L-Asparaginase of Saccharomyces cerevisiae: an extracellular Enzyme.

During recent studies conducted with suspensions of three strains of Saccharomyces cerevisiae, it was observed that ammonia was rapidly liberated when L-asparagine was added to the medium. Subsequent investigation has revealed that these strains of S. cerevisiae have an externally active asparaginase as well as an internally active one. The appearance of the external asparaginase is stimulated by nitrogen starvation, requires an available energy source, and is prevented by cycloheximide. The internal enzyme appears to be constitutive. The external activity is relatively insensitive to para-hydroxymercuribenzoate inhibition, whereas the internal activity is highly inhibited by this compound.     

Evolutionary Patterns of Morphology and Behavior as Inferred from a Molecular Phylogeny of New World Emballonurid Bats (Tribe Diclidurini)

A molecular phylogeny of New World emballonurid bats based on parsimony and Bayesian analyses of loci from the three different nuclear genetic transmission pathways in mammals (autosomal, X, and Y chromosomes) is well supported and independently corroborated by each individual gene tree. This is in contrast to a single most parsimonious but poorly supported tree based on morphological data, which has only one intergeneric or higher relationship shared with the molecular phylogeny. Combining the morphological and molecular data partitions results in a tree similar to the molecular tree suggesting a high degree of homoplasy and low phylogenetic signal in the morphological data set. Behavioral data are largely incomplete and likewise produce a poorly resolved tree. Nonetheless, patterns of evolution in morphology and behavior can be investigated by using the molecular tree as a phylogenetic framework. Character optimization of the appearance of dorsal fur and preferred roosting sites maps consistently and are correlated on the phylogeny. This suggests an association of camouflage for bats with unusual appearance (two dorsal stripes in Rhynchonycteris and Saccopteryx, or pale fur in Cyttarops and Diclidurus) and roosting in exposed sites (tree trunks or under palm leaves). In contrast, the ancestral states for Old and New World emballonurids are typically uniform brown or black, and they usually roost in sheltered roosts such as caves and tree hollows. Emballonuridae is the only family of bats that has a sac-like structure in the wing propatagium, which is found in four New World genera. Mapping the wing sac character states onto the phylogeny indicates that wing sacs evolved independently within each genus and that there may be a phylogenetic predisposition for this structure. Ear orientation maps relatively consistently on the molecular phylogeny and is correlated to echolocation call parameters and foraging behavior, suggesting a phylogenetic basis for these character systems.     

Tunable Stochastic Pulsing in the Escherichia coli Multiple Antibiotic Resistance Network from Interlinked Positive and Negative Feedback Loops

Cells live in uncertain, dynamic environments and have many mechanisms for sensing and responding to changes in their surroundings. However, sudden fluctuations in the environment can be catastrophic to a population if it relies solely on sensory responses, which have a delay associated with them. Cells can reconcile these effects by using a tunable stochastic response, where in the absence of a stressor they create phenotypic diversity within an isogenic population, but use a deterministic response when stressors are sensed. Here, we develop a stochastic model of the multiple antibiotic resistance network of Escherichia coli and show that it can produce tunable stochastic pulses in the activator MarA. In particular, we show that a combination of interlinked positive and negative feedback loops plays an important role in setting the dynamics of the stochastic pulses. Negative feedback produces a pulsatile response that is tunable, while positive feedback serves to amplify the effect. Our simulations show that the uninduced native network is in a parameter regime that is of low cost to the cell (taxing resistance mechanisms are expressed infrequently) and also elevated noise strength (phenotypic variability is high). The stochastic pulsing can be tuned by MarA induction such that variability is decreased once stresses are sensed, avoiding the detrimental effects of noise when an optimal MarA concentration is needed. We further show that variability in the expression of MarA can act as a bet hedging mechanism, allowing for survival in time-varying stress environments, however this effect is tunable to allow for a fully induced, deterministic response in the presence of a stressor.     

