Effect of acyl-CoA oxidase activity on the accumulation of gamma-decalactone by the yeast Yarrowia lipolytica: a factorial approach

beta-Oxidation is a cyclic pathway involved in the degradation of lipids. In yeast, it occurs in peroxisomes and the first step is catalyzed by an acyl-CoA oxidase (Aoxp). The yeast Yarrowia lipolytica possesses several genes (POX) coding for Aoxps. This study is based on the factorial analysis of results obtained with the many POX derivative strains that have been constructed previously. The effect of interactions between Aoxps on the acyl-CoA oxidase (Aox) activity was important even at the second order. We then investigated the effect of Aox activity on growth and lactone production. Aox activity was correlated with acidification of the medium by cells and with cellular growth but not with lactone production, although Aox activity on short chains was inversely correlated with lactone accumulation. Due to the poor correlation between Aox activity and lactone production, the modeling of this parameter gave no satisfactory results but growth depending on Aox activity was modeled.     

Decreased expression of glutamate transporters in genetic absence epilepsy rats before seizure occurrence

In absence epilepsy, epileptogenic processes are suspected of involving an imbalance between GABAergic inhibition and glutamatergic excitation. Here, we describe alteration of the expression of glutamate transporters in rats with genetic absence (the Genetic Absence Epilepsy Rats from Strasbourg: GAERS). In these rats, epileptic discharges, recorded in the thalamo-cortical network, appear around 40 days after birth. In adult rats no alteration of the protein expression of the glutamate transporters was observed. In 30-day-old GAERS protein levels (quantified by western blot) were lower in the cortex by 21% and 35% for the glial transporters GLT1 and GLAST, respectively, and by 32% for the neuronal transporter EAAC1 in the thalamus compared to control rats. In addition, the expression and activity of GLAST were decreased by 50% in newborn GAERS cortical astrocytes grown in primary culture. The lack of modification of the protein levels of glutamatergic transporters in adult epileptic GAERS, in spite of mRNA variations (quantified by RT-PCR), suggests that they are not involved in the pathogeny of spike-and-wave discharges. In contrast, the alteration of glutamate transporter expression, observed before the establishment of epileptic discharges, could reflect an abnormal maturation of the glutamatergic neurone-glia circuitry.     

Astrocytes,cells involved in neuro-immune interactions in the central nervous system

The astrocyte, the major glial cell in the central nervous system, may influence many aspects of inflammation and immune reactivity within the brain. We have established a model of chronically activated T lymphocytes, interacting with neural cells of diverse origin to study the complex immune regulatory system suspected to lead to neuroinflammatory diseases. We show that human astrocytes became reactive following T cell contact, secreting proinflammatory cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinase (TIMP). The altered MMP/TIMP system was shown to be involved in deleterious effects displayed by activated T cells towards human multipotent neural precursers by controlling their sensitivity to T cell-induced Fas-mediated apoptosis. MMP/TIMP was suspected to stabilize Fas at the cell membrane. In a model of mixed rat glial cells in primary culture (astrocytes, oligodendrocytes), activated T lymphocytes induced the collapse of processes and the death of immature oligodendrocytes. These effects were associated with upregulation of Fas at the cell surface of oligodendrocytes and secretion of MMP and TIMP by astrocytes. By amplifying the expression of inflammatory molecules including the MMP/TIMP system, astrocytes appear to be a crucial relay in the deleterious molecular cascade triggered by activated T lymphocytes. Detection of altered MMP/TIMP in patients suffering from myelopathy associated with retroviral infection (HTLV-1) strongly suggests its involvement in the physiopathological process of the disease.     

