Analysis of the molecular variation between and within cultivated and wild Pistacia species using AFLPs

Knowledge of pistachio genetic diversity is necessary for the formulation of appropriate management strategies for the conservation of these species. We analysed amplified fragment length polymorphisms in a total of 216 pistachio accessions, which included seven populations from three wild species (Pistacia vera, Pistacia khinjuk and Pistacia atlantica subsp. kurdica) and most of the important cultivars from Iran, together with some foreign cultivars. High levels of genetic diversity were detected within the Iranian cultivars, and they showed a clear separation from foreign cultivars, as revealed by unweighted pair group method with arithmetic averaging and supported by analysis of molecular variance. The lowest amount of polymorphism was observed in P. atlantica subsp. kurdica, which showed the lowest number of total bands as compared to the other species. This revealed strong genetic erosion of P. atlantica subsp. kurdica, which reflected a severe decline in habitat and over-exploitation. Based on these findings, strategies are proposed for the genetic conservation and management of pistachio species and cultivars.     

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment

Tolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin‐regulated water reabsorption via aquaporin‐2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2‐mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin–cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP‐induced increase in ser256‐AQP2 and osmotic water permeability. A similar effect on ser256‐AQP2 was found in V1aR ?/? mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan–V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256‐AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium‐inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance.     

Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida

? Expansins are cell wall proteins required for cell enlargement and cell wall loosening during many developmental processes. The involvement of the Petunia hybrida expansin A1 (PhEXPA1) gene in cell expansion, the control of organ size and cell wall polysaccharide composition was investigated by overexpressing PhEXPA1 in petunia plants. ? PhEXPA1 promoter activity was evaluated using a promoter-GUS assay and the protein's subcellular localization was established by expressing a PhEXPA1-GFP fusion protein. PhEXPA1 was overexpressed in transgenic plants using the cauliflower mosaic virus (CaMV) 35S promoter. Fourier transform infrared (FTIR) and chemical analysis were used for the quantitative analysis of cell wall polymers. ? The GUS and GFP assays demonstrated that PhEXPA1 is present in the cell walls of expanding tissues. The constitutive overexpression of PhEXPA1 significantly affected expansin activity and organ size, leading to changes in the architecture of petunia plants by initiating premature axillary meristem outgrowth. Moreover, a significant change in cell wall polymer composition in the petal limbs of transgenic plants was observed. ? These results support a role for expansins in the determination of organ shape, in lateral branching, and in the variation of cell wall polymer composition, probably reflecting a complex role in cell wall metabolism.     

Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells

Increasing evidence suggests that bone marrow-derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor-associated neo-angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF-α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF-α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF-α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF-α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin-2, granulocyte-colony stimulating factor, hepatocyte growth factor, interleukin (IL)-6, IL-8, and platelet-derived growth factor-BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF-α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF-α increased the ability of MSCs to induce migration of MCF-7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo-angiogenesis and tumor cell migration.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.     

Mitochondrial respiratory chain super-complex I–III in physiology and pathology

Recent investigations by native gel electrophoresis showed the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis demonstrated that Complex I and Complex III in mammalian mitochondria kinetically behave as a single unit with control coefficients approaching unity for each component, suggesting the existence of substrate channeling within the super-complex. The formation of this supramolecular unit largely depends on the lipid content and composition of the inner mitochondrial membrane. The function of the super-complexes appears not to be restricted to kinetic advantages in electron transfer: we discuss evidence on their role in the stability and assembly of the individual complexes, particularly Complex I, and in preventing excess oxygen radical formation. There is increasing evidence that disruption of the super-complex organization leads to functional derangements responsible for pathological changes, as we have found in K-ras-transformed fibroblasts.     

Roles of ring-hydroxylating dioxygenases in styrene and benzene catabolism in Rhodococcus jostii RHA1

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ΔbphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the β-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.     

Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system

Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli.     

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells.

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.     

Production of glycerides from glycerol and fatty acid by immobilized lipases in non-aqueous media

Immobilized Mucor miehei lipase catalyzes synthesis reactions between glycerol and oleic acid. No organic solvent is necessary to solubilize the substrates, which allows for the use of a reaction medium solely composed of the necessary substrates. Water produced in the reaction evaporates due to the high temperature used for the process. A conversion of 86% of oleic acid into triolein is obtained when using the substrates in stoichiometric amounts. Varying the ratio of glycerol over oleic acid allows for the preferential synthesis of one of the glycerides. Some batch reactors have been set up using different means of removing the water: spontaneous evaporation, molecular sieves, vacuum, and dry air bubbling.