Loading fluorescent Ca²⁺ indicators into living cells

Small-molecule fluorescent Ca2+ reporters are the most widely used tools in the field of Ca2+ signaling. The excellent spatial and temporal resolution afforded by fluorescent reporters has driven the understanding of Ca2+ as a messenger in many different cell types. In many situations, the cellular loading and monitoring of fluorescent Ca2+ indicators is quite trivial. However, there are numerous pitfalls that require consideration to ensure that optimal data are recorded. Fluorescent Ca2+ indicators have carboxylic acid groups for binding of Ca2+. Because these “free-acid” forms of the indicators are hydrophilic they cannot readily cross cell membranes and need to be introduced into cells using techniques such as microinjection, pinocytosis, or diffusion from a patch pipette. However, the most convenient and widely used method for loading indicators into cells is as hydrophobic compounds in which the carboxylic acid groups are esterified (commonly as acetoxymethyl [AM] or acetate esters). The ester versions of the indicators permeate the plasma membrane. The Ca2+-sensitive, free-acid form of the indicator is liberated following hydrolysis of the ester groups by intracellular esterases

Local and global spontaneous calcium events regulate neurite outgrowth and onset of GABAergic phenotype during neural precursor differentiation

Neural stem cells can generate in vitro progenitors of the three main cell lineages found in the CNS. The signaling pathways underlying the acquisition of differentiated phenotypes in these cells are poorly understood. Here we tested the hypothesis that Ca2+ signaling controls differentiation of neural precursors. We found low-frequency global and local Ca2+ transients occurring predominantly during early stages of differentiation. Spontaneous Ca2+ signals in individual precursors were not synchronized with Ca2+ transients in surrounding cells. Experimentally induced changes in the frequency of local Ca2+signals and global Ca2+ rises correlated positively with neurite outgrowth and the onset of GABAergic neurotransmitter phenotype, respectively. NMDA receptor activity was critical for alterations in neuronal morphology but not for the timing of the acquisition of the neurotransmitter phenotype. Thus, spontaneous Ca2+ signals are an intrinsic property of differentiating neurosphere-derived precursors. Their frequency may specify neuronal morphology and acquisition of neurotransmitter phenotype

NMR solution structure of a cold-adapted thiol-disulfide oxidoreductase

Psychrophilic enzymes produced by cold-adapted micro-organisms have successfully overcome the low temperature challenge and adapted to maintain high catalytic rates in their permanently cold environments. The current consensus is that this high activity at low temperatures is mainly achieved through an increase in the flexibility of the protein structure, thereby allowing for the molecular motions necessary for activity in their low thermal energy environment. The actual molecular basis for the adaptation is still however only poorly understood and direct evidence of the proposed increased flexibility is scant, with previous attempts to demonstrate this leading to conflicting results. In an attempt to better understand strategies of cold adaptation we have determined the NMR solution structure of the reduced form of a cold adapted thiol disulphide oxidoreductase (DsbA) isolated from an Antarctic bacterium. While a number of crystal structures for cold adapted enzymes have been published, this is the first report of an NMR structure of these enzymes and thereby opens up a new dimension in the study of cold adaptation. In particular, the potential power of NMR to monitor both local and global motions over a large range of time scales should allow for a better understanding of the role of dynamics in protein adaptation to temperature. The gene encoding the cold-adapted enzyme has been isolated and the protein overexpressed in E. coli with both unlabelled and labelled (15N13C, 15N) protein being purified from the periplasmic extracts. NMR data were acquired on a Bruker AvanceII+ 800 MHz spectrometer and the solution structure of the reduced form of this cold adapted oxidoreductase determined and compared to that of its mesophilic homolog from Vibrio cholerae. In addition, the temperature dependence of activity and stability of both the psychrophile and mesophile have been ascertained and compared. Here, the results of the NMR structure determination and the comparative structural and physicochemical studies of the cold adapted DsbA with its mesophilic homolog will be presented.European Molecular Biology Organisation (EMBO). Fundação para a Ciência e a Tecnologia (FCT) via Programa Ciência 2008

Ariel - Volume 12 Number 2

Executive Editors David G. Polin Larry H. Pastor Business Manager Alex Macones Jean Lien Editorial Page Editor Deepak Kapoor Sports Editor Todd Hoover Photography Editors Lois Leach Ken Yonemur

Key stress factors and parameters for batch production optimisation of silk-elastin-like proteins in E. coli

