Environmental filtering determines community patterns in temporary wetlands: a multi-taxon approach

Climate characteristics appear to play a key role in filtering organisms based on their biological traits. If this trait filtering by climate indeed occurs, it should have effects on the composition, dynamics, taxonomic relatedness and co-occurrence patterns of local assemblages, regardless of the taxonomic group considered. This preliminary study aimed to assess the extent to which environmental variables might determine these patterns in local communities and to evaluate whether the ultimate cross-taxon congruence relationships are consistent across, or dependent on, the selected region. To this end, we studied the bryophyte, macrophyte, macroinvertebrate, and amphibian communities in two clusters of temporary wetlands on the NE Iberian Peninsula under mesothermal and semiarid climates. We observed effects of environmental filtering, with the communities differing between the climatic regions not only in their compositions but also in their dynamics and taxonomic relatedness patterns. Although the cross-taxon congruence in terms of species richness was high in the mesothermal climate, most of the congruent relationships were disrupted in the semiarid environment. Overall, because climate-dependent patterns appear to prevail over climate-consistent ones, we suggest that the use of surrogate taxa may be of limited value when aiming to assess wetland biodiversity across large areas.     

SAR study and conformational analysis of a series of novel peptide G protein‐coupled receptor kinase 2 inhibitors

G protein‐coupled receptor kinase 2 (GRK2) plays a central role in the cellular transduction network. In particular, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. Thereby, its inhibition offers a potential therapeutic solution to several pathological conditions. In the present study, we performed a SAR study and a NMR conformational analysis of peptides derived from HJ loop of GRK2 and able to selectively inhibit GRK2. From Ala‐scan and d ‐Ala point replacement, we found that Arg residues don't affect the inhibitory properties, while a d ‐amino acid at position 5 is key to the activity. Conformational analysis identified two β‐turns that involve N‐terminal residues, followed by a short extended region. These information can help the design of peptides and peptido‐mimetics with enhanced GRK2 inhibition properties. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 121–128, 2014.     

Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III

Dipeptidyl peptidases III (DPPs III) form a distinct metallopeptidase family characterized by the unique HEXXGH motif. High susceptibility to inactivation by organomercurials suggests the presence of a reactive cysteine residue(s) in, or close to, their active site. Yeast DPP III contains five Cys, none of which is absolutely conserved within the family. In order to identify reactive residue(s), site-directed mutagenesis on yeast His₆-tagged DPP III was employed to substitute specifically all five cysteine residues to serine. The variant enzymes thus obtained were enzymatically active and showed an overall structure not greatly affected by the mutations as judged by circular dichroism. Analysis by native and SDS-PAGE under non-reducing conditions revealed the existence of a monomeric and dimeric form in all DPP III proteins except in the C130S, implying that dimerization of yeast DPP III is mediated by the surface-exposed cysteine 130.     

Development of a repressible mycobacterial promoter system based on two transcriptional repressors

Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. While several regulated bacterial promoters have been characterized, few of them have been successfully used in mycobacteria. In this article we describe the development of a novel repressible promoter system effective in both fast- and slow-growing mycobacteria based on two chromosomally encoded repressors, dependent on tetracycline (TetR) and pristinamycin (Pip), respectively. This uniqueness results in high versatility and stringency. Using this method we were able to obtain an ftsZ conditional mutant in Mycobacterium smegmatis and a fadD32 conditional mutant in Mycobacterium tuberculosis, confirming their essentiality for bacterial growth in vitro. This repressible promoter system could also be exploited to regulate gene expression during M. tuberculosis intracellular growth.     

