Francisella tularensis regulates the expression of the amino acid transporter SLC1A5 in infected THP‐1 human monocytes

Francisella tularensis, a Gram‐negative bacterium that causes the disease tularemia in a large number of animal species, is thought to reside preferentially within macrophages in vivo. F. tularensis has developed mechanisms to rapidly escape from the phagosome into the cytoplasm of infected cells, a habitat with a rich supply of nutrients, ideal for multiplication. SLC1A5 is a neutral amino acid transporter expressed by human cells, which serves, along with SLC7A5 to equilibrate cytoplasmic amino acid pools. We herein analysed whether SLC1A5 was involved in F. tularensis intracellular multiplication. We demonstrate that expression of SLC1A5 is specifically upregulated by F. tularensis in infected THP‐1 human monocytes. Furthermore, we show that SLC1A5 downregulation decreases intracellular bacterial multiplication, supporting the involvement of SLC1A5 in F. tularensis infection. Notably, after entry of F. tularensis into cells and during the whole infection, the highly glycosylated form of SLC1A5 was deglycosylated only by bacteria capable of cytosolic multiplication. These data suggest that intracellular replication of F. tularensis depends on the function of host cell SLC1A5. Our results are the first, which show that Francisella intracellular multiplication in human monocyte cytoplasm is associated with a post‐translational modification of a eukaryotic amino acid transporter.     

Genetic diversity and structure of local apple cultivars from Northeastern Spain assessed by microsatellite markers

A set of 493 old and local Spanish accessions of apple (Malus x domestica Borkh) maintained at three collections in Northeastern Spain was studied using 16 simple sequence repeats in order to estimate their genetic diversity and to identify the genetic structure and relationships among their accessions. An additional diverse set of 45 apple cultivars, including old Spanish and international cultivars, was added as reference. Genetic analyses performed by Bayesian model-based clustering revealed a very strong differentiation of two major groups. The first one clustered 159 individuals (52?% of unique genotypes) including local accessions and six old Spanish cultivars. The second major group was formed by 145 individuals, including 38 international reference cultivars and one old Spanish cultivar. Nested Bayesian clustering was applied to those two groups and two and four sub-groups were found at each one, respectively. The identification of private and unique alleles, and the remarkable differences in allelic richness among groups and sub-groups constitute further evidence of a clear genetic structure. The results obtained through the factorial correspondence and analyses of molecular variance confirmed those obtained by Bayesian analyses, revealing moderate but significant differentiation among the two major groups (F _ST?=?0.076) and the six sub-groups (F _ST?=?0.111). Our results highlight that the genetic diversity encompassed by currently cultivated apple accounts only for a small fraction of that existing within the species, and that an important part (??60?%) of the local material analyzed constitutes a good example of genetic distinctness with respect to the main cultivars used in European orchards.     

Pten positively regulates brown adipose function, energy expenditure, and longevity

Aging in worms and flies is regulated by the PI3K/Akt/Foxo pathway. Here we extend this paradigm to mammals. Pten(tg) mice carrying additional genomic copies of Pten are protected from cancer and present a significant extension of life span that is independent of their lower cancer incidence. Interestingly, Pten(tg) mice have an increased energy expenditure and protection from metabolic pathologies. The brown adipose tissue (BAT) of Pten(tg) mice is hyperactive and presents high levels of the uncoupling protein Ucp1, which we show is a target of Foxo1. Importantly, a synthetic PI3K inhibitor also increases energy expenditure and hyperactivates the BAT in mice. These effects can be recapitulated in isolated brown adipocytes and, moreover, implants of Pten(tg) fibroblasts programmed with Prdm16 and Cebpβ form subcutaneous brown adipose pads more efficiently than wild-type fibroblasts. These observations uncover a role of Pten in promoting energy expenditure, thus decreasing nutrient storage and its associated damage.     

Potassium starvation in yeast: mechanisms of homeostasis revealed by mathematical modeling

The intrinsic ability of cells to adapt to a wide range of environmental conditions is a fundamental process required for survival. Potassium is the most abundant cation in living cells and is required for essential cellular processes, including the regulation of cell volume, pH and protein synthesis. Yeast cells can grow from low micromolar to molar potassium concentrations and utilize sophisticated control mechanisms to keep the internal potassium concentration in a viable range. We developed a mathematical model for Saccharomyces cerevisiae to explore the complex interplay between biophysical forces and molecular regulation facilitating potassium homeostasis. By using a novel inference method ("the reverse tracking algorithm") we predicted and then verified experimentally that the main regulators under conditions of potassium starvation are proton fluxes responding to changes of potassium concentrations. In contrast to the prevailing view, we show that regulation of the main potassium transport systems (Trk1,2 and Nha1) in the plasma membrane is not sufficient to achieve homeostasis.     

Prionemia and leukocyte-platelet-associated infectivity in sheep transmissible spongiform encephalopathy models

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.     

Tri-domain bifunctional inhibitor of metallocarboxypeptidases A and serine proteases isolated from marine annelid Sabellastarte magnifica

This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar K(i) values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature.