Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase from Escherichia coli

The UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase was over-produced and purified from two plasmid-harbouring strains of Escherichia coli. The first strain, E. coli JM83(pHE5), gave a 15-fold over-production relative to parental strain. The enzyme could be partially purified (8.8-fold) by ion-exchange chromatography. With the second strain, E. coli JM83(pMLD25), a very strong over-production was obtained, since the enzyme represented about 20% of the cytoplasmic proteins. Purification yielded 77% protein homogeneity. However, the enzymatic activity, which was very unstable, was lost during the purification procedure. Several properties of the enzyme were studied. The enzyme gave maximal activity around pH 8. The isoelectric point was 5.2. The activity was increased by potassium phosphate. Reverse and exchange reactions could be catalysed. The N-terminal sequence of the protein was determined and correlated with the nucleotide sequence of the murE gene. The actual initiation codon was assigned.     

Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat Model of Huntington Disease

In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons’ capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease.     

Molecular characterization of the mouse agouti locus.

The agouti (a) locus acts within the microenvironment of the hair follicle to regulate coat color pigmentation in the mouse. We have characterized a gene encoding a novel 131 amino acid protein that we propose is the one gene associated with the agouti locus. This gene is normally expressed in a manner consistent with a locus function, and, more importantly, its structure and expression are affected by a number of representative alleles in the agouti dominance hierarchy. In addition, we found that the pleiotropic effects associated with the lethal yellow (Ay) mutation, which include pronounced obesity, diabetes, and the development of neoplasms, are accompanied by deregulated overexpression of the agouti gene in numerous tissues of the adult animal.     

Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

The expression of clinically useful proteins in plants has been bolstered by the development of high-yielding systems for transient protein expression using agroinfiltration. There is a need now to know more about how host plant development and metabolism influence the quantity and quality of recombinant proteins. Endogenous proteolysis is a key determinant of the stability and yield of recombinant proteins in plants. Here we characterised cysteine (C1A) and aspartate (A1) protease profiles in leaves of the widely used expression host Nicotiana benthamiana, in relation with the production of a murine IgG, C5-1, targeted to the cell secretory pathway. Agroinfiltration significantly altered the distribution of C1A and A1 proteases along the leaf age gradient, with a correlation between leaf age and the level of proteolysis in whole-cell and apoplast protein extracts. The co-expression of tomato cystatin SlCYS8, an inhibitor of C1A proteases, alongside C5-1 increased antibody yield by nearly 40% after the usual 6-days incubation period, up to ∼3 mg per plant. No positive effect of SlCYS8 was observed in oldest leaves, in line with an increased level of C1A protease activity and a very low expression rate of the inhibitor. By contrast, C5-1 yield was greater by an additional 40% following 8- to 10-days incubations in younger leaves, where high SlCYS8 expression was maintained. These findings confirm that the co-expression of recombinant protease inhibitors is a promising strategy for increasing recombinant protein yields in plants, but that further opportunity exists to improve this approach by addressing the influence of leaf age and proteases of other classes.     

Clinico‐molecular analysis of eleven patients with Hermansky–Pudlak type 5 syndrome,a mild form of HPS

Hermansky–Pudlak syndrome (HPS), first described in 1959, is a rare form of syndromic oculocutaneous albinism associated with bleeding diathesis and in some cases pulmonary fibrosis and granulomatous colitis. All 10 HPS types are caused by defects in vesicle trafficking of lysosome‐related organelles (LRO) proteins. The HPS5 protein associates with HPS3 and HPS6 to form the biogenesis of lysosome‐related organelles complex‐2 (BLOC‐2). Here, we report the clinical and genetic data of 11 patients with HPS‐5 analyzed in our laboratory. We report 11 new pathogenic variants. The 11 patients present with ocular features that are typical for albinism, with mild hypopigmentation, and with no other major complication, apart from a tendency to bleed. HPS‐5 therefore appears as a mild form of HPS, which is often clinically undistinguishable from mild oculocutaneous or ocular forms of albinism. Molecular analysis is therefore required to establish the diagnosis of this mild HPS form, which has consequences in terms of prognosis and of clinical management of the patients.     

Functional redundancy of the B9 proteins and nephrocystins in Caenorhabditis elegans ciliogenesis

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS.     

Proteomic characterization of mouse cytosolic and membrane prostate fractions: high levels of free SUMO peptides are androgen-regulated

The prostate is a relatively homogeneous tissue that is highly specialized in synthetic and secretory functions. The frequency of malignant growth explains its great clinical significance. We used here a combination of subcellular fractionation, 1-DE (one-dimensional gel electrophoresis) protein separation and mass spectrometry, to establish a prostate protein expression profile in mice. Analysis of proteins present in cytosolic (C) and membrane (P) prostate fractions led to the identification of 619 distinct proteins. A majority of abundant proteins were found to compose the metabolism and protein synthesis machinery. Those identified also correspond to known endoplasmic reticulum and Golgi residents, chaperones and anterograde cargos. They included a series of proteins involved in exocytic/endocytic trafficking. Among the signaling proteins, we identified the ubiquitin-like peptides smt3. We showed that both free small ubiquitin-related modifier SUMO-2/3 and SUMO-1 levels are subject to tight control by the androgen 5alpha-dihydrotestosterone (DHT). By contrast with SUMO-2/3, free SUMO-1 peptides are particularly abundant in the prostate when compared with other tissues. Therefore, we report prostate protein expression profiles of cytosolic and membrane fractions in mice. Our data suggest that the identified free SUMO peptides play an important role in this secretory tissue.     

Distribution of dimethylsulfide and dimethylsulfoniopropionate and its relation with phytoneuston in the surface microlayer of the western North Atlantic during summer

One of the key steps towards predicting dimethylsulfide (DMS) emission to the atmosphere is to understand the distribution and cycling of biogenic sulfur in the microlayer. In this study, we examined the distribution of DMS and dissolved and particulate fractions of dimethylsulfoniopropionate (DMSPd and DMSPp) in the surface microlayer and bulk water of the western North Atlantic during July 2003. DMS concentrations in the bulk water varied from 0.71 to 7.65 nM. In contrast, DMS concentrations in the surface microlayer were fairly low (0.17–1.33 nM). Average concentrations of DMSPd and DMSPp in the bulk water were 2.09 (1.87–6.25) and 44.1 (8.06–119.8) nM, respectively, and those in the surface microlayer were 15.4 (4.06–54.3) and 29.9 (7.32–97.0) nM. In general, DMS was depleted in the microlayer (mean concentration: 0.60 nM) relative to the bulk water (mean concentration: 2.38 nM) with enrichment factors (the ratio of the microlayer concentration to bulk water concentration) ranging from 0.13 to 0.54. There was no consistent enrichment of DMSPp and chlorophyll a in the microlayer. On the contrary, DMSPd appeared to be highly enriched in the microlayer with an average EF of 4.89. The concentration of phaeopigments was also generally greater in the microlayer than in the bulk water, presumably due to enhanced photo-oxidation of chlorophyll a under high surface light intensities in the microlayer. In the study area, the concentration of DMSPp was significantly correlated with the abundance of dinoflagellates in the microlayer. Moreover, a significant correlation between the distributions of DMS, DMSPp, chlorophyll a and phaeopigment concentrations in the microlayer and the bulk water demonstrated that the biogenic materials in the microlayer come primarily from the bulk water below.