Heterologous Expression of a Plant Small Heat-Shock Protein Enhances Escherichia Coli Viability under Heat And Cold Stress Ref.: 1

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress

Effects of water potential on spore germination and viability of Fusarium species

Germination of macroconidia and/or microconidia of 24 strains of Fusarium solani, F. chlamydosporum, F. culmorum, F. equiseti, F. verticillioides, F. sambucinum, F. oxysporum and F. proliferatum isolated from fluvial channels and sea beds of the south-eastern coast of Spain, and three control strains (F. oxysporum isolated from affected cultures) was studied in distilled water in response to a range of water potentials adjusted with NaCI. (0, -13.79, -41.79, -70.37, -99.56 and -144.54 bars). The vialibility (UFC/ml) of suspension was also tested in three time periods (0,24 and 48h). Conidia always germinated in distilled water. The pattern of conidial germination obseved of F. verticillioides, F. oxysporum, F. proliferatum, F. chlamydosporum and F. culmorum was similar. A great diminution of spore germination was found in -13.79 bars solutions. Spore germination percentage for F. solani isolates was maximal at 48 h. and -13.79 bars with 21.33% spore germination, 16% higher than germination in distilled water. F. equiseti shows the maximum germination percentage in -144.54 bars solution in 24 h time with 12.36% germination. These results did not agree with those obtained in the viability test where maximum germination was found in distilled water. The viability analysis showed the great capacity of F. verticilloides strains to form viable colonies, even in such extreme conditions as -144,54 bars after 24 h F. proliferatum colony formation was prevented in the range of -70.37 bars. These results show the clear affectation of water potential to conidia germination of Fusaria. The ability of certain species of Fusarium to develop a saprophytic life in the salt water of the Mediterraneam Sea could be certain. Successful germination, even under high salty media conditions, suggests taht Fusarium spp. could have a competitive advantage over other soil fungi in crops irrigated with saline water. In the specific case of F. solani, water potential of -13.79 bars affected germination positively. It could indicate that F. solani has an special physiological mechanism of survival in low water potential environments.

Viability of the biochar production from different manure wastes in the Amblés Valley (Ávila, Spain)

In the last years, intensive animal husbandry production has led to a large concentration of animals in small areas. This has resulted in the production of excessive amounts of manures with insufficient nearby land for application. One of this areas is the Amblés Valley located in the centre of Spain, near to Ávila city, with an extension of 167472 ha of which 88.9% is agricultural land. This valley has an important livestock focused on pig, cattle, chicken production which is associated with the generation of more than 200,000 t/year of manure. There are a number of environmental problems associated with these intensive agricultural systems, including N and P pollution of water bodies, methane emissions and odour pollution. These serious environmental threats are called for innovative environmental management approaches. A feasible technology for the management of manures, offering a potential to valorise these wastes, is pyrolysis, which results in the production of biochar. The objective of this work is evaluated the technical and economic feasibility of the production of biochar in Amblés Valley (Spain).

Growth and viability of macrophages continuously stimulated to produce nitric oxide

Deregulated production of nitric oxide (NO) has been implicated in the development of certain human diseases, including cancer. We sought to assess the damaging potential of NO produced under long-term conditions through the development of a suitable model cell culture system. In this study, we report that when murine macrophage-like RAW264.7 cells were exposed continuously to bacterial lipopolysaccharide (LPS) or mouse recombinant interferon-γ (IFN-γ) over periods of 21–23 days, they continued to grow, but with doubling times 2 to 4 times, respectively, longer than the doubling time of unstimulated cells. Stimulated cells produced NO at rates of 30 to 70 nmol per million cells per day throughout the stimulation period. Within 24 hr after removal of stimulant, cells resumed exponential growth. Simultaneous exposure to LPS and IFN-γ resulted in decreased cell number, which persisted for 2 days after removal of the stimulants. Exponential growth was attained only after an additional 4 days. Addition of N-methyl-l-arginine (NMA), an NO synthase inhibitor, to the medium inhibited NO production by 90% of all stimulated cells, partially reduced doubling time of cells stimulated with LPS or IFN-γ, and partially increased viability and growth rates in those exposed to both LPS and IFN-γ. However, when incubated with LPS and IFN-γ at low densities both in the presence and in the absence of NMA, cells grew at a rate slower than that of unstimulated cells, with no cell death, and they resumed exponential growth 24 hr after removal of stimulants. Results from cell density experiments suggest that macrophages are protected from intracellularly generated NO; much of the NO damaging activity occurred outside of the producer cells. Collectively, results presented in this study suggest that the type of cellular toxicity observed in macrophages is markedly influenced by rate of exposure to NO: at low rates of exposure, cells exhibit slower growth; at higher rates, cells begin to die; at even higher rates, cells undergo growth arrest or die. The ability of RAW264.7 cells to produce NO over many cell generations makes the cell line a useful system for the study of other aspects of cellular damage, including genotoxicity, resulting from exposure to NO under long-term conditions.

