Cellulase gene expression in ripening avocado fruit: The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

Summary A cDNA library was constructed from poly(A)⁺RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)⁺-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.Abbreviations poly(A)⁺ polyadenylated - DS sodium dodecyl sulfate - D kilodalton - bp base pairs Supported by Research Grant GM 19807 from the United States Public Health Service and by additional funds from the University of California Research Council.     

Effects of warming on annual production and nutrient‐use efficiency of aquatic mosses in a high Arctic lake

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The Apolipoprotein M/S1P Axis Controls Triglyceride Metabolism and Brown Fat Activity

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In Vitro Comparison of the Effects of Probiotic,Commensal and Pathogenic Strains on Macrophage Polarization

Macrophages are important with respect to both innate and adaptive immune responses and are known to differentiate into pro-inflammatory M1- or anti-inflammatory M2-phenotypes following activation. In order to study how different bacteria affect macrophage polarization, we exposed murine RAW 264.7 macrophages to sixteen different strains representing probiotic strains, pathogens, commensals and strains of food origin. Increased inducible nitric oxide synthase (iNOS) or arginase-1 gene expression indicates M1 or M2 polarization, respectively, and was quantified by qRT-PCR. Strains of Escherichia and Salmonella elevated iNOS expression more so than strains of Enterococcus, Lactobacillus and Lactococcus, indicating that Gram-negative strains are more potent M1 inducers. However, strain-specific responses were observed. For instance, Escherichia coli Nissle 1917 was a poor inducer of iNOS gene expression compared to the other E. coli strains, while Enterococcus faecalis Symbioflor-1 was more potent in this respect compared to all the eleven Gram-positive strains tested. Macrophage polarization was further characterized by quantifying secreted pro- and anti-inflammatory cytokines. Exposure to the pathogen E. coli 042 produced a cytokine profile indicating M1 differentiation, which is in accordance with the PCR data. However, exposure to most strains resulted in either high or low secretion levels of all cytokines tested, rather than a clear M1 or M2 profile. In general, the Gram-negative strains induced high levels of cytokine secretion compared to the Gram-positive strains. Interestingly, strains of human origin had a higher impact on macrophages compared to strains of food origin.     

Implications of Climate-enforced Temperature Increases on Freshwater Pico- and Nanoplankton Populations Studied in Artificial Ponds During 16 Months

Global warming scenarios foresee increases in air temperatures of 3–5 °C in Northern European regions within the next 70 years. To evaluate the potential effects of global warming on shallow eutrophic lakes, a flow-through experiment combining three temperature scenarios and two nutrient levels was conducted in 24 outdoor mesocosms. Eight mesocosms were unheated and acted as controls, while sixteen were heated – eight according to the Intergovernmental Panel on Climate Change’s (IPCC) climate scenario A2 down-scaled to regional level (2.5–4.4 °C, depending on season) and eight according to scenario A2+ with an additional 50% temperature increase. Half of the mesocosms were enriched with nitrogen and phosphorus to simulate increased runoff from terrestrial sources due to the increased precipitation predicted by the A2 scenario. The other half were un-enriched and received only natural nutrient input from the groundwater that fed all the mesocosms. The abundance and development pattern of the microbial communities within the mesocosms were tracked during a 16-month period. Generally, the results showed that the abundances of picoalgae, bacteria and heterotrophic nanoflagellates changed in a similar manner over time; abundances being lower in winter than in summer. Warming in itself had no effect on abundance, albeit it significantly modified the positive effect of the nutrients. Only at ambient temperatures did the whole microbial assemblage respond positively to nutrients. In the A2 scenario, only picoalgae responded to nutrients, while in the A2+ scenario all but the heterotrophic nanoflagellates showed a response. Elevated winter temperatures seemed not to be more important for the microbial assemblage than elevated summer temperatures. Our results demonstrate that the direct effects of warming were far less important than the nutrient effect. The results furthermore reveal that warming and nutrients in combination set off complex interactions. In consequence, global warming may possibly have pronounced effects on aquatic ecosystems if accompanied by increased nutrient loading.     

Effects of cell-bound microcystins on survival and feeding of Daphnia spp

The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones and Daphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to all Daphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among Daphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.     

Pelagic food web processes in an oligotrophic lake

Major pelagic carbon pathways, including primary production, release of extracellular products (EOC), bacterial production and zooplankton grazing were measured in oligotrophic Lake Almind (Denmark) and in enclosures (7 m³) subjected to artificial eutrophication. Simultaneous measurements at three days interval of carbon exchange rates and pools allowed the construction of carbon flow scenarios over a nineteen day experimental period.The flow of organic carbon was dominated by phytoplankton EOC release, which amounted from 44 to 58% of the net fixation of inorganic carbon. Gross bacterial production accounted for 33 to 75% of the primary production. The lower values of EOC release (44%) and bacterial production (33%) were found in the enclosures with added nutrients. The release of recently fixed photosynthetic products was the most important source of organic carbon to the bacterioplankton. Uptake of dissolved free amino acids was responsible for 52 to 62% of the gross bacterial production. Thus, amino acids constituted a significant proportion of the EOC. Zooplankton (< 50 µm) grazing on algae and bacteria accounted only for a minor proportion of the particulate production in May. Circumstantial evidence is presented that suggests the chrysophycean alga Dinobryon was the most important bacterial remover.The results clearly demonstrated EOC release and bacterial metabolism to be key processes in pelagic carbon cycling in this oligotrophic lake.     

Structural and functional insight into how the Plasmodium falciparum VAR2CSA protein mediates binding to chondroitin sulfate A in placental malaria

Malaria is a major global health problem. Pregnant women are susceptible to infection regardless of previously acquired immunity. Placental malaria is caused by parasites capable of sequestering in the placenta. This is mediated by VAR2CSA, a parasite antigen that interacts with chondroitin sulfate A (CSA). One vaccine strategy is to block this interaction with VAR2CSA-specific antibodies. It is a priority to define a small VAR2CSA fragment that can be used in an adhesion blocking vaccine. In this, the obvious approach is to define regions of VAR2CSA involved in receptor binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with nanomolar affinity, and that the CSA-binding site lies in the N-terminal part of the protein. In this study we define the minimal binding region by truncating VAR2CSA and analyzing CSA binding using biosensor technology. We show that the core CSA-binding site lies within the DBL2X domain and parts of the flanking interdomain regions. This is in contrast to the idea that single domains do not possess the structural requirements for specific CSA binding. Small-angle x-ray scattering measurements enabled modeling of VAR2CSA and showed that the CSA-binding DBL2X domain is situated in the center of the structure. Mutating classic sulfate-binding sites in VAR2CSA, along with testing dependence of ionic interactions, suggest that the CSA binding is not solely dependent on the sulfated CSA structure. Based on these novel PfEMP1 structure-function studies, we have constructed a small VAR2CSA antigen that has the capacity to induce highly adhesion-blocking antibodies.     

Revision of the Classical Dopamine D2 Agonist Pharmacophore Based on an Integrated Medicinal Chemistry,Homology Modelling and Computational Docking Approach

The scientific advances during the 1970ies and 1980ies within the field of dopaminergic neurotransmission enabled the development of a pharmacophore that became the template for design and synthesis of dopamine D₂ agonists during the following four decades. A major drawback, however, is that this model fails to accommodate certain classes of restrained dopamine D₂ agonists including ergoline structures. To accommodate these, a revision of the original model was required. The present study has addressed this by an extension of the original model without compromising its obvious qualities. The revised pharmacophore contains an additional hydrogen bond donor feature, which is required for it to accommodate ergoline structures in a low energy conformation and in accordance with the steric restrictions dictated by the original model. The additional pharmacophore feature suggests ambiguity in the binding mode for certain compounds, including a series of ergoline analogues, which was reported recently. The ambiguity was confirmed by docking to a homology model of the D₂ receptor as well as by pharmacological characterization of individual enantiomers of one of the analogues. The present research also addresses the potential of designing ligands that interact with the receptor in a large, distal cavity of the dopamine D₂ receptor that has not previously been studied systematically. The pharmacological data indicate that this area may be a major determinant for both the dopamine D₂ affinity and efficacy, which remains to be explored in future studies.     

Temporal trends and variability in a high-arctic ecosystem in Greenland: multidimensional analyses of limnic and terrestrial ecosystems

The high arctic is undergoing a faster change in climate than most other regions of the planet, with already observed ecological consequences. Combined with the characteristics of high-arctic ecosystems, such as low species redundancy, high seasonality and weather extremes, shifts in individual species performance and phenology may lead to altered interaction dynamics through trophic mismatch and cascades. An ecosystem approach is therefore desirable in the attempt to understand the multidimensional impacts of climate. Here, we present ecosystem-wide trend analyses of a long-term dataset on terrestrial and limnic biota with focus on the distribution of observed trends and associated variation across the ecosystem. We used 114 time series drawn from 11 abiotic variables, 19 terrestrial and 7 limnic biotic species/taxa and compared temporal trends, changes and abrupt shifts in the variation within and across the two biota. A total of 36 % of the time series analysed showed a significant trend during the study period with a higher frequency of trends occurring within performance variables. Overall, the changes tended to be negative, indicating advances in phenology but reduced species performance. General system variance was also higher in the limnic biota than in the terrestrial biota, both exhibiting increasing variance up through the trophic system. Overall, our results suggest that multiple biotic responses to the climatic changes in this high-arctic ecosystem are not synchronised across trophic levels and may differ qualitatively and quantitatively between terrestrial and limnic biota.