Screening of an intragenic second-site suppressor of purine-cytosine permease from Saccharomyces cerevisiae. Possible role of Ser272 in the base translocation process.

The purine-cytosine permease from Saccharomyces cerevisiae mediates the active transport through the plasma membrane of adenine, hypoxanthine, guanine and cytosine using the proton electrochemical potential difference as an energy source. Analysis of the activity of strains mutated in a hydrophilic segment (371-377) of the polypeptidic chain has shown the involvement of this segment in the maintenance of the active three-dimensional structure of the carrier. In an attempt to identify permease domains that could interact functionally and/or physically with this segment, we looked for second-site mutations that could suppress the effects of amino acid changes in this region. This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the N374I change (fcy2-20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for adenine, cytosine and hypoxanthine. The second-site mutation corresponds to the replacement of the Ser272 residue by Leu. Its suppressive effect is shown to be a partial restoration of the binding of cytosine and hypoxanthine to the permease. To test whether this second-site mutation is specific for the fcy2-20 allele, two double mutants were constructed (Fcy2pT213I, S272L and Fcy2pS272L, N377G). Results obtained with these two double mutants showed that the suppressive effect of S272 L replacement was not specific for the original N374I change. To understand the general effect of this amino acid replacement for the three distinct double mutants, a strain overexpressing Fcy2pS272I, was constructed. Kinetic analysis of this strain showed that, by itself, the S272 L change induced an improvement in the base-binding step that could account for its global suppressive effect. Moreover, S272 L induced a decrease in the turnover of the permease, thus showing the involvement of S272 in the translocation process. Taking into account the topological model of the permease proposed here, this Ser residue is probably located in a transmembrane amphipathic alpha-helix (TM5). The location and the observed decrease in the turnover of the carrier observed with the S272 L change lead us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the proton.     

Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis

Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions to further elucidate the role of the GSH synthesis pathway. Two strategies were used: the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Over time of the bioprocess, a correlation between intracellular GSH and product titer has been observed. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and nontreated cells has highlighted a slowdown of the tricarboxylic acid cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the GSH-related proteome has been observed with an upregulation of the regulatory subunit of glutamate–cysteine ligase and a downregulation of a specific GSH transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global downregulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it could be the missing piece of the puzzle to finally elucidate the link between GSH synthesis and productivity.     

Influence of Region-Specific Alterations of Neuropeptidase Content on the Catabolic Fates of Neuropeptides in Alzheimer's Disease

Abstract: We established the cartography of 11 exo- and endopeptidases in the frontal and parietal cortices and in the cerebellum of brains of patients diagnosed with a senile dementia of the Alzheimer's type (SDAT). Comparison with those of four subjects who had died without known neurologic or psychiatric illness indicated that there existed a region-specific alteration of the peptidase contents in the disease. In the frontal area of SDAT brains, postproline dipeptidyl aminopeptidase and aminopeptidase M activities were significantly reduced. In the parietal cortex of SDAT brain, activities of three additional endopeptidases—angiotensin-converting enzyme, proline endopeptidase, and endopeptidase 24.15—were also drastically reduced. In contrast, the cerebellum displayed a set of proteolytic activities that remained unaffected in SDAT brain. The putative influence of the disease on the catabolic fates of neurotensin, neuropeptide Y, and somatostatin(1–14) was investigated. Neurotensin was catabolized at identical rates in the frontal and parietal cortices in nondemented and SDAT brains. In contrast, neuropeptide Y metabolism was slowed down in SDAT brains in the frontal but not in the parietal cortex. Finally, the degradation velocities of somatostatin(1–14) were lowered in both cortical areas of SDAT brains. It is interesting that, by means of specific peptidase inhibitors, we demonstrated that endopeptidase 24.15 participated in somatostatin(1–14) inactivation in the parietal but not in the frontal cortex. It is suggested that the lowering of the rate of somatostatin(1–14) inactivation in the parietal cortex of SDAT brains likely results from the depletion of endopeptidase 24.15 in this brain region.     

Non-cryopreserved hematopoietic stem cells in autograft patients with lymphoma: a matched-pair analysis comparing a single center experience with the use of cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry

Background aimsAround 50 000 autologous stem cell transplantations are done each year worldwide using cryopreserved peripheral blood stem cells (PBSCs). Cryopreservation is time-consuming and expensive. Since 2007, several retrospective studies have shown that PBSCs can be stored at 4°C for 2–3 days, allowing autologous stem cell transplantation in patients with multiple myeloma receiving high-dose melphalan. Data with non-cryopreserved PBSCs in patients autografted for lymphoma following longer pre-conditioning regimens are limited. In addition, no controlled comparison has been able to detect unforeseen differences.MethodsThe authors compared outcomes of 94 consecutive adult patients with lymphoma (66 with Hodgkin lymphoma) autografted in our department in Oran (Algeria) using PBSCs stored at 4°C, from 2009 to 2018, with patients receiving cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry. Patients autografted in Oran were matched with patients receiving cryopreserved PBSCs in the registry (four controls per patient in Oran).ResultsNeutrophil engraftment was significantly faster with cryopreserved PBSCs (P = 0.003). By day 10, only 17% of patients receiving non-cryopreserved PBSCs engrafted versus 48% for cryopreserved PBSCs. Likewise, platelet recovery to 20 000/mm³ was significantly faster in patients receiving cryopreserved PBSCs (P = 0.01). However, all patients in both groups had recovered by day 20. There were no significant differences in non-relapse mortality (9% versus 7%, P = 0.4), relapse incidence (22% versus 32%, P = 0.13), progression-free survival (70% versus 61%, P = 0.4) or overall survival (85% versus 75%, P = 0.3).ConclusionsThis analysis suggests that, in patients with lymphoma receiving pre-transplant regimens such as carmustine, etoposide, cytarabine and melphalan, PBSCs stored at 4°C for up to 6 days can be used safely in centers with no cryopreservation facility. However, the kinetics of hematopoietic recovery showed a significant, albeit small, delay in engraftment for both neutrophils and platelets, which favors the use of cryopreservation if available.