European Commission via the 7th Framework Programme Project EcoPlast. Fundação para a Ciência e a Tecnologia (FCT) via Ciência 2008

Key stress factors and parameters for production optimisation of silk-elastin-like proteins in E. coli BL21(DE3)

Silk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. We have recently synthesised a series of novel silk-elastin-like proteins and here we will describe the optimisation of the production of these with the pET-E. coli BL21(DE3) expression system. Both batch production in shake flasks and fed-batch production approaches were investigated. Furthermore, a comprehensive empirical approach examining all process variables (media, medium composition, inducer, induction time and period, temperature, pH, aeration, agitation, pre- and post-induction growth rates) and a detailed characterisation of the bioprocesses were carried out in an attempt to maximise production and to identify the factors limiting higher production levels. Using the optimised conditions, approximately 0.5 g/l of purified SELP was obtained in shake flasks and as much as 4 g/L was obtained when using the fed-batch approach. These represent, respectively, approximately 10 and almost 100-fold increases on that previously reported for SELPs.Fundação para a Ciência e a Tecnologia (FCT)This work was financed by the European Commission via the 7th Framework Programme Project EcoPlast (FP7-NMP-2009-SME-3, collaborative project number 246176)

Backbone and side chain 1H, 15N and 13C assignments for a thiol-disulphide oxidoreductase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Enzymes produced by psychrophilic organisms have successfully overcome the low temperature challenge and evolved to maintain high catalytic rates in their permanently cold environments. As an initial step in our attempt to elucidate the cold-adaptation strategies used by these enzymes we report here the 1H, 15N and 13C assignments for the reduced form of a thiol-disulphide oxidoreductase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.The NMR spectrometers are part of The National NMR Network (REDE/1517/RMN/2005), supported by ‘‘Programa Operacional Ciência e Inovação (POCTI) 2010’’ and Fundação para a Ciência e a Tecnologia (FCT). This work was funded by FCT, POCTI and FEDER; Projects POCI/BIA-PRO/57263/2004 and PTDC/BIO/70806/2006. TC is holder of a long term EMBO fellowship. MM is thankful to the Fundação para a Ciência e Tecnologia for its support through Programa Ciência 2007.info:eu-repo/semantics/publishedVersio

ENSO in a changing climate

The El Niño–Southern Oscillation (ENSO) phenomenon is a naturally occurring climate fluctuation, which originates in the tropical Pacific region and affects ecosystems, agriculture, freshwater supplies, hurricanes and other severe weather events worldwide (Goddard and Dilley 2005; McPhaden et al. 2006). Despite considerable progress in our understanding of the impact of climate change on many of the processes that contribute to ENSO variability (e.g., Collins et al. 2010), it is not yet possible to say whether ENSO activity will be enhanced or damped, or if the frequency or character of events will change in the coming decades (Vecchi and Wittenberg 2010). As changes in ENSO have the potential to be one of the largest manifestations of anthropogenic climate change, this status has profound impacts on the reliability of regional attribution of climate variability and change. Two main reasons can be invoked for these shortcomings. First there is a lack of long and comprehensive enough observations of the various ENSO processes to be able to detect past changes. It may be that we need to observe ENSO for another several decades to detect and attribute significant ENSO changes (Wittenberg 2009; Stevenson et al. 2012). Second, as ENSO involves a complex interplay of numerous ocean and atmospheric processes, accurately modeling this climate phenomenon with CGCMs, an

Optimising a cell factory system for the bioproduction of silk-elastin-like polymers.

Silk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the production optimisation for a novel recently described SELP in the pETE. coli BL21(DE3) expression system. Both batch production in shake flasks and fed-batch production in fermenters were investigated and optimised. A comprehensive empirical approach optimising all process variables for both processes, in addition to molecular biology approaches for improving performance of the production plasmid were used to maximise production levels. Typical volumetric productivities reported for SELPs are approximately 30 mg/L but here we have increased production levels up to approximately 4 g/L, representing the highest reported SELP productivity to date.Fundação para a Ciência e a Tecnologia (FCT)European CommissionThis work was financed by the European Commission, via the 7th Framework Programme Project EcoPlast (FP7-NMP-2009-SME-3, collaborative project number 246176), by FEDER through POFC – COMPETE and by national funds from Fundação para a Ciência e Tecnologia (FCT) through PEst project C/BIA/UI4050/2011C/BIA/UI4050/2011