Site-directed mutagenesis of mouse thymidylate synthase: alteration of Arg44 to Val44 in a conserved loop guarding the active site has striking effects on catalysis and nucleotide binding

The arginine located at position 44 of mouse thymidylate synthase is in a highly conserved loop that is in close proximity to the active site cleft of the enzyme. Structural analyses have suggested that this arginine forms hydrogen bonds with the alpha-carboxylate of the C-terminal amino acid and the phosphate of the substrate analog, FdUMP (D. A. Matthews, K. Appelt and S. J. Oatley, (1989) Adv. Enz. Reg., 29, 47-60). We have used protein engineering techniques to change this amino acid residue to valine. This alteration leads to large reductions in the ability of the enzyme to form covalent complexes with substrate (dUMP) or inhibitor (FdUMP) and at least a 100-fold reduction in catalytic activity. These observations show that this arginine plays an important role in maintaining catalytic activity of the enzyme.     

A cell number-counting factor regulates levels of a novel protein, SslA, as part of a group size regulation mechanism in Dictyostelium

Developing Dictyostelium cells form aggregation streams that break into groups of approximately 2 x 10(4) cells. The breakup and subsequent group size are regulated by a secreted multisubunit counting factor (CF). To elucidate how CF regulates group size, we isolated second-site suppressors of smlA(-), a transformant that forms small groups due to oversecretion of CF. smlA(-) sslA1(CR11) cells form roughly wild-type-size groups due to an insertion in the beginning of the coding region of sslA1, one of two highly similar genes encoding a novel protein. The insertion increases levels of SslA. In wild-type cells, the sslA1(CR11) mutation forms abnormally large groups. Reducing SslA levels by antisense causes the formation of smaller groups. The sslA(CR11) mutation does not affect the extracellular accumulation of CF activity or the CF components countin and CF50, suggesting that SslA does not regulate CF secretion. However, CF represses levels of SslA. Wild-type cells starved in the presence of smlA(-) cells, recombinant countin, or recombinant CF50 form smaller groups, whereas sslA1(CR11) cells appear to be insensitive to the presence of smlA(-) cells, countin, or CF50, suggesting that the sslA1(CR11) insertion affects CF signal transduction. We previously found that CF reduces intracellular glucose levels. sslA(CR11) does not significantly affect glucose levels, while glucose increases SslA levels. Together, the data suggest that SslA is a novel protein involved in part of a signal transduction pathway regulating group size.     

Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis

Human neurocysticercosis (NC) is caused by the establishment of Taenia solium larvae in the central nervous system. NC is a severe disease still affecting the population in developing countries of Latin America, Asia, and Africa. While great improvements have been made on NC diagnosis, treatment, and prevention, the management of patients affected by extraparenchymal parasites remains a challenge. The development of a T. solium NC experimental model in pigs that will allow the evaluation of new therapeutic alternatives is herein presented. Activated oncospheres (either 500 or 1000) were surgically implanted in the cerebral subarachnoid space of piglets. The clinical status and the level of serum antibodies in the animals were evaluated for a 4-month period after implantation. The animals were sacrificed, cysticerci were counted during necropsy, and both the macroscopic and microscopic characteristics of cysts were described. Based on the number of established cysticerci, infection efficiency ranged from 3.6% (1000 oncospheres) to 5.4% (500 oncospheres). Most parasites were caseous or calcified (38/63, 60.3%) and were surrounded by an exacerbated inflammatory response with lymphocyte infiltration and increased inflammatory markers. The infection elicited specific antibodies but no neurological signs. This novel experimental model of NC provides a useful tool to evaluate new cysticidal and anti-inflammatory approaches and it should improve the management of severe NC patients, refractory to the current treatments.     

Vertebrate metacommunity structure along an extensive elevational gradient in the tropics: a comparison of bats,rodents and birds

Aim We evaluated the structure of metacommunities for each of three vertebrate orders (Chiroptera, Rodentia and Passeriformes) along an extensive elevational gradient. Using elevation as a proxy for variation in abiotic characteristics and the known elevational distributions of habitat types, we assessed the extent to which variation in those factors may structure each metacommunity based on taxon‐specific characteristics. Location Manu Biosphere Reserve in the Peruvian Andes. Methods Metacommunity structure is an emergent property of a set of species distributions across geographic or environmental gradients. We analysed elements of metacommunity structure (coherence, range turnover and range boundary clumping) to determine the best‐fit structure for each metacommunity along an elevational gradient comprising 13 250‐m elevational intervals and 58 species of rodent, 92 species of bat or 586 species of passerine. Results For each taxon, the environmental gradient along which the metacommunity was structured was highly correlated with elevation. Clementsian structure (i.e. groups of species replacing other such groups along the gradient) characterized rodents, with a group of species that was characteristic of rain forests and a group of species that was characteristic of higher elevation habitats (i.e. above 1500 m). Distributions of bats were strongly nested, with more montane communities comprising subsets of species at lower elevations. The structure of the passerine metacommunity was complex and most consistent with a quasi‐Clementsian structure. Main conclusions Each metacommunity exhibited a different structure along the same elevational gradient, and each structure can be accounted for by taxon‐specific responses to local environmental factors that vary predictably with elevation. The structures of rodent and bird metacommunities suggest species sorting associated with habitat specializations, whereas structure of the bat metacommunity is probably moulded by a combination of species‐specific tolerances to increasingly cold, low‐productivity environs of higher elevations and the diversity and abundance of food resources associated with particular habitat types.     

