A comparative proteomic analysis of skin secretions of the tammar wallaby (Macropus eugenii) and the wombat (Vombatus ursinus)

The secretome of the pouch skin of the model marsupial the tammar wallaby, Macropus eugenii has been investigated using techniques of two-dimensional gel electrophoresis, in-gel trypsin digestion followed by nanoliquid chromatography coupled tandem mass spectrometry (LC-MS/MS). Differences in the patterns of secreted proteins were observed in the female pouch at three stages of maturity — reproductively immature; reproductively mature and active and mature, postreproductively active. Skin from the underarm area of mature females had a markedly different secreted protein profile. The greatest diversity of proteins was seen in the mature reproductive pouch and from an opportunistic sample collected from the pouch another mature female marsupial, the common wombat, Vombatus ursinus. A total of 20 proteins were confidently identified from the pouch skin secretions of the tammar wallaby and wombats, whilst 20 proteins were tentatively identified. In all skin secretomes, globins were the most abundant proteins whilst the antimicrobial, dermcidin was detected in the wombat sample. Some proteins such as keratin and actin could be sourced to sloughed and degraded skin cells. A number of proteins were present at such low concentrations that confident identification was not possible. This was compounded by the lack of a comprehensive database of marsupial proteins which constrains the reliability of automated identification protocols.     

Exploration of Ellsworth Subglacial Lake: a concept paper on the development, organisation and execution of an experiment to explore, measure and sample the environment of a West Antarctic subglacial lake

Antarctic subglacial lakes have, over the past few years, been hypothesised to house unique forms of life and hold detailed sedimentary records of past climate change. Testing this hypothesis requires in situ examinations. The direct measurement of subglacial lakes has been considered ever since the largest and best-known lake, named Lake Vostok, was identified as having a deep water-column. The Subglacial Antarctic Lake Environments (SALE) programme, set up by the Scientific Committee on Antarctic Research (SCAR) to oversee subglacial lakes research, state that prior exploration of smaller lakes would be a “prudent way forward”. Over 145 subglacial lakes are known to exist in Antarctica, but one lake in West Antarctica, officially named Ellsworth Subglacial Lake (referred to hereafter as Lake Ellsworth), stands out as a candidate for early exploration. A consortium of over 20 scientists from seven countries and 14 institutions has been assembled to plan the exploration of Lake Ellsworth. An eight-year programme is envisaged: 3 years for a geophysical survey, 2 years for equipment development and testing, 1 year for field planning and operation, and 2 years for sample analysis and data interpretation. The science experiment is simple in concept but complex in execution. Lake Ellsworth will be accessed using hot water drilling. Once lake access is achieved, a probe will be lowered down the borehole and into the lake. The probe will contain a series of instruments to measure biological, chemical and physical characteristics of the lake water and sediments, and will utilise a tether to the ice surface through which power, communication and data will be transmitted. The probe will pass through the water column to the lake floor. The probe will then be pulled up and out of the lake, measuring its environment continually as this is done. Once at the ice surface, any water samples collected will be taken from the probe for laboratory analysis (to take place over subsequent years). The duration of the science mission, from deployment of the probe to its retrieval, is likely to take between 24 and 36 h. Measurements to be taken by the probe will provide data about the following: depth, pressure, conductivity and temperature; pH levels; biomolecules (using life marker chips); anions (using a chemical analyzer); visualisation of the environment (using cameras and light sources); dissolved gases (using chromatography); and morphology of the lake floor and sediment structures (using sonar). After the probe has been retrieved, a sediment corer may be dropped into the lake to recover material from the lake floor. Finally, if time permits, a thermistor string may be left in the lake water to take time-dependent measurements of the lake’s water column over subsequent years. Given that the comprehensive geophysical survey of the lake will take place in two seasons during 2007–2009, a two-year instrument and logistic development phase from 2008 (after the lake’s bathymetry has been assessed) makes it possible that the exploration of Lake Ellsworth could take place at the beginning of the next decade.     

A field portable method for the semi‐quantitative estimation of dehydration tolerance of photosynthetic tissues across distantly related land plants

Desiccation tolerant (DT) plants withstand complete cellular dehydration, reaching relative water contents (RWC) below 30% in their photosynthetic tissues. Desiccation sensitive (DS) plants exhibit different degrees of dehydration tolerance (DHT), never surviving water loss >70%. To date, no procedure for the quantitative evaluation of DHT extent exists that is able to discriminate DS species with differing degrees of DHT from truly DT plants. We developed a simple, feasible and portable protocol to differentiate between DT and different degrees of DHT in the photosynthetic tissues of seed plants and between fast desiccation (< 24 h) tolerant (FDT) and sensitive (FDS) bryophytes. The protocol is based on (1) controlled desiccation inside Falcon tubes equilibrated at three different relative humidities that, consequently, induce three different speeds and extents of dehydration and (2) an evaluation of the average percentage of maximal photochemical efficiency of PSII (F_v/fm) recovery after rehydration. Applying the method to 10 bryophytes and 28 tracheophytes from various locations, we found that (1) imbibition of absorbent material with concentrated salt‐solutions inside the tubes provides stable relative humidity and avoids direct contact with samples; (2) for 50 ml capacity tubes, the optimal plant amount is 50–200 mg fresh weight; (3) the method is useful in remote locations due to minimal instrumental requirements; and (4) a threshold of 30% recovery of the initial F_v/fm upon reaching RWC ≤ 30% correctly categorises DT species, with three exceptions: two poikilochlorophyllous species and one gymnosperm. The protocol provides a semi‐quantitative expression of DHT that facilitates comparisons of species with different morpho‐physiological traits and/or ecological attributes.     

