The complete mitochondrial and plastid genomes of the invasive marine red alga Caulacanthus okamurae (Caulacanthaceae, Rhodophyta) from Moss Landing, California, USA.

Caulacanthus okamurae is an invasive red alga that forms extensive mats in sheltered marine habitats around the world. To determine its genomic structure and genetic relationship to native and other non-native populations of C. okamurae, high-throughput sequencing analysis was performed on an introduced specimen from Bennett Slough, Moss Landing, California, USA. Assembly of 23,146,595 filtered 150 bp paired-end Illumina sequencing reads yielded its complete mitogenome (GenBank accession MT193839) and plastid genome (GenBank accession MT193838). The mitogenome is 25,995 bp in length and contains 50 genes. The plastid genome is 173,516 bp and contains 234 genes. Comparison of the organellar chromosomes to other Gigartinales revealed a high-level of gene synteny. BLAST analysis of marker sequences (rbcL, cox1, cox2) of C. okamurae from Moss Landing identified four identical DNA sequences: one from a specimen from a native population of C. okamurae from South Korea and three from specimens representing invasive populations from France, Spain, and the USA. These genetic results confirm the presence of C. okamurae in central California, USA, and represent the first complete mitogenome and plastid genome from the Caulacanthaceae.

Hyperphosphatemia induces senescence in human endothelial cells by increasing endothelin-1 production.

Hyperphosphatemia is related to some pathologies, affecting vascular cell behavior. This work analyzes whether high concentration of extracellular phosphate induces endothelial senescence through up-regulation of endothelin-1 (ET-1), exploring the mechanisms involved. The phosphate donor ß-glycerophosphate (BGP) in human endothelial cells increased ET-1 production, endothelin-converting enzyme-1 (ECE-1) protein, and mRNA expression, which depend on the AP-1 activation through ROS production. In parallel, BGP also induced endothelial senescence by increasing p16 expression and the senescence-associated ß-galactosidase (SA-ß-GAL) activity. ET-1 itself was able to induce endothelial senescence, increasing p16 expression and SA-ß-GAL activity. In addition, senescence induced by BGP was blocked when different ET-1 system antagonists were used. BGP increased ROS production at short times, and the presence of antioxidants prevented the effect of BGP on AP1 activation, ECE-1 expression, and endothelial senescence. These findings were confirmed in vivo with two animal models in which phosphate serum levels were increased: seven/eight nephrectomized rats as chronic kidney disease models fed on a high phosphate diet and aged mice. Both models showed hyperphosphatemia, higher levels of ET-1, and up-regulation in aortic ECE-1, suggesting a direct relationship between hyperphosphatemia and ET-1. Present results point to a new and relevant role of hyperphosphatemia on the regulation of ET-1 system and senescence induction at endothelial level, both in endothelial cells and aorta from two animal models. The mechanism involved showed a higher ROS production, which probably activates AP-1 transcription factor and, as a result, ECE-1 expression, increasing ET-1 synthesis, which in consequence induces endothelial senescence.

Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography.

The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.

Evaluation of a polynephron dialysis membrane considering new aspects of biocompatibility.

PURPOSE: The biocompatibility of dialyzers may influence the inflammatory state of hemodialysis patients. This study compares the effect of a high-flux polynephron membrane with other high-flux membranes, helixone and polyamide, on some inflammation biomarkers based on the analysis of circulating mononuclear cells (MC). METHODS: The study included 47 patients on hemodialysis with helixone and polyamide; 9 formed the control group, without changes in their dialyzers throughout the study, and 38 formed the intervention group, in which their dialyzers were replaced by polynephron. In both groups, blood samples were taken at the beginning of the study before and after hemodialysis session, and at the end of the study 4 months later. In each extraction, biochemical parameters were determined, and MC isolated using Ficoll gradient. Production of reactive oxygen species and the percentage of activated MC (CD14+CD16+) were measured by flow cytometry, and protein levels of heat-shock proteins (Hsp70/Hsp90) studied by Western blot. RESULTS: After 1 hemodialysis session with different membranes, no significant differences were observed in the different parameters considered. After 4 months of dialysis with polynephron, a significant reduction in the percentage of CD14+CD16+ and in the ß2-microglobulin reduction ratio were found, with respect to helixone and polyamide, without changes in the other parameters analyzed. CONCLUSIONS: The use of polynephron for 4 months reduces the percentage of CD14+CD16+ compared to helixone and polyamide, suggesting a better profile regarding activation of the inflammatory response. These findings could be explained by a better biocompatibility or an increased reduction of medium-sized toxic molecules.

Ilk conditional deletion in adult animals increases cyclic GMP-dependent vasorelaxation.

AIMS: Integrin-linked kinase (ILK) regulates proliferation, differentiation, cell adhesion, and motility in many cell types and has been related to cancer progression, fibrosis, and vascular diseases. We designed the present study to directly explore the effect of ILK deletion on the regulation of vascular tone through the soluble guanylate cyclase (sGC) /protein kinase G (PKG) pathway in healthy adult mice. METHODS AND RESULTS: Experiments were carried out using a tamoxifen-inducible CRE-LOX system to conditionally delete the ILK gene in adult mice. Mice lacking ILK expression (cKO) presented increased vascular content and increased activity of sGC and PKG, resulting in a more intense vasodilatory response to a single dose of a nitric oxide (NO) donor [sodium nitroprusside (SNP)] or PKG agonist [8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (8-Br)]. Five minutes after SNP or 8-Br administration the reduction in the systolic arterial pressure was enhanced in cKO mice (SNP WT: -7.4 ± 1.2 mmHG; SNP cKO: -14.0 ± 2.5; 8-Br WT: -2.9 ± 1.5 mmHG; 8-Br cKO: -10.0 ± 3.4 mmHG). ILK deletion restored the vascular response to SNP after chronic oral nitrite administration. In addition, ILK deletion also increased hypotensive SNP effect in angiotensin II-treated animals, suggesting a role for ILK in basal and pathological states. CONCLUSION: Deletion of ILK in adult animals increased the vascular response to NO. These findings show, for the first time, a requirement for ILK in regulating sGC-PKG expression in vivo.