Do soil enzymes respond to long-term grazing in an arid ecosystem?

Background and aims

Our objective was to assess the effects of long-term continuous grazing on soil enzyme activities in relation to shifts in plant litter attributes and soil resources in an arid ecosystem, considering both spatial and temporal variations.

Methods

We randomly extracted soil samples with the respective litter cover at 5 modal size plant-covered patches (PCP) and the nearest inter-canopy areas (IC) at Patagonian Monte sites with low, medium and high grazing intensity in winter and summer from 2007 to 2009. We analyzed enzyme activities (dehydrogenase, ß-glucosidase, protease, alkaline and acid phosphatase), microbial biomass-C, organic-C, total soil-N, and moisture in soil and mass and quality in plant litter. We assessed faeces density and plant cover in the field.

Results and conclusions

Grazing led to reduced grass cover, decreasing plant litter mass with increasing soluble phenolics, and reduced phosphatases, ß-glucosidase and microbial biomass-C at PCP. A localized nutrient input from animal excreta seems to promote microbial biomass-C, alkaline phosphatase and dehydrogenase activities but only at IC from the site with high grazing intensity. Plant heterogeneous distribution, plant litter quantity and quality, nutrient inputs from grazers and seasonal variation in soil moisture, also affecting soil resources and microbial biomass, modulate soil enzyme responses to long-term grazing in the arid Patagonian Monte.     

Spatial distribution, connectivity, and the influence of scale: habitat availability for the endangered Mona Island rock iguana

The Caribbean region is one of the five leading biodiversity hotspots in the world. Analysis of the spatial structure of critical habitats and how it affects endemic species in this region is essential baseline information for biodiversity monitoring and management. We quantified and evaluated the spatial structure and connectivity of depression forests on Mona Island and their potential impact on Mona Island rock iguana habitat, as a framework to assess spatial distribution, connectivity, and the issue of scale in small and widely dispersed habitats. Using IKONOS imagery, we mapped and delineated depression forests at four different scales (minimum mapping units: <100, 100, 500, and 1,000 m), and calculated landscape metrics describing their spatial structure, and connectivity, for each map resolution. Our approach resulted in a more detailed map than previously described maps, providing better information on habitat connectivity for iguanas. The comparison of the island landscape mapped at different scales provided evidence on how changing scales affect the output of spatial metrics and may have a significant impact when planning decisions and assigning conservation priorities. It also highlighted the importance of adequate ecological scales when addressing landscape management and conservation priorities. The analysis of landscapes at multiple scales provided a mechanism to evaluate the role of patch detection and its effect on the interpretation of connectivity and spatial structure of suitable areas for species with small and widely dispersed habitats. These methodologies can be applied other species, in different environments, with similar limitations related to connectivity and habitat availability.     

Planning adaptation to climate change in fast-warming marine regions with seafood-dependent coastal communities

Many coastal communities rely on living marine resources for livelihoods and food security. These resources are commonly under stress from overfishing, pollution, coastal development and habitat degradation. Climate change is an additional stressor beginning to impact coastal systems and communities, but may also lead to opportunities for some species and the people they sustain. We describe the research approach for a multi-country project, focused on the southern hemisphere, designed to contribute to improving fishing community adaptation efforts by characterizing, assessing and predicting the future of coastal-marine food resources, and co-developing adaptation options through the provision and sharing of knowledge across fast-warming marine regions (i.e. marine ‘hotspots’). These hotspots represent natural laboratories for observing change and concomitant human adaptive responses, and for developing adaptation options and management strategies. Focusing on adaptation options and strategies for enhancing coastal resilience at the local level will contribute to capacity building and local empowerment in order to minimise negative outcomes and take advantage of opportunities arising from climate change. However, developing comparative approaches across regions that differ in political institutions, socio-economic community demographics, resource dependency and research capacity is challenging. Here, we describe physical, biological, social and governance tools to allow hotspot comparisons, and several methods to evaluate and enhance interactions within a multi-nation research team. Strong partnerships within and between the focal regions are critical to scientific and political support for development of effective approaches to reduce future vulnerability. Comparing these hotspot regions will enhance local adaptation responses and generate outcomes applicable to other regions.     

Effects of chronic immobilization stress on biokinetics and dosimetry of 67 Ga in a murine model

The aim of this work is to determine the effect of chronic immobilization stress on kinetics and dosimetry of 67Ga in a mouse model. A control group (CG) and a stress group (SG), each with 15 mice, were included in the study, and the latter group was subjected to a chronic immobilization stress model 2 h daily for 14 consecutive days. At day 13, 67Ga-citrate was administered intraperitoneally (11.24 ± 0.44 MBq) to each mouse. Then, sets of three mice were obtained sequentially at 24, 36, 48, 60 and 72 h, in which the radionuclide activity was measured with an activity counter. The 67Ga biokinetic data showed a fast blood clearance in the SG, with a mean residence time of 0.06 h. The calculated mean radiation absorbed doses were: liver (2.45 × 10−03 Gy), heart (3.17 × 10−04 Gy) and kidney (1.88 × 10−04 Gy) in the SG. The results show that stress reduced weight gain by approximately 13% and also increased adrenal gland weight by 26%. On the other hand, chronic stress accelerates 67Ga clearance after 24 h compared to normal conditions. It is concluded that murine organisms under chronic immobilization stress have higher gallium-67 clearance rates, decreasing the cumulated activity and absorbed dose in all organs.