All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae

The cyclophilins and FK506 binding proteins (FKBPs) bind to cyclosporin A, FK506, and rapamycin and mediate their immunosuppressive and toxic effects, but the physiological functions of these proteins are largely unknown. Cyclophilins and FKBPs are ubiquitous and highly conserved enzymes that catalyze peptidyl-prolyl isomerization, a rate-limiting step during in vitro protein folding. We have addressed their functions by a genetic approach in the yeast Saccharomyces cerevisiae. Five cyclophilins and three FKBPs previously were identified in yeast. We identified four additional enzymes: Cpr6 and Cpr7, which are homologs of mammalian cyclophilin 40 that have also recently been independently isolated by others, Cpr8, a homolog of the secretory pathway cyclophilin Cpr4, and Fpr4, a homolog of the nucleolar FKBP, Fpr3. None of the eight cyclophilins or four FKBPs were essential. Surprisingly, yeast mutants lacking all 12 immunophilins were viable, and the phenotype of the dodecuplet mutant resulted from simple addition of the subtle phenotypes of each individual mutation. We conclude that cyclophilins and FKBPs do not play an essential general role in protein folding and find little evidence of functional overlap between the different enzymes. We propose that each cyclophilin and FKBP instead regulates a restricted number of unique partner proteins that remain to be identified.

Trigger factor is induced upon cold shock and enhances viability of Escherichia coli at low temperatures

Trigger factor (TF) in Escherichia coli is a molecular chaperone with remarkable properties: it has prolyl-isomerase activity, associates with nascent polypeptides on ribosomes, binds to GroEL, enhances GroEL’s affinity for unfolded proteins, and promotes degradation of certain polypeptides. Because the latter effects appeared larger at 20°C, we studied the influence of temperature on TF expression. Unlike most chaperones (e.g., GroEL), which are heat-shock proteins (hsps), TF levels increased progressively as growth temperature decreased from 42°C to 16°C and even rose in cells stored at 4°C. Upon temperature downshift from 37°C to 10°C or exposure to chloramphenicol, TF synthesis was induced, like that of many cold-shock proteins. We therefore tested if TF expression might be important for viability at low temperatures. When stored at 4°C, E. coli lose viability at exponential rates. Cells with reduced TF content die faster, while cells overexpressing TF showed greater viability. Although TF overproduction protected against cold, it reduced viability at 50°C, while TF deficiency enhanced viability at this temperature. By contrast, overproduction of GroEL/ES, or hsps generally, while protective against high temperatures, reduced viability at 4°C, which may explain why expression of hsps is suppressed in the cold. Thus, TF represents an example of an E. coli protein which protects cells against low temperatures. Moreover, the differential induction of TF at low temperatures and hsps at high temperatures appears to provide selective protection against these opposite thermal extremes.

Profilin Binding to Poly-l-Proline and Actin Monomers along with Ability to Catalyze Actin Nucleotide Exchange Is Required for Viability of Fission Yeast

We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-l-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2–5% wild-type affinity for actin or poly-l-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-l-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast.

Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F1 genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F1 ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F1 ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F1 ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F1 ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F1 ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.

Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes

Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not.

A cell cycle-regulated bacterial DNA methyltransferase is essential for viability.

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.

Identification of a viability domain in the granulocyte/macrophage colony-stimulating factor receptor beta-chain involving tyrosine-750.

The granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. In this study we have investigated domains of the GMR beta-chain (GMR beta) involved in maintaining cellular viability. Using a series of nested GMR beta deletion mutants, we demonstrate that there are at least two domains of GMR beta that contribute to viability signals. Deletion of amino acid residues 626-763 causes a viability defect that can be rescued with fetal calf serum (FCS). Deletion of residues 518-626, in contrast, causes a further decrement in viability that can be only partially compensated by the addition of FCS. GMR beta truncated proximal to amino acid 517 will not support long-term growth under any conditions. Site-directed mutagenesis of tyrosine-750 (Y750), which is contained within the distal viability domain, to phenylalanine eliminates all demonstrable tyrosine phosphorylation of GMR beta. Cell lines transfected with mutant GMR beta (Y750-->F) have a viability disadvantage when compared to cell lines containing wild-type GMR that is partially rescued by the addition of FCS. We studied signal transduction in mutant cell lines in an effort to identify pathways that might participate in the viability signal. Although tyrosine phosphorylation of JAK2, SHPTP2, and Vav is intact in Y750-->F mutant cell lines, Shc tyrosine phosphorylation is reduced. This suggests a potential role for Y750 and potentially Shc in a GM-CSF-induced signaling pathway that helps maintain cellular viability.

Role of heme regulated eIF2α kinase in pancreatic beta cell function and viability

The eukaryotic translation initiation factor 2 alpha (eIF2α) is part of the initiation complex that drives the initiator amino acid methionine to the ribosome, a crucial step in protein translation. In stress conditions such as virus infection, endoplasmic reticulum (ER) stress, amino acid or heme deficiency eIF2α can be phosphorylated and thereby inhibit global protein synthesis. This adaptive mechanism prevents protein accumulation and consequent cytotoxic effects. Heme-regulated eIF2α kinase (HRI) is a member of the eIF2α kinase family that regulates protein translation in heme deficiency conditions. Although present in all tissues, HRI is predominantly expressed in erythroid cells where it remains inactive in the presence of normal heme concentrations. In response to heme deficiency, HRI is activated and phosphorylates eIF2α decreasing globin synthesis. This mechanism is important to prevent accumulation of heme-free globin chains which cause ER stress and apoptosis. RNA sequencing data from our group showed that in human islets and in primary rat beta cells HRI is the most expressed eIF2α kinase compared to the other family members. Despite its high expression levels, little is known about HRI function in beta cells. The aim of this project is to identify the role of HRI in pancreatic beta cells. This was investigated taking a loss-of-function approach. HRI knock down (KD) by RNA interference induced beta cell apoptosis in basal condition. HRI KD potentiated the apoptotic effects of palmitate or proinflammatory cytokines, two in vitro models for type 2 and type 1 diabetes, respectively. Increased cytokine-induced apoptosis was also observed in HRI-deficient primary rat beta cells. Unexpectedly, we observed a mild increase in eIF2α phosphorylation in HRI-deficient cells. The levels of mRNA or protein expression of C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) were not modified. HRI KD cells have decreased spliced X-box binding protein 1 (XBP1s), an important branch of the ER stress response. However, overexpression of XBP1s by adenovirus in HRI KD cells did not protect from HRI siRNA-induced apoptosis. HRI deficiency decreased phosphorylation of Akt and its downstream targets glycogen synthase kinase 3 (GSK3), forkhead box protein O1 (FOXO1) and Bcl-2-associated death promoter (BAD). Overexpression of a constitutively active form of Akt by adenovirus in HRI-deficient beta cells partially decreased HRI KD-mediated apoptosis. Interestingly, BAD silencing protected from apoptosis caused by HRI deficiency. HRI silencing in beta cells also induced JNK activation. These results suggest an important role of HRI in beta cell survival through modulation of the Akt/BAD pathway. Thus, HRI may be an interesting target to modulate beta cell fate in diabetic conditions.

Análisis de las propuestas de reforma del sistema electoral en España

En este estudio se analiza el sistema electoral español tratando de diagnosticar las causas de su falta de proporcionalidad entre votos y escaños. Se muestra que la causa principal de ello es la pequeña magnitud de las circunscripciones en España y se desmonta el mito de la barrera legal como impedimento para acceder al Congreso, señalando que es mucho más efectiva la barrera natural que aparece al tener España un número muy elevado de circunscripciones de baja magnitud. Posteriormente se analizan las distintas propuestas de reforma del sistema electoral encaminadas a aumentar su proporcionalidad extrapolándolas de los resultados electorales de las elecciones generales de 2015. También se analizan las dificultades para sacar adelante dichas propuestas, siendo la mayor de ellas que muchas de las propuestas exigen un cambio constitucional. Sin embargo, también se señala que algunas fórmulas que no exigen cambio constitucional pueden alcanzar incluso mayor proporcionalidad. Finalmente se comparan las distintas propuestas de reforma del sistema electoral y se advierte del peligro que conlleva una proporcionalidad muy elevada. Ésta, en general, da origen a un parlamento permanentemente fragmentado y puede generar una gran inestabilidad en los gobiernos.