Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1

The toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain. This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol. The polypeptide is initially unfolded by binding to the reduced form of PDI. We show that upon oxidation of the COOH-terminal disulfide bond in PDI by the enzyme Ero1, the A1 chain is released. Both yeast Ero1 and the mammalian Ero1alpha isoform are active in this reaction. Ero1 has a preference for the PDI-toxin complex. We further show that the complex is transferred to a protein at the lumenal side of the ER membrane, where the unfolded toxin is released from PDI by the action of Ero1. Taken together, our results identify Ero1 as the enzyme mediating the release of unfolded CT from PDI and characterize an additional step in retrotranslocation of the toxin.     

Antennal resolution of pulsed pheromone plumes in three moth species

Male antennae of Cadra cautella,Pectinophora gossypiella, and Spodoptera exigua were presented with 20-ms-duration pulses of their two-component pheromone at rates of 1 to 33 Hz. Fourier analyses of electroantennograms resolved the temporal structure of trains of pheromone filaments delivered at up to 33 Hz for C. cautella and S. exigua and 25 Hz for P. gossypiella. Pheromone components tested separately for each species were generally equivalent in filament resolution to complete blends. Ambient temperatures of 18, 23 and 28 °C affected filament resolution only slightly, with poorer ability to discriminate rapidly pulsed signals at 18 °C. The question of how, or indeed if, such frequencies are conserved beyond the peripheral nervous system, remains.     

Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP

Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.     

Muscovite (mica) allows the characterisation of supported bilayers by ellipsometry and confocal fluorescence correlation spectroscopy

We demonstrate for the first time that ellipsometry and confocal fluorescence correlation spectroscopy (FCS) are complementary methods for the characterisation of supported planar phospholipid bilayers (SPBs) formed on mica, a mineral used in atomic force microscopy investigations of SPBs. Addition of small unilamellar vesicles containing 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC) to an oxidised borosilicate surface, on the other hand, results in a planar lipid system characterised by lateral diffusion coefficients which are three time smaller than those obtained for SPBs. Moreover, seven labelled phospholipids were tested for their suitability in the FCS characterisation of vesicles as well as of SPBs.     

Arginine in burn injury improves cardiac performance and prevents bacterial translocation

Horton, Jureta W., Jean White, David Maass, and BillySanders. Arginine in burn injury improves cardiac performance andprevents bacterial translocation. J. Appl.Physiol. 84(2): 695-702, 1998.This studyexamined the effects of arginine supplement of fluid resuscitation fromburn injury on cardiac contractile performance and bacterialtranslocation after a third-degree burn comprising 43% of the totalbody surface area in adult rats. Before burn injury, rats wereinstrumented to measure blood pressure; after burn (or sham injury),paired groups of sham-burned and burned rats were given vehicle(saline), L-arginine,D-arginine, orN-methyl-L-arginine(300 mg/kg in 0.3 ml of saline 30 min, 6 h, and 23 h postburn) plusfluid resuscitation; sham-burned rats received drug only.Twenty-four hours after burn trauma, hemodynamics were measured; theanimals were then killed and randomly assigned to Langendorff heartstudies or to studies examining translocation of gut bacteria. Burnrats treated with vehicle, D-arginine, orN-methyl-L-argininehad well-defined cardiocirculatory responses that included hypotension,tachycardia, respiratory compensation for metabolic acidosis,hypocalcemia, cardiac contractile depression, and significant bacterialtranslocation. Compared with values measured in vehicle-treated burnrats, L-arginine given afterburn improved blood pressure, prevented tachycardia, reduced serumlactate levels, improved cardiac performance, and significantly reducedbacterial translocation, confirming that L-arginine administration afterburn injury provided significant cardiac and gastrointestinalprotection. Circulating neutrophil counts fell after burn trauma andserum glucagon levels rose, but these changes were not altered bypharmacological intervention. Our finding of significantly highercoronary perfusate guanosine 3,5-cyclic monophosphateconcentration inL-arginine-treated burn ratssuggests that the beneficial effects ofL-arginine were mediated bynitric oxide production.

     

HERITABLE VARIATION IN ETHANOL TOLERANCE AND ITS ASSOCIATION WITH BIOCHEMICAL TRAITS IN DROSOPHILA MELANOGASTER

To help elucidate mechanisms of larval ethanol tolerance seven isochromosomal lines of Drosophila melanogaster with different second chromosomes were fed a growth-limiting concentration of ethanol (4.5% v/v) and examined for associations between growth traits and biochemical characteristics that had previously been implicated in the determination of tolerance variation. Repeated measures of survival and development time over four generations verified the inherited nature of these traits. Significant variation among the lines were evident for flux from ethanol into lipid, for activity levels of alcohol dehydrogenase and glycerol-3-phosphate oxidase (GPO), and for levels of long chain and unsaturated fatty acids. A high degree of positive association occurred among the variables. A partial correlation analysis controlling for performance of the lines on ethanol-free medium revealed a strong association between the degree of long chain fatty acid content and line survival when ethanol was fed. The correlation between GPO activity and survival in an ethanol environment appeared to depend on the association of GPO activity with long chain fatty acid content. The positive correlations of flux from ethanol into lipid with many of the other variables suggested that the ADH pathway influenced the level of ethanol tolerance. These associations are all consistent with the hypothesis that the lipid content of body tissues, especially the levels of long chain and unsaturated fatty acids in cell membranes, may have an important influence on both spatial and interspecific variation in the ethanol tolerance of larvae.     

In Vitro Reconstitution of Microtubule Plus End-directed, GTPγS-sensitive Motility of Golgi Membranes

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5′-adenylylimidodiphosphate, and 100 μM but not 20 μM vanadate. Motility was also blocked by GTPγS or AlF₄⁻ but was insensitive to AlCl₃, NaF, staurosporin, or okadaic acid. The targets for GTPγS and AlF₄⁻ were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.     

Effect of sodium transport inhibition on active phosphate transport by toad bladder

Summary The relationship between active Na transport (estimated by the short-circuit (SCC)) and active inorganic phosphate (P_i) transport was studied in the toad bladder. When SCC was inhibited by amiloride, ouabaim, or removal of K from the serosal bathing solution, active P_i transport was totally inhibited. When Na was replaced isotonically by choline in either the mucosal bathing solution or both the mucosal and serosal bathing solutions, there was no measurable SCC or active P_i transport. These experiments are compatible with the hypothesis that active P_i transport occurs only in the presence of active Na transport.     

Pigmentation and Antibacterial Activity of Fast Neutron- and X-Ray-induced Strains of Monascus purpureus Went

Seven new strains of Monascus purpureus Went were induced by neutron and x-ray irradiation. The quantity and quality of pigments produced by these strains differed. Strains N4S and N11S produced twice as much pigment as normal, while another strain, N14S, was albino. An unknown orange pigment was found in young colonies of the N11S strain. This orange pigment reacted with alcohols and malt extract medium to form red pigments. Strains N4S, N11S, X2P, and wild type inhibited the growth of certain bacteria, especially the Bacillus species. Strain N11S had more antibacterial activity than wild type. A major active compound was isolated with an ultraviolet absorption spectrum that was related to those of the red pigments found in this fungus. The active compound(s) was named monascidin.