Endoplasmic Reticulum Stress Induces a Caspase-dependent N-terminal Cleavage of RBX1 Protein in B Cells

Endoplasmic reticulum (ER) stress develops when the ER is overloaded with too many proteins to fold. This elicits a signaling pathway called the unfolded protein response. The unfolded protein response is physiologically required for the terminal development of B cells into antibody-secreting plasma cells. Ring Box Protein 1 (RBX1) is a 14-kDa protein necessary for ubiquitin ligation activity of the multimeric cullin ring ubiquitin ligases (CRLs). As RBX1 is shared by a large number of CRLs, alterations in its activity may lead to global changes in protein stability. We discovered that RBX1 is cleaved in the course of LPS-induced plasma cell differentiation and in multiple myeloma cell lines upon induction of pharmacological ER stress. The cleavage is executed by several caspase proteases that cleave RBX1 eight amino acids from the N terminus. To address the possible implication of RBX1 cleavage for CRL activity, we replaced the endogenous RBX1 homolog of the yeast Saccharomyces cerevisiae, Roc1, with the wild type or the N-terminal Δ8 mutant human RBX1. We show that yeast expressing the cleaved RBX1 are hypersensitive to ER stress and are impaired in CRL-mediated ubiquitination and degradation. We propose a model by which N-terminal cleavage of RBX1 impairs its activity and promotes susceptibility to ER stress induction.     

Cellulolytic bacteria from soils in harsh environments

It is believed that the exposure of organisms to harsh climate conditions may select for differential enzymatic activities, making the surviving organisms a very promising source for bioprospecting. Soil bacteria play an important role in degradation of organic matter, which is mostly due to their ability to decompose cellulose-based materials. This work focuses on the isolation and identification of cellulolytic bacteria from soil found in two environments with stressful climate conditions (Antarctica and the Brazilian semi-arid caatinga). Cellulolytic bacteria were selected using enrichments at high and low temperatures (4 or 60°C) in liquid media (trypic soy broth—TSB and minimum salt medium—MM) supplemented with cellulose (1%). Many of the isolates (119 out of 254—46.9%) displayed the ability to degrade carboxymethyl-cellulose, indicating the presence of endoglucolytic activity, while only a minority of these isolates (23 out of 254—9.1%) showed exoglucolytic activity (degradation of avicel). The obtained isolates revealed a preferential endoglucolytic activity according to the temperature of enrichments. Also, the identification of some isolates by partial sequencing of the 16S rRNA gene indicated that the Bacteroidetes (e.g., Pedobacter, Chryseobacterium and Flavobacterium) were the main phylum of cellulolytic bacteria isolated from soil in Antarctica; the Firmicutes (e.g., Bacillus) were more commonly isolated from samples from the caatinga; and Actinobacteria were found in both types of soil (e.g., Microbacterium and Arthrobacter). In conclusion, this work reports the isolation of bacteria able to degrade cellulose-based material from soil at very low or very high temperatures, a finding that should be further explored in the search for cellulolytic enzymes to be used in the bioenergy industry.     

Studying and modelling dynamic biological processes using time-series gene expression data

Biological processes are often dynamic, thus researchers must monitor their activity at multiple time points. The most abundant source of information regarding such dynamic activity is time-series gene expression data. These data are used to identify the complete set of activated genes in a biological process, to infer their rates of change, their order and their causal effects and to model dynamic systems in the cell. In this Review we discuss the basic patterns that have been observed in time-series experiments, how these patterns are combined to form expression programs, and the computational analysis, visualization and integration of these data to infer models of dynamic biological systems.     

1-substituted cyclopropenes: Effective Blocking Agents for Ethylene Action in Plants

A series of 1-alkane substituted cyclopropenes has been prepared and tested as ethylene antagonists using banana fruits as an assay system. 1-Methyl-, 1-ethyl-, 1-propyl-, 1-butyl-, 1-pentyl-, 1-hexyl-, 1-heptyl-, 1-octyl-, 1-nonyl-, and 1-decylcyclopropene were all very active compounds. 1-Methylcyclopropene protected bananas from ethylene with a minimum concentration of 0.7 nl.l^–1 after a 24 h exposure. As the carbon chain length was extended the minimum requirement increased some, but starting with 1-butylcyclopropene, the minimum concentration requirement declined and many cyclopropenes were required in lower concentrations than 1-methylcyclopropene. The time of protection at ambient temperature (22–23 °C) was 12 d for 1-methyl-, 1-ethyl-, 1-propyl-, and 1-butylcyclopropene. 1-Pentylcyclopropene protected bananas for 14 d, 1-hexylcyclopropene for 20 d, 1-heptylcyclopropene for 21 d, 1-octylcyclopropene for 25 d, 1-nonylcyclopropene for 35 d, and 1-decylcyclopropene for 36 d.     

Detection of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> by PCR on Field Strains from Healthy and Diseased Pigs

We investigated whether primers able to specifically amplify a 0.7-kb DNA fragment from the conserved cpx genes could be applied to analyze A. pleuropneumoniae field isolates. The specific cpx primers were tested on 120 strains of A. pleuropneumoniae and other NAD-dependent field isolates from healthy and diseased animals to analyze A. pleuropneumoniae isolates from pigs in Brazil. We found that PCR and hybridization were able to discriminate between isolates of A. pleuropneumoniae and other bacteria. The 0.7-kb cpx DNA fragments were amplified from all 63 A. pleuropneumoniae isolates from herds with clinical symptoms and were isolated from lesions of acute cases of swine pleuropneumonia, both serotypable and nonserotypable. The PCR was also applied to 57 field isolates obtained from animals of apparently healthy herds, and the amplified cpx product was present in four serotypable and only two out of eleven A. pleuropneumoniae nonserotypable isolates. All nonserotypable A. pleuropneumoniae isolates revealed the apxA amplification pattern compatible with previously known serotypes. Some nonserotypable isolates might represent a population of isolates that originally were serotypable but lost the ability to react with serotype-specific antisera or might belong to novel serotypes. The PCR method applied is highly sensitive for serotypable A. pleuropneumoniae strains and for nonserotypable strains isolated from acute cases of swine pleuropneumoniae in Brazil. Received: 13 June 2002 / Accepted: 5 August 2002     

Determination of dependence of binding parameters on receptor occupancy

Measurements of the binding of ligand to receptors that are macromolecules, either free or components of biomembranes, often show deviation from what is expected of a simple reaction described by an association and a dissociation rate constant. A more versatile model and more discriminating experiments are required for a satisfactory explanation. This paper is based on a general model of the binding reaction in which the rate constants and equilibrium constant are dependent upon occupancy of receptors. The analysis of the model leads to three kinds of experiments: (1) equilibrium measurements which permit quantitative determination of a dissociation equilibrium parameter as a function of receptor occupancy; (2) measurements prior to equilibrium which yield the same information; and (3) measurements prior to equilibrium which reveal quantitatively the dependence of both association and dissociation rate parameters separately, on occupancy.