do not specifically interact with uncoated vesicles. The electron micrographs reveal single fusion events. Studies on the kinetics are consistent with a fusional mechanism of the negatively charged lipid vesicles with uncoated vesicles.     

Purification and partial characterization of the (H+,K+)-transporting adenosinetriphosphatase from fundic mucosa

The microsomal (H+,K+)-ATPase systems from dog and pig fundic mucosa were purified to homogeneity and partially characterized. The method involves sodium dodecyl sulfate (SDS) (0.033% w/v) extraction of the microsomal non-ATPase proteins under appropriate conditions followed by sucrose density gradient centrifugation. Two distinct membrane bands of low (buoyant density = 1.08 g/mL) and high (buoyant density = 1.114 g/mL) densities having distinct enzymatic and chemical composition were harvested. The low-density membrane was highly enriched in Mg2+- or Ca2+-stimulated ATPase and 5'-nucleotidase activities but totally devoid of (H+,K+)-ATPase and K+-p-nitrophenylphosphatase activities. The latter two activities were found exclusively in the high-density membrane. SDS-polyacrylamide gel electrophoresis revealed the high-density membranes to consist primarily of a major 100-kilodalton (kDa) protein and a minor 85-kDa glycoprotein, the former being the catalytic subunit of the (H+,K+)-ATPase. The amino acid composition of the pure dog (H+,K+)-ATPase revealed close similarities with that from pig. The N-terminal amino acid was identified to be lysine as the sole residue. Similar to the high-density membrane-associated pure (H+,K+)-ATPase, the low-density membranes containing high Mg2+-ATPase activity also contained a 100-kDa peptide and a 85-kDa glycopeptide in addition to numerous low molecular weight peptides. Also, similar to the pure (H+,K+)-ATPase, the Mg2+-ATPase-rich fraction produced an E approximately P unstable to hydroxylamine and partially (about 25%) sensitive to K+ but having a slow turnover. The levels of E approximately P produced by the pure (H+,K+)-ATPase- and Mg2+-ATPase-rich fractions were 1400 and 178 pmol/mg of protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of labeled clathrin and coated vesicles

Clathrin (8 S) and coated vesicles have been covalently labeled by using the sulfhydryl-labeling fluorescent probe N-(1-anilinonaphthalene)maleimide. A large increase in energy transfer from Trp to anilinonaphthalene (AN) residues was observed in clathrin in the pH range approximately 6.5-6.0, where the rate of clathrin self-association increased rapidly. The change in energy transfer was indicative of a conformational rearrangement, which could be responsible for the initiation of the clathrin self-association reaction to form coat structure. The AN label was found in both the coat and membrane proteins after dissociation of coated vesicles at pH 8.5. The labeled coat and membrane proteins readily recombined to form coated vesicles after reducing the pH to 6.5, indicating that the labeling did not interfere with the ability of clathrin to self-associate and interact with uncoated vesicles to form coat structure. A comparison of the AN fluorescence with the Coomassie blue pattern after electrophoresis in sodium dodecyl sulfate-gels revealed that a 180,000-Da protein (clathrin) was mainly labeled in coated vesicles, while a 110,000-Da protein was also strongly labeled in uncoated vesicles. AN-labeled baskets and coated vesicles have been prepared. Trypsin digestion reduced the sedimentation rate of baskets from 150 S to 120 S and of coated vesicles from 200 S to 150 S. Gel electrophoresis of baskets and coated vesicles showed extensive conversion of clathrin (Mr 180,000) to a product of Mr approximately equal to 110,000, suggesting equivalent structural organization of the coat in coated vesicles as in baskets. In both cases, the peptide(s) released from the vesicles by digestion were essentially free of fluorescent label. In the case of the uncoated vesicles, tryptic digestion released most of the proteins remaining after coat removal.