Table 3: Reproductive viability of Hemichloris antarctica after cycles of cooling und warming

Laboratory experiments show that undercooling to about -5°C occurs in colonized Beacon sandstones of the Ross Desert, Antarctica. High-frequency temperature oscillations between 5°C and -5°C or -10°C (which occur in nature on the rock surface) did not damage Hemichloris antarctica. In a cryomicroscope, H. antarctica appeared to be undamaged after slow or rapid cooling to -50°C. l4CO2 incorporation after freezing to -20°C was unaffected in H. antarctica or in Trebouxia sp. but slightly depressed in Stichococcus sp. (isolated from a less extreme Antarctic habitat). These results suggest that the freezing regime in the Antarctic desert is not injurious to endolithic algae. It is likely that the freezing-point depression inside the rock makes available liquid water for metabolic activity at subzero temperatures. Freezing may occur more frequently on the rock surface and contribute to the abiotic nature of the surface.

Effects of Ocean Acidification and Warming on Sperm Activity and Early Life Stages of the Mediterranean Mussel (Mytilus galloprovincialis)

Larval stages are among those most vulnerable to ocean acidification (OA). Projected atmospheric CO2 levels for the end of this century may lead to negative impacts on communities dominated by calcifying taxa with planktonic life stages. We exposed Mediterranean mussel (Mytilus galloprovincialis) sperm and early life stages to pHT levels of 8.0 (current pH) and 7.6 (2100 level) by manipulating pCO2 level (380 and 1000 ppm). Sperm activity was examined at ambient temperatures (16-17 °C) using individual males as replicates. We also assessed the effects of temperature (ambient and = 20 °C) and pH on larval size, survival, respiration and calcification of late trochophore/early D-veliger stages using a cross-factorial design. Increased pCO2 had a negative effect on the percentage of motile sperm (mean response ratio R= 71%) and sperm swimming speed (R= 74%), possibly indicating reduced fertilization capacity of sperm in low concentrations. Increased temperature had a more prominent effect on larval stages than pCO2, reducing performance (RSize = 90% and RSurvival = 70%) and increasing energy demand (RRespiration = 429%). We observed no significant interactions between pCO2 and temperature. Our results suggest that increasing temperature might have a larger impact on very early larval stages of M. galloprovincialis than OA at levels predicted for the end of the century.

New Constructive Solutions to Improve Energy Efficiency in Existing Buildings and their Economic Viability

The improvement of energy efficiency in existing buildings is always a challenge due to their particular, and sometimes protected, constructive solutions. New constructive regulations in Spain leave a big undefined gap when a restoration is considered because they were developed for new buildings. However, rehabilitation is considered as an opportunity for many properties because it allows owners to obtain benefits from the use of the buildings. The current financial and housing crisis has turned society point of view to existing buildings and making them more efficient is one of the Spanish government’s aims. The economic viability of a rehabilitation action should take all factors into account: both construction costs and the future operative costs of the building must be considered. Nevertheless, the application of these regulations in Spain is left to the designer’s opinion and always under a subjective point of view. With the research work described in this paper and with the help of some case-studies, the cost of adapting an existing building to the new constructive regulations will be studied and Energetic Efficiency will be evaluated depending on how the investment is recovered. The interest of the research is based on showing how new constructive solutions can achieve higher levels of efficiency in terms of energy, construction and economy and it will demonstrate that Life Cycle Costing analysis can be a mechanism to find the advantages and disadvantages of using these new constructive solutions. Therefore, this paper has the following objectives: analysing constructive solutions in existing buildings - to establish a process for assessing total life cycle costs (LCC) during the planning stages with consideration of future operating costs - to select the most advantageous operating system – To determine the return on investment in terms of construction costs based on new techniques, the achieved energy savings and investment payback periods.

Viability study of sailing propulsion combined with a conventional system

For many years now, sails have been used as a propulsion system. At present, they are restricted to recreational/sport crafts since the appearance of the first steam vessels in the beginning of the 19 th century. But in the last years, due to the increase of fuel price and the pollution of the environment, it is being studied the possibility to introduce again the sail as a propulsive method combined with other conventional systems. In this paper, it is studied the viability of using a sail as a propellant with other conventional systems of propulsion. After considering the concept of apparent wind, the range of use of this complementary propulsion is presented. The calculation methodology, the numerical simulations and the wind inputs from a specific route are also included.

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).

