Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca²⁺]_cyt response. Cyclic-nucleotide-mediated elevations in [Ca²⁺]_cyt involved both internal and external Ca²⁺ stores. Both cAMP- and cGMP-mediated [Ca²⁺]_cyt elevations could be inhibited by the Ca²⁺-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca²⁺]_cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca²⁺]_cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca²⁺ signaling.     

Metabolic adaptations in goat mammary tissue during pregnancy and lactation

Metabolic adaptations of goat mammary tissue during pregnancy and lactation were monitored in serial biopsies of the tissue. Changes in the synthetic capacity of secretory cells were studied by combining measurements of enzyme activities with short-term culture of mammary explants to measure lactose, casein and total protein synthesis. By these criteria, the main phase of mammary differentiation began in late pregnancy and was essentially complete by Week 5 of lactation, coinciding with the achievement of peak milk yield. While milk yield declined after Week 5, the activities of key enzymes expressed per mg DNA and the rates of lactose and casein synthesis in mammary explants were maintained over a considerable period. The results suggest that changes in the synthetic capacity of epithelial cells may account for much of the rise in milk yield in early lactation, but are not responsible for the declining phase of milk production characteristic of lactation in ruminants.     

Comparison of multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) for genetic typing of Staphylococcus aureus

We compared multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) for typing of Staphylococcus aureus and show that the methods yield similar results, although with differences in resolving power and reproducibility. Epidemiological conditions should determine which is the optimal typing method to be used.     

Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination

Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.     

Improving the long‐term storage of a mammalian biosensor cell line via genetic engineering

The unique properties of mammalian cells make them valuable for a variety of applications in medicine, industry, and diagnostics. However, the utility of such cells is restricted due to the difficulty in storing them non‐frozen for an extended time and still maintaining their stability and responsiveness. In order to extend the active life span of a mammalian biosensor cell line at room and refrigerated temperatures, we have over expressed genes that are reported to provide protection from apoptosis, stress, or oxidation. We demonstrated that over expression of genes from the extremophile, Artemia franciscana, as well as GADD45β, extends room‐temperature storage of fully active cells 3.5‐fold, while over production of several anti‐apoptotic proteins extended 4°C storage 2‐ to 3‐fold. Methodologies like these that improve the stability of mammalian‐cell‐based technologies in the absence of freezers may enable widespread use of these tools in applications that have been considered impractical based solely on limited storage characteristics. Biotechnol. Bioeng. 2010; 106: 474–481. © 2010 Wiley Periodicals, Inc.     

Twenty-four hours in the life of Zingel asper

For the first time, radio-tracking observations were made over 24 h cycles during the growing season to quantify the habitat use and home range of Zingel asper , a critically endangered fish species, endemic to the Rhône River with a high risk of extinction (IUCN). New advances in radio telemetry with regard to the size of tags provided an opportunity to collect behavioural and habitat use information while remaining non-lethal. Zingel asper was characterized by small ranges of habitat use and, contrary to previous assumptions, the species was diurnal as illustrated by a smaller home range during the night than during daytime.