Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by an stain for primary and secondary amino group (e.g. ninydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in higly yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.     

Phytoplankton of rhithral rivers: Its origin,diversity and possible use for quality-assessment

While phytoplankton studies on large potamal rivers have increased in number in recent years, upper river sections have received considerably less attention. However, in order to better understand processes that govern the development of dominance of euplanktonic elements in the lower river sections, detailed studies of the upstream areas are necessary. We studied the composition, diversity and recruitment of the planktonic algal communities in the River Sajó and in its main tributary River Hernád, two characteristic rhithral river systems in Central Europe. Results revealed that diatoms are dominant elements of the phytoplankton in the upper river segments both in terms of taxa richness, and relative abundance. We found that composition of the phytoplankton showed the closest resemblance to that of benthic communities of soft substrates, highlighting the key role of riverbed characteristics in river phytoplankton recruitment. The occurrence of euplanktonic elements was not restricted to human impacted river segments, these taxa also occurred in pristine headwaters. Accordingly, planktonic algae potentially colonise small headwater streams naturally, although their dominance is expected to occur only at downstream reaches following impounded river segments. Diatom metrics used in ecological state assessment were calculated for the rithroplankton. These metrics fell in the range of values calculated for diatom flora on the hard substrates. However, the potential use of rithroplankton for quality assessment might be limited, because the high variability of index values at site level would result in misclassification of the ecological status.     

A twofold role for global energy gradients in marine biodiversity trends

Explanations for major biodiversity patterns have not achieved a consensus, even for the latitudinal diversity gradient (LDG), but most relate to patterns of solar energy influx into Earth systems, and its effects on temperature (as biochemical activity rates are temperature sensitive) and photosynthesis (which drives nearly all of the productivity that fuels ecosystems). Marine systems break some of the confounding correlations among temperature, latitude and biodiversity that typify the terrestrial systems that have dominated theoretical discussions and large‐scale analyses. High marine diversities occur not only in warm shallow seas where productivity may be either low or high, depending on regional features, but also in very cold deep‐sea regions, indicating that diversity is promoted by stability in temperature and in trophic resources (nutrients and food items), and more specifically by their interaction, rather than by high mean values of either variable. The common association of high diversity with stable but low to moderate annual productivity suggests that ecological specialization underlies the similarly high diversities in the shallow tropics and deep sea. Recent work on shallow‐marine bivalves is consistent with this view of decreasing specialization in less stable habitats. Lower diversities in shallow seas are associated with either high thermal seasonality (chiefly in temperate latitudes) or highly seasonal trophic supplies (at any latitude), which exclude species that are adapted to narrow ranges of those variables.     

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.     

Habitat ecological integrity and environmental impact assessment of anthropic activities: A GIS-based fuzzy logic model for sites of high biodiversity conservation interest

In literature, an effective method enabling the classification, based on a single indicator, of habitats that need a priority protection intervention has not been identified yet. Moreover, the excessive number of landscape metrics, used to quantify integrity of habitats, can cause confusion, often providing redundant and inconsistent results.The aim of this work is to develop a method for evaluating the ecological vulnerability of the habitats in sites of high biodiversity conservation interest. In the first phase, we selected and analyzed, by using principal component analysis (PCA) and fuzzy logic, the landscape metrics, in order to obtain the map of the intrinsic ecological vulnerability index. In the second step, the result of this intrinsic vulnerability was connected, through another fuzzy model, to anthropogenic impacts, obtaining the integrated ecological vulnerability index. We developed specific spatial indicators (landscape metrics), which can examine the mutual position and morphology of the habitats present, along with indicators of human pressure, related to the type and intensity of use of the anthropic territory, with reference to the habitat itself as well as to the areas immediately adjacent. The developed fuzzy models are innovative, compared to the current ecological studies, and examine landscape metrics as well as the impact of human activities.The case study is the “Val Basento-Ferrandina Scalo” Site of Community Importance, Ferrandina-SCI (Basilicata Region, Southern Italy). The results allowed us to build a rank of the habitats based on their intrinsic and integrated ecological vulnerability. Moreover, the results show that, in the Ferrandina-SCI, the most important source of concern is not human activities, but rather the inherent risk of ecological fragility caused by geographical and landