Biochemical and genetic evidence for the presence of multiple phosphatidylinositol- and phosphatidylinositol 4,5-bisphosphate-specific phospholipases C in Tetrahymena

Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], produce the Ca(2+)-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca(2+). One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca(2+), modestly activated by GTPγS in vitro, and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P(2) levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P(3)-Ca(2+) regulatory axis in ciliates.     

Nanoscopic cationic methacrylate star homopolymers: synthesis by group transfer polymerization, characterization and evaluation as transfection reagents

Seven star polymers with degrees of polymerization (DPs) of the arms from 10 to 100 and dimensions in the nanometer range were prepared using sequential group transfer polymerization of 2-(dimethylamino)ethyl methacrylate (DMAEMA, hydrophilic positively ionizable monomer) and ethylene glycol dimethacrylate (hydrophobic neutral cross-linker). The polymers were characterized in tetrahydrofuran by gel permeation chromatography and static light scattering to determine the molecular weights and the weight-average number of arms for each sample. The number of arms of the star polymers varied from 20 to 72. Aqueous solutions of the star polymers were studied by turbidimetry, hydrogen ion titration, and dynamic light scattering to determine their cloud points, pKs, and hydrodynamic diameters. The cloud points of the larger star polymers, with arm DP 30-100, were found to be 29-34 degrees C, almost independent of the DP of the arms. Similarly, the pKs of all star polymers were calculated to range between 6.7 and 7.0, again independent of the arm DP. In contrast, the hydrodynamic diameters of the star polymers strongly depended on the DP of the arms. In particular, by increasing the DP of the arms from 20 to 100, the hydrodynamic diameters in water increased from 7 to 31 nm. All star polymers were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 with the enhanced green fluorescent protein as the reporter gene. Our results showed that as the DP of the arms of the DMAEMA star homopolymers increased from 10 to 100, the overall transfection efficiency decreased, with the star polymer with DP of the arms of 10 emerging as the best transfection reagent. Systematic variation of the amounts of star polymer and plasmid DNA used in the transfections led to an optimization of the performance of this star polymer, yielding overall transfection efficiencies of 15%, comparable to the optimum overall transfection efficiency of the commercially available transfection reagent SuperFect of 13%.     

Identifying the “demon whale-biter”: Patterns of scarring on large whales attributed to a cookie-cutter shark Isistius sp

The presence of crater-like wounds on cetaceans and other large marine vertebrates and invertebrates has been attributed to various organisms. We review the evidence for the identity of the biting agent responsible for crater wounds on large whales, using data collected from sei (Balaenoptera borealis), fin (B. physalus), inshore and offshore Bryde’s (B. brydeii sp) and sperm whales (Physeter macrocephalus) examined at the Donkergat whaling station, Saldanha Bay, South Africa between March and October 1963. We then analyse the intensity and trends in its predation on large whales. Despite the scarcity of local records, we conclude that a cookie-cutter shark Isistius sp is the most likely candidate. We make inferences about the trends in (1) total counts of unhealed bitemarks, and (2) the proportion of unhealed bitemarks that were recent. We use day of the year; reproductive class, social grouping or sex; depth interval and body length as candidate covariates. The models with highest support for total counts of unhealed bitemarks involve the day of the year in all species. Depth was an important predictor in all species except offshore Bryde’s whales. Models for the proportion of recent bites were only informative for sei and fin whales. We conclude that temporal scarring patterns support what is currently hypothesized about the distribution and movements of these whale species, given that Isistius does not occur in the Antarctic and has an oceanic habitat. The incidence of fresh bites confirms the presence of Isistius in the region. The lower numbers of unhealed bites on medium-sized sperm whales suggests that this group spends more time outside the area in which bites are incurred, providing a clue to one of the biggest gaps in our understanding of the movements of mature and maturing sperm males.     

Spatial variation in maximum dive depth in gray seals in relation to foraging

Habitat preference maps are a way of representing animals' space use in two dimensions. For marine animals, the third dimension is an important aspect of spatial ecology. We used dive data from seven gray seals Halichoerus grypus (a primarily benthic forager) collected with GPS phone tags (Sea Mammal Research Unit) to investigate the distribution of the maximum depth visited in each dive. We modeled maximum dive depth as a function of spatiotemporal covariates using a generalized additive mixed model (GAMM) with individual as a random effect. Bathymetry, horizontal displacement, latitude and longitude, Julian day, sediment type, and light conditions accounted for 37% of the variability in the data. Persistent patterns of autocorrelation in the raw data suggest that individual intrinsic rhythm might be an important factor, not captured by external covariates. The strength of using this statistical method to generate spatial predictions of the distribution of maximum dive depth is its applicability to other plunge and pursuit divers. Despite being predictions of a point estimate, these maps provide some insight into the third dimension of habitat use in marine animals. The capacity to predict this aspect of vertical habitat use may help avoid conflict between animal habitat and coastal or offshore developments.     

IGF-I affects glycosaminoglycan/proteoglycan synthesis in breast cancer cells through tyrosine kinase-dependent and -independent pathways

The insulin-like growth factor I (IGF-I) has been implicated in breast cancer development acting through insulin-like growth factor I receptor (IGF-IR), but also through estrogen receptor (ER). The effect of IGF on proteoglycan (PG) synthesis by two human breast cancer epithelial cell lines, the ER-positive MCF-7 and the ER-negative BT-20, was studied alone and in combination with genistein. Both cell lines synthesise hyaluronan (HA), matrix secreted and cell membrane-associated galactosaminoglycan containing proteoglycans (GalAGPGs) and heparan sulphate proteoglycans (HSPGs) in variable amounts. IGF-I affects the synthesis of PGs by BT-20 cells by decreasing the amounts of HA and secreted GalAGPGs and HSPGs and upregulates the expression of cell membrane-associated GalAGPGs and HSPGs. IGF-I exerts this effect on BT-20 cells acting mainly through receptors with protein tyrosine kinase activity (PTK). In contrast, IGF-I stimulates the synthesis of secreted GalAGPGs and HSPGs by MCF-7 cells, exhibiting only a slight suppression on synthesis of cell-associated GalAGPGs and HSPGs. The regulatory effect of IGF-I on PGs distribution in MCF-7 cells is mediated through a mix of pathways, which involves both receptors with PTK activity and PTK-independent signalling. It is suggested that the effects of IGF-I on the synthesis and distribution of PGs by epithelial breast cancer cells also depend on the presence or the absence of ER. The result of the IGF-I action is the balanced biosynthesis between the matrix and cell-associated PGs in both cell lines, approaching a common biosynthetic phenotype.     

Biotechnology Towards Energy Crops

New crops are gradually establishing along with cultivation systems to reduce reliance on depleting fossil fuel reserves and sustain better adaptation to climate change. These biological assets could be efficiently exploited as bioenergy feedstocks. Bioenergy crops are versatile renewable sources with the potential to alternatively contribute on a daily basis towards the coverage of modern society’s energy demands. Biotechnology may facilitate the breeding of elite energy crop genotypes, better suited for bio-processing and subsequent use that will improve efficiency, further reduce costs, and enhance the environmental benefits of biofuels. Innovative molecular techniques may improve a broad range of important features including biomass yield, product quality and resistance to biotic factors like pests or microbial diseases or environmental cues such as drought, salinity, freezing injury or heat shock. The current review intends to assess the capacity of biotechnological applications to develop a beneficial bioenergy pipeline extending from feedstock development to sustainable biofuel production and provide examples of the current state of the art on future energy crops.