Both transmembrane domains of SecG contribute to signal sequence recognition by the Escherichia coli protein export machinery

A chimeric protein containing the uncleaved signal sequence of plasminogen activators inhibitor-2 (PAI2) fused to alkaline phosphatase (AP) interferes with Escherichia coli protein export and arrests growth. Suppressors of this toxicity include secG mutations that define the Thr-41-Leu-42-Phe-43 (TLF) domain of SecG. These mutations slow down the export of PAI2-AP. Another construct encoding a truncated PAI2 signal sequence (hB-AP) is also toxic. Most suppressors exert their effect on both chimeric proteins. We describe here five secG suppressors that only suppress the toxicity of hB-AP and selectively slow down its export. These mutations do not alter the TLF domain: three encode truncated SecG, whereas two introduce Arg residues in the transmembrane domains of SecG. The shortest truncated protein only contains 13 residues of SecG, suggesting that the mutation is equivalent to a null allele. Indeed, a secG disruption selectively suppresses the toxicity of hB-AP. However, the missense mutations are not null alleles. They allow SecG binding to SecYE, although with reduced affinity. Furthermore, these mutated SecG are functional, as they facilitate the export of endogenous proteins. Thus, SecG participates in signal sequence recognition, and both transmembrane domains of SecG contribute to ensure normal signal sequence recognition by the translocase.     

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI₂ levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI₂, in T1DM mice.     

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: Implication of their membrane fluidizing action

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called “green notes”. The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.     

Expression and function of cyclooxygenase and lipoxygenase enzymes in human islets of Langerhans

Arachidonic acid (AA) is generated in pancreatic beta-cells through the activation of Ca2+-dependent cytosolic phospholipase A2 (cPLA2) and the consequent hydrolysis of membrane phospholipids in the sn-2 position of the glycerophospholipid backbone. AA acts as a second messenger in beta-cells to elevate cytosolic Ca2+ levels and stimulate insulin secretion, but it is not clear whether these are direct effects of AA or are dependent on its metabolism by cyclooxygenase (COX) and/or lipoxygenase (LOX) enzymes. In addition, much of the published data in this area have been generated using insulin-secreting cell lines or rodent islets, with very little information on AA generation and metabolism in human islets of Langerhans. This short review examines cPLA2, COX and LOX expression and function in insulin- secreting cell lines and rodent and human islets.     

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.     

Regulation of blood volume during weightlessness simulation of long duration

To study the effects of microgravity on the mechanisms involved in the regulation of body hydrous status, total body water (TBW), plasma volume (PV), and its main regulating hormones (plasma renin, aldosterone, atrial natriuretic peptide (ANP), anti-diuretic hormone (ADH)) were determined, by isotopic dilution, Dill and Costill's formula, and radio-immunologic dosages, in 9 male subjects submitted to a 90-d head-down bed rest (HDBR). ADH was determined in 24 h urinary collection as well as osmolality, sodium, and potassium. Body mass decreased (-2.8 +/- 0.8 kg) as well as TBW(-7.2% +/- 0.9%, i.e., -2.6 +/- 0.7 kg) and PV (-4.7% +/- 1.8%). Renin and aldosterone were enhanced (+109.0% +/- 15.4% and +87.2% +/- 38.9%, respectively). Simultaneously, we observed a decrease in ANP (-33.2% +/- 20.4%). Other variables, including ADH, were not affected by HDBR. Body mass and TBW decrease (and consequently lean body mass) are associated with muscle atrophy. Renin, aldostrerone, and ANP modifications are well explained by the decrease in PV, which was not enough to induce ADH changes. It suggests that in man, the main regulatory factor for ADH secretion is osmolality, when PV is modestly and progressively decreased without arterial pressure modification, which was the case in the present protocol.     

A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration

The semaphorin-signaling transducer collapsin response mediator protein 2 (CRMP2) has been identified in the nervous system where it mediates Sema3A-induced growth cone navigation. In the present study, we provide first evidence that CRMP2 is present in the immune system and plays a critical role in T lymphocyte function. CRMP2 redistribution at the uropod in polarized T cells, a structural support of lymphocyte motility, suggests that it may regulate T cell migration. This was evidenced in primary T cells by small-interfering RNA-mediated CRMP2 gene silencing and blocking Ab, as well as CRMP2 overexpression in Jurkat T cells tested in a chemokine- and semaphorin-mediated transmigration assay. Expression analysis in PBMC from healthy donors showed that CRMP2 is enhanced in cell subsets bearing the activation markers CD69+ and HLA-DR+. Heightened expression in T lymphocytes of patients suffering from neuroinflammatory disease with enhanced T cell-transmigrating activity points to a role for CRMP2 in pathogenesis. The elucidation of the signals and mechanisms that control this pathway will lead to a better understanding of T cell trafficking in physiological and pathological situations.