Xenopus Cdc6 confers sperm binding competence to oocytes without inducing their maturation

Amphibian eggs normally require meiotic maturation to be competent for fertilization. A necessary prerequisite for this event is sperm binding, and we show that under normal physiological conditions this property is acquired at, but not before, meiotic maturation. Immature oocytes do not bind sperm, but injection of total egg poly(A)+ mRNA into immature oocytes confers sperm binding in the absence of meiotic maturation. Using an expression cloning approach we have isolated a single cDNA from egg poly(A)+ mRNA that can induce sperm binding in immature oocytes. The cDNA was found to encode Xenopus Cdc6, a protein that previously has been shown to function in initiation of DNA replication and cell cycle control. This unanticipated finding provides evidence of a link between a regulator of the cell cycle and alterations in cell surface properties that affect gamete binding.

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.

Reduced fertility in mice deficient for the POU protein sperm-1

Members of the POU-homeodomain gene family encode transcriptional regulatory molecules that play important roles in terminal differentiation of many organ systems. Sperm-1 (Sprm-1) is a POU domain factor that is exclusively expressed in the differentiating male germ cell. We show here that the Sprm-1 protein is expressed in the haploid spermatid and that 129/Sv Sprm-1(−/−) mice are subfertile when compared with wild-type or heterozygous littermates yet exhibit normal testicular morphology and produce normal numbers of mobile spermatozoa. Our data suggest that the Sprm-1 protein plays a discrete regulatory function in the haploid spermatid, which is required for the optimal function, but not the terminal differentiation, of the male germ cell.

Activation of mouse sperm T-type Ca2+ channels by adhesion to the egg zona pellucida

The sperm acrosome reaction is a Ca2+-dependent exocytotic event that is triggered by adhesion to the mammalian egg’s zona pellucida. Previous studies using ion-selective fluorescent probes suggested a role of voltage-sensitive Ca2+ channels in acrosome reactions. Here, whole-cell patch clamp techniques are used to demonstrate the expression of functional T-type Ca2+ channels during mouse spermatogenesis. The germ cell T current is inhibited by antagonists of T-type channels (pimozide and amiloride) as well as by antagonists whose major site of action is the somatic cell L-type Ca2+ channel (1,4-dihydropyridines, arylalkylamines, benzothiazapines), as has also been reported for certain somatic cell T currents. In sperm, inhibition of T channels during gamete interaction inhibits zona pellucida-dependent Ca2+ elevations, as demonstrated by ion-selective fluorescent probes, and also inhibits acrosome reactions. These studies directly link sperm T-type Ca2+ channels to fertilization. In addition, the kinetics of channel inhibition by 1,4-dihydropyridines suggests a mechanism for the reported contraceptive effects of those compounds in human males.

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.

Molecular Cloning and Characterization of a Radial Spoke Head Protein of Sea Urchin Sperm Axonemes: Involvement of the Protein in the Regulation of Sperm Motility

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40°C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form α-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Assembly of Lampbrush Chromosomes from Sperm Chromatin

We have examined the behavior of demembranated sperm heads when injected into the germinal vesicle (GV) of amphibian oocytes. Xenopus sperm heads injected into Xenopus GVs swelled immediately and within hours began to stain with an antibody against RNA polymerase II (Pol II). Over time each sperm head became a loose mass of chromosome-like threads, which by 24–48 h resolved into individually recognizable lampbrush chromosomes (LBCs). Although LBCs derived from sperm are unreplicated single chromatids, their morphology and immunofluorescent staining properties were strikingly similar to those of the endogenous lampbrush bivalents. They displayed typical transcriptionally active loops extending from an axis of condensed chromomeres, as well as locus-specific “landmarks.” Experiments with [3H]GTP and actinomycin D demonstrated that transcription was not necessary for the initial swelling of the sperm heads and acquisition of Pol II but was required for maintenance of the lampbrush loops. Splicing was not required at any stage during formation of sperm LBCs. When Xenopus sperm heads were injected into GVs of the newt Notophthalmus, the resulting sperm LBCs displayed very long loops with pronounced Pol II axes, like those of the endogenous newt LBCs; as expected, they stained with antibodies against newt-specific proteins. Other heterologous injections, including sperm heads of the frog Rana pipiens and the zebrafish Danio rerio in Xenopus GVs, confirm that LBCs can be derived from taxonomically distant organisms. The GV system should help identify both cis- and trans-acting factors needed to convert condensed chromatin into transcriptionally active LBCs. It may also be useful in producing cytologically analyzable chromosomes from organisms whose oocytes do not go through a typical lampbrush phase or cannot be manipulated by current techniques.