An overview of loggerhead sea turtle (<Emphasis Type="Italic">Caretta caretta</Emphasis>) bycatch and technical mitigation measures in the Mediterranean Sea

This paper reviews the gear parameters responsible for loggerhead sea turtle (Caretta caretta) capture and mortality while taking into account the mitigation measures tested in the Mediterranean Sea. Incidental catch is considered as one of the major threats for turtle survival; however, the loggerhead bycatch estimated in different areas seems to be unrealistic, which highlights the need of a method for homogenising the estimates. Drifting longlines and bottom trawls have the greatest impact on Mediterranean turtle populations, respectively in pelagic and demersal phase, while passive nets (gillnets and trammel nets) seem to be responsible for the highest direct mortality, due to drowning. Most of the experiments available for the Mediterranean are focused on drifting longline. The longline parameters, hook shape and size, bait type, setting position and the reaction to sensory stimuli, strongly affect the sea turtle bycatch and mortality. Circle hooks have the potential to reduce turtle mortality only in certain fisheries and areas; larger hooks are less likely to be swallowed by turtles due to physical constraints of the mouth, reducing the mortality rate and the catch of juveniles; branchlines, once ingested, appear to be one of the major causes of sea turtle mortality; squid bait, which consistently catches more turtles than mackerel, and lightsticks, which strongly attract turtles, should be banned, at least in some areas and seasons. On the contrary only two bottom trawl studies are available from the Mediterranean. Turtle excluder devices have been tested with promising results in Turkey and Italy, even if the loss of large fish should be carefully investigated. For set nets no practical solutions are available at this time. The analysis allows the conclusion that technical parameters affecting turtle bycatch and mortality should only be studied one at a time, in order to avoid inconclusive results, studies on post-release mortality should be implemented and finally fishermen cooperation is paramount in reducing turtle bycatch and mortality.     

Locomotor anatomy and biomechanics of the Dmanisi hominins

The Dmanisi hominins inhabited a northern temperate habitat in the southern Caucasus, approximately 1.8 million years ago. This is the oldest population of hominins known outside of Africa. Understanding the set of anatomical and behavioral traits that equipped this population to exploit their seasonal habitat successfully may shed light on the selection pressures shaping early members of the genus Homo and the ecological strategies that permitted the expansion of their range outside of the African subtropics. The abundant stone tools at the site, as well as taphonomic evidence for butchery, suggest that the Dmanisi hominins were active hunters or scavengers. In this study, we examine the locomotor mechanics of the Dmanisi hind limb to test the hypothesis that the inclusion of meat in the diet is associated with an increase in walking and running economy and endurance. Using comparative data from modern humans, chimpanzees, and gorillas, as well as other fossil hominins, we show that the Dmanisi hind limb was functionally similar to modern humans, with a longitudinal plantar arch, increased limb length, and human-like ankle morphology. Other aspects of the foot, specifically metatarsal morphology and tibial torsion, are less derived and similar to earlier hominins. These results are consistent with hypotheses linking hunting and scavenging to improved walking and running performance in early Homo. Primitive retentions in the Dmanisi foot suggest that locomotor evolution continued through the early Pleistocene.     

Rational design of novel mutants of fungal 17beta-hydroxysteroid dehydrogenase

Reduction of 17-ketosteroids is a biocatalytic process of economic significance for the production of steroid drugs. This reaction can be catalyzed by different microbial 17beta-hydroxysteroid dehydrogenases (17beta-HSD), like the 17beta-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3beta,17beta-HSD from Pseudomonas testosteroni. In addition to the bacterial 3beta,17beta-HSD the 17beta-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17beta-HSD that has been expressed as a recombinant protein and fully characterized. On the basis of its modeled 3D structure, we selected several positions for the replacement of amino acids by site-directed mutagenesis to change substrate specificity, alter coenzyme requirements, and improve overall catalytic activity. Replacement of Val161 and Tyr212 in the substrate-binding region by Gly and Ala, respectively, increased the initial rates for the conversion of androstenedione to testosterone. Replacement of Tyr49 within the coenzyme binding site by Asp changed the coenzyme specificity of the enzyme. This latter mutant can convert the steroids not only in the presence of NADP(+) and NADPH, but also in the presence of NADH and NAD(+). The replacement of His164, located in the non-flexible part of the 'lid' covering the active center resulted in a conformation of the enzyme that possessed a higher catalytic activity.     