Early Proliferation Does Not Prevent the Loss of Oligodendrocyte Progenitor Cells during the Chronic Phase of Secondary Degeneration in a CNS White Matter Tract

Partial injury to the central nervous system (CNS) is exacerbated by additional loss of neurons and glia via toxic events known as secondary degeneration. Using partial transection of the rat optic nerve (ON) as a model, we have previously shown that myelin decompaction persists during secondary degeneration. Failure to repair myelin abnormalities during secondary degeneration may be attributed to insufficient OPC proliferation and/or differentiation to compensate for loss of oligodendrocyte lineage cells (oligodendroglia). Following partial ON transection, we found that sub-populations of oligodendroglia and other olig2+ glia were differentially influenced by injury. A high proportion of NG2+/olig2–, NG2+/olig2+ and CC1−/olig2+ cells proliferated (Ki67+) at 3 days, prior to the onset of death (TUNEL+) at 7 days, suggesting injury-related cues triggered proliferation rather than early loss of oligodendroglia. Despite this, a high proportion (20%) of the NG2+/olig2+ OPCs were TUNEL+ at 3 months, and numbers remained chronically lower, indicating that proliferation of these cells was insufficient to maintain population numbers. There was significant death of NG2+/olig2– and NG2−/olig2+ cells at 7 days, however population densities remained stable, suggesting proliferation was sufficient to sustain cell numbers. Relatively few TUNEL+/CC1+ cells were detected at 7 days, and no change in density indicated that mature CC1+ oligodendrocytes were resistant to secondary degeneration in vivo. Mature CC1+/olig2– oligodendrocyte density increased at 3 days, reflecting early oligogenesis, while the appearance of shortened myelin internodes at 3 months suggested remyelination. Taken together, chronic OPC decreases may contribute to the persistent myelin abnormalities and functional loss seen in ON during secondary degeneration.     

Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants

Objectives

To develop and validate a microdilution method for measuring the minimum inhibitory concentration (MIC) of biosurfactants.

Results

A standardized microdilution method including resazurin dye has been developed for measuring the MIC of biosurfactants and its validity was established through the replication of tetracycline and gentamicin MIC determination with standard bacterial strains.

Conclusion

This new method allows the generation of accurate MIC measurements, whilst overcoming critical issues related to colour and solubility which may interfere with growth measurements for many types of biosurfactant extracts.

     

The effect of close approaches for tagging activities by small research vessels on the behavior of humpback whales (Megaptera novaeangliae)

Small research vessels are often used as platforms for tagging activities to collect behavioral data on cetaceans and they have the potential to disturb that group or individual. If this disturbance is ignored, results and conclusions produced by that study could be inaccurate. Here land‐based behavioral data of migrating humpback whales (Megaptera novaeangliae) (n = 29) were used to determine the effect of close approaches for tagging by research vessels on their diving, movement and surface behaviors. Groups of whales were tagged, using digital recording tags, by small research vessels, as part of a behavioral response study. In groups that were approached for tagging, temporary changes in movement behaviors during close approaches were found, with subsequent recovery to “pre‐approach” levels. In female‐calf groups more long‐term changes in travel speed were found. Results suggest that, although close approaches for tagging by small vessels may cause behavioral changes in humpback whales, this change may be small and temporary. However, in female‐calf groups, the behavioral change may be greater and longer lasting. This study shows that when using small vessels for behavioral research, disturbance, and recovery should be measured to ensure integrity of data used for other analyses.     

Expression of human BK ion channels in Sf9 cells, their purification using metal affinity chromatography, and functional reconstitution into planar lipid bilayers

This report describes a procedure for purification of large conductance calcium-activated potassium (BK, maxi-K) channels using immobilised metal affinity chromatography (IMAC) under non-denaturing conditions. An amino-terminal histidine fusion tag was added to hSlo, the human BK channel, and expressed in Sf9 insect cells. Following IMAC purification and production of proteoliposomes, protein function was assessed electrophysiologically in planar bilayer lipid membranes. Single channel openings had conductances of 250-300 pS and were inhibited by paxilline, demonstrating that the BK channels remained functional following IMAC purification. This method to obtain functional human ion channels will be useful in assays to screen potential pharmaceuticals.