Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.     

A functional comparison of ovine and porcine trypsins

Trypsin was isolated from ovine and porcine pancreas using affinity chromatography on immobilized p-aminobenzamidine. Molecular masses of the two proteins were 23900 and 23435 Da, determined by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry. The purified trypsins were compared using the kinetic properties K(m) and k(cat) which were determined at pH 8.0 and between 25 and 55 degrees C. Comparison of the Michaelis constants for ovine and porcine trypsins toward N-alpha-benzoyl-arginine-p-nitroanilide (BapNA) indicated that ovine trypsin had higher affinity for this substrate than the porcine enzyme. The rates of the reactions catalysed by the two enzymes correlated strongly over the range of temperatures and substrate concentrations tested, as did the k(cat) values. The specific activity of ovine trypsin for BapNA was, on average, approximately 10% higher than that of the porcine enzyme over the range of conditions tested. Porcine trypsin was less susceptible to denaturation at low pH or high temperature than was ovine trypsin. Porcine and ovine trypsin produced seven identically sized fragments from auto-catalytic hydrolysis. Proposed regions of identity between ovine and porcine trypsins were I(54)-K(77), L(98)-R(107), S(134)-K(178) and N(209)-K(116). Hydrolysis of beta-lactoglobulin, egg white lysozyme or casein by ovine or porcine trypsin yielded virtually identical patterns of fragments although the rate at which fragments were produced, in the case of beta-lactoglobulin, differed between the two enzymes. On balance the two enzymes appear to be functionally identical in their action.     

Regulation of the acid-labile subunit in sustained endotoxemia

The effect of sustained endotoxemia on expression of the acid-labile subunit (ALS) in relation to hepatic markers of altered GH and insulin sensitivity was examined. Juvenile rats were injected with endotoxin twice daily for 48 h, causing reduced food intake and attenuated growth. In pair-fed controls, food restriction caused marked suppression of ALS gene expression and circulating levels within 12 h, and endotoxemia augmented this effect. This acute effect of endotoxin corresponded temporally with transient induction of suppressor of cytokine signaling (SOCS)-3, cytokine-inducible SH2-containing protein (CIS), phosphoenolpyruvate carboxykinase (PEPCK), and insulin-like growth factor-binding protein (IGFBP)-1 and suppression of GH receptor (GHR). During the subsequent 36 h of sustained endotoxin treatment, expression of ALS recovered to, and then rose above, that of their pair-fed controls. This effect was paralleled by other ternary complex components. The inductive effect of sustained endotoxemia relative to pair-fed controls could not be explained by differences in expression of GHR, SOCS-3, or CIS but coincided with normalized PEPCK and IGFBP-1 levels, suggesting better hepatic insulin sensitivity in these animals. These data may indicate that, in sustained endotoxemia, ALS levels are regulated through modulation of hepatic insulin sensitivity.     

Early postnatal death and motor disorders in mice congenitally deficient in calnexin expression

Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific.     

A discrete domain of the human TrkB receptor defines the binding sites for BDNF and NT-4

TrkB is a member of the Trk family of tyrosine kinase receptors. In vivo, the extracellular region of TrkB is known to bind, with high affinity, the neurotrophin protein brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). We describe the expression and purification of the second Ig-like domain of human TrkB (TrkBIg(2)) and show, using surface plasmon resonance, that this domain is sufficient to bind BDNF and NT-4 with subnanomolar affinity. BDNF and NT-4 may have therapeutic implications for a variety of neurodegenerative diseases. The specificity of binding of the neurotrophins to their receptor TrkB is therefore of interest. We examine the specificity of TrkBIg(2) for all the neurotrophins, and use our molecular model of the BDNF-TrkBIg(2) complex to examine the residues involved in binding. It is hoped that the understanding of specific interactions will allow design of small molecule neurotrophin mimetics.     

Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2)

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H₂O₂ have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H₂O₂ and menadione, a compound known to release H₂O₂ intracellularly, were used to examine the phospholipases A₂ (PLA₂) responsible for AA release from primary murine astrocytes. Both H₂O₂ and menadione dose-dependently stimulated AA release, and the release mediated by H₂O₂ was completely inhibited by catalase. H₂O₂ also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). However, complete inhibition of cPLA₂ phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H₂O₂-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA₂ and the Ca²⁺-independent iPLA₂, nearly completely inhibited H₂O₂-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA₂, only inhibited H₂O₂-mediated AA release by 40%. Along with the observation that H₂O₂-mediated AA release was only partially inhibited upon chelating intracellular Ca²⁺ by BAPTA, these results indicate the involvement of both cPLA₂ and iPLA₂ in H₂O₂-mediated AA release in murine astrocytes.     

Mutational spectrum in the cardioauditory syndrome of Jervell and Lange-Nielsen

Jervell and Lange-Nielsen syndrome (JLNS) is an autosomal recessive syndrome characterised by profound congenital sensorineural deafness and prolongation of the QT interval on the electrocardiogram, representing abnormal ventricular repolarisation. In a study of ten British and Norwegian families with JLNS, we have identified all of the mutations in the KCNQ1 gene, including two that are novel. Of the nine mutations identified in this group of 10 families, five are nonsense or frameshift mutations. Truncation of the protein proximal to the recently identified C-terminal assembly domain is expected to preclude assembly of KCNQ1 monomers into tetramers and explains the recessive inheritance of JLNS. However, study of a frameshift mutation, with a dominant effect phenotypically, suggests the presence of another assembly domain nearer to the N-terminus.     

Sister chromatid cohesion in mitosis

Sister chromatid cohesion is essential for accurate chromosome segregation during the cell cycle. Newly identified structural proteins are required for sister chromatid cohesion and there may be a link in some organisms between the processes of cohesion and condensation. Proteins that induce and regulate the separation of sister chromatids have also been recently identified.