Single chain antibody fragments for ocular use produced at high levels in a commercial wheat variety

We are investigating the use of single chain antibody fragments (scFv) in eye drops for diagnosis and treatment of eye diseases. For ocular use, recombinant proteins must be free of bacterial endotoxin that causes inflammation in the eye. We required a means of generating high yields of scFvs with little endotoxin contamination. Using microprojectile bombardment we produced transgenic lines of the commercial wheat variety, Westonia, that express two scFvs that bind to CD4 or CD28 on the surface of rat thymocytes. A high level of expression of active scFv in the range 50-180 microg/g was measured by quantitative flow cytometry in crude extracts made from mature seeds. The levels of expression were stable over four generations of transgenic plants and mature seeds were stored for one year with little loss of scFv activity. Substantial purification of scFv was achieved by immobilised metal affinity chromatography. Compared to bacterial extracts, crude transgenic seed extracts contained only a small amount of endotoxin (150 EU/ml) that will be easily removed by purification. The transgenic wheat lines express functional scFv at levels comparable to production in bacteria and promise to be superior to bacteria for production of scFv pharmaceuticals for ocular use.     

His164 regulates accessibility to the active site in fungal 17beta-hydroxysteroid dehydrogenase

17beta-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/ reductase superfamily. To study the catalytic properties of this enzyme, we prepared several specific mutations of 17beta-HSDcl (Tyr167Phe, His164Trp/Gly, Tyr212Ala). Wild-type 17beta-HSDcl and the 17beta-HSDcl mutants were evaluated by chromatographic, kinetic and thermodynamic means. The Tyr167Phe mutation resulted in a complete loss of enzyme activity, while substitution of His164 with Trp and Gly both resulted in higher specificity number (V/K) for the steroid substrates, which are mainly a consequence of easier accessibility of steroid substrates to the active-site hollow under optimized conditions. The Tyr212Ala mutant showed increased activity in the oxidative direction, which appears to be a consequence of increased NADPH dissociation. The kinetic characterizations and thermodynamic analyses also suggest that His164 and Tyr212 in 17beta-HSDcl have a role in the opening and closing of the active site of this enzyme and in the discrimination between oxidized and reduced coenzyme.     

Sensitivity of the invasive geophyte Oxalis pes-caprae to nutrient availability and competition

BACKGROUND AND AIMS: Invasion by alien plants may be partially related to disturbance-related increases in nutrient availability and decreases of competition with native species, and to superior competitive ability of the invader. Oxalis pes-caprae is an invasive winter geophyte in the Mediterranean Islands that reproduces vegetatively via bulbs. An investigation was made into the relative responses of O. pes-caprae and the native annual grass Lolium rigidum to nutrient availability and to competition with each other in order to understand patterns of invasion in the field. Because Oxalis accumulates oxalic acid in its leaves, which could ameliorate soil phosphorous availability, field observations were made to determine whether the presence of Oxalis alters soil P availability. METHODS: A full-factorial glasshouse experiment was conducted with nutrient availability (high and low) and competition (Lolium alone, Oxalis alone, and Lolium and Oxalis together). Plant performance was assessed by determining (1) above- and below-ground biomass at the time of Oxalis maximum biomass and (2) reproductive output of Oxalis and Lolium at the end of their respective growth cycles. Measurements were also taken for leaf N and P content. Soil samples were taken in the field from paired Oxalis-invaded and non-invaded plots located in Menorca (Balearic Islands) and available P was determined. KEY RESULTS: High nutrient availability increased Oxalis and Lolium vegetative biomass and reproductive output to a similar degree. Competition with Lolium had a much stronger negative effect on Oxalis bulb production than reduced nutrients. Lolium was a superior competitor than Oxalis; the latter did not affect Lolium maximum biomass and spike production. Significantly greater soil-P availability in Oxalis-invaded field soils relative to paired non-invaded soils suggest that Oxalis influences soil P cycling. CONCLUSIONS: Oxalis is a poor competitor. This is consistent with the preferential distribution of Oxalis in disturbed areas such as ruderal habitats, and might explain its low influence on the cover of native species in invaded sites. The results also suggest that certain disturbances (e.g. autumn ploughing) may greatly enhance Oxalis invasion.