A novel CRM1-dependent nuclear export signal in adenoviral E1A protein regulated by phosphorylation.

Adenoviral E1A is a versatile protein that can reprogram host cells for efficient viral replication. The nuclear import of E1A is mediated by a nuclear localization signal; however, whether E1A can be actively exported from the nucleus is unknown. We first reported a CRM1-dependent nuclear export signal (NES) in E1A that is conserved in the group C adenoviruses. We showed that CRM1 and E1A coimmunoprecipitated and that blockage of CRM1 function by leptomycin B or small interfering RNA resulted in the nuclear localization of E1A. Through mutational analyses, we identified an active canonical NES element within the E1A protein spanning amino acids 70-80. We further demonstrated that phosphorylation of adjacent serine (S)89 resulted in the cytoplasmic accumulation of E1A. Interestingly, coincident with the accumulation of cells in the S/G2/M phase and histone H1 phosphorylation, E1A was relocated to the cytoplasm at the late stage of the viral cycle, which was blocked by the CDC2/CDK2 inhibitor roscovitine. Importantly, titration of the progenies of the viruses in infected cells showed that the replication efficiency of the NES mutant adenovirus was up to 500-fold lower than that of the wild-type adenovirus. Collectively, our data demonstrate the existence of a NES in E1A that is modulated by the phosphorylation of the S89 residue and the NES plays a role for an efficient viral replication in the host cells.

Downmodulation of E1A protein expression as a novel strategy to design cancer-selective adenoviruses.

Oncolytic adenoviruses are being tested as potential therapies for human malignant tumors, including gliomas. Here we report for the first time that a mutation in the E1A gene results in low levels of E1A protein, conditioning the replication of mutant adenoviruses specifically to cancer cells. In this study, we compared the oncolytic potencies of three mutant adenoviruses encompassing deletions within the CR1 (Delta-39), CR2 (Delta-24) regions, or both regions (Delta-24/39) of the E1A protein. Delta-39 and Delta-24 induced a cytopathic effect with similar efficiency in glioma cells and a comparable capacity for replication. Importantly, the activity of Delta-39 was significantly attenuated compared to Delta-24 in proliferating normal human astrocytes. Direct analyses of the activation of E2F-1 promoter demonstrated the inability of Delta-39 to induce S-phase-related transcriptional activity in normal cells. Interestingly, E1A protein levels in cells infected with Delta-39 were remarkably downmodulated. Furthermore, protein stability studies revealed enhanced degradation of CR1 mutant E1A proteins, and inhibition of the proteasome activity resulted in the striking rescue of E1A levels. We conclude that the level of E1A protein is a critical determinant of oncolytic phenotype and we propose a completely novel strategy for the design and construction of conditionally replicative adenoviruses.

Comparative effect of oncolytic adenoviruses with E1A-55 kDa or E1B-55 kDa deletions in malignant gliomas.

Replication-competent oncolytic adenoviruses hold considerable promise for treating malignant gliomas. The toxicity of the clinically tested E1B-55 kDa mutant virus is negligible; however, its full clinical potential is still being evaluated. The purpose of the present study is to compare the antiglioma activity in vitro and in vivo between Delta-24, an E1A mutant adenovirus, and RA55, an E1B-55 kDa mutant adenovirus. We selected human glioma cell lines that were tumorigenic in nude mice and express wild-type p53 (U-87 MG, D54 MG) or mutant p53 (U-251 MG, U-373 MG) protein. Our studies demonstrated that Delta-24 induced a more potent antiglioma effect in vitro than RA55. Moreover, Delta-24 replicated markedly more efficiently than RA55 in both wild-type and mutant p53 scenarios. Importantly, direct intratumoral injection of Delta-24, but not RA55, significantly suppresses tumor growth in intracranial (U-87 MG, U-251 MG) or subcutaneous (D54 MG) animal models. Staining for hexon protein detected replicating adenoviruses in xenografts infected with Delta-24, but not with RA55. Collectively, these data indicate that E1A mutant adenoviruses targeting the Rb pathway are more powerful putative agents for antiglioma therapy than E1B mutant adenoviruses, and suggest that E1A mutant adenoviruses should be tested in the clinical setting for patients with malignant gliomas.

A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus.

Tomato plants harboring the ripening-inhibitor (rin) mutation yield fruits that fail to ripen. Additionally, rin plants display enlarged sepals and loss of inflorescence determinacy. Positional cloning of the rin locus revealed two tandem MADS-box genes (LeMADS-RIN and LeMADS-MC), whose expression patterns suggested roles in fruit ripening and sepal development, respectively. The rin mutation alters expression of both genes. Gene repression and mutant complementation demonstrate that LeMADS-RIN regulates ripening, whereas LeMADS-MC affects sepal development and inflorescence determinacy. LeMADS-RIN demonstrates an agriculturally important function of plant MADS-box genes and provides molecular insight into nonhormonal (developmental) regulation of ripening.