The crystal structure of C-terminal merozoite surface protein 1 at 1.8 A resolution, a highly protective malaria vaccine candidate

The C-terminal proteolytic processing product of merozoite surface protein 1 (MSP1) appears essential for successful erythrocyte invasion by the malarial parasite, Plasmodium. We have determined the crystal structure at 1.8 A resolution of a soluble baculovirus-recombinant form of the protein from P. cynomolgi, which confers excellent protective efficacy in primate vaccination trials. The structure comprises two EGF-like domains, and sequence comparisons strongly suggest that the same conformation is present in all species of Plasmodium, including P. falciparum and P. vivax, which are pathogenic in man. In particular, conserved interdomain contacts between the two EGF modules should preserve the compact form of the molecule in all species. Implications of the crystal structure for anti-malarial vaccine development are discussed.     

pPAC-ResQ: A yeast-bacterial shuttle vector for capturing inserts from P1 and PAC clones by recombinogenic targeted cloning

We have developed a method to capture inserts from P1 and P1 artificial chromosome (PAC) clones into a yeast-bacteria shuttle vector by using recombinogenic targeting. We have engineered a vector, pPAC-ResQ, a derivative of pClasper, which was previously used to capture inserts from yeast artificial chromosome clones. pPAC-ResQ contains DNA fragments flanking the inserts in P1 and PAC vectors as recombinogenic ends. When linearized pPAC-ResQ vector and P1 or PAC DNA are cotransformed into yeast, recombination between the two leads to the transfer of inserts into pPAC-ResQ. pPAC-ResQ clones thus obtained can be further modified in yeast for functional analysis and shuttled to Escherichia coli to produce large quantities of cloned DNA. This approach provides a rapid method to modify P1/PAC clones for functional analysis.     

SCP1, a 356,023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2)

The sequencing of the entire genetic complement of Streptomyces coelicolor A3(2) has been completed with the determination of the 365,023 bp sequence of the linear plasmid SCP1. Remarkably, the functional distribution of SCP1 genes somewhat resembles that of the chromosome: predicted gene products/functions include ECF sigma factors, antibiotic biosynthesis, a gamma-butyrolactone signalling system, members of the actinomycete-specific Wbl class of regulatory proteins and 14 secreted proteins. Some of these genes are among the 18 that contain a TTA codon, making them targets for the developmentally important tRNA encoded by the bldA gene. RNA analysis and gene fusions showed that one of the TTA-containing genes is part of a large bldA-dependent operon, the gene products of which include three proteins isolated from the spore surface by detergent washing (SapC, D and E), and several probable metabolic enzymes. SCP1 shows much evidence of recombinational interactions with other replicons and transposable elements during its history. For example, it has two sets of partitioning genes (which may explain why an integrated copy of SCP1 partially suppressed the defective partitioning of a parAB-deleted chromosome during sporulation). SCP1 carries a cluster of probable transfer determinants and genes encoding likely DNA polymerase III subunits, but it lacks an obvious candidate gene for the terminal protein associated with its ends. This may be related to atypical features of its end sequences.     

Bioinformatic identification of novel regulatory DNA sequence motifs in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Background  

Streptomyces coelicolor is a bacterium with a vast repertoire of metabolic functions and complex systems of cellular development. Its genome sequence is rich in genes that encode regulatory proteins to control these processes in response to its changing environment. We wished to apply a recently published bioinformatic method for identifying novel regulatory sequence signals to gain new insights into regulation in S. coelicolor.     

How many species are infected with Wolbachia? Cryptic sex ratio distorters revealed to be common by intensive sampling.

Inherited bacterial symbionts from the genus Wolbachia have attracted much attention by virtue of their ability to manipulate the reproduction of their arthropod hosts. The potential importance of these bacteria has been underlined by surveys, which have estimated that 17% of insect species are infected. We examined whether these surveys have systematically underestimated the proportion of infected species through failing to detect the low-prevalence infections that are expected when Wolbachia distorts the sex ratio. We estimated the proportion of species infected with Wolbachia within Acraea butterflies by testing large collections of each species for infection. Seven out of 24 species of Acraea were infected with Wolbachia. Four of these were infected with Wolbachia at high prevalence, a figure compatible with previous broad-scale surveys, whilst three carried low-prevalence infections that would have had a very low likelihood of being detected by previous sampling methods. This led us to conclude that sex-ratio-distorting Wolbachia may be common in insects that have an ecology and/or genetics that permit the invasion of these parasites and that previous surveys may have seriously underestimated the proportion of species that are infected.     

Effects of in situ cobalt ion addition on the activity of a gfp-oph fusion protein: the fermentation kinetics

The effects of cobalt ion addition and inducer concentration were studied in the fermentation of E. coli BL21 expressing a GFP (green fluorescent protein)-OPH (organophosphorus hydrolase) fusion protein. It was found that cobalt ion addition improved the OPH activity significantly. When 2 mM of CoCl(2) was supplied during the IPTG-induction phase, OPH activity was enhanced approximately 10-fold compared to the case without cobalt or by the addition of cobalt to the cell extracts. Results indicate, therefore, that incorporation of the cobalt during synthesis is needed for enhanced activity. Also, the maximum OPH activity was not linearly related to inducer concentration. A mathematical model was then constructed to simulate these phenomena. Model parameters were determined by constrained least-squares and optimal IPTG and cobalt addition concentrations were obtained, pinpointing the conditions for the maximum productivity. Finally, the GFP fluorescence intensity was found linear to the OPH activity in each fermentation, demonstrating the function of GFP for monitoring its fusion partner's quantity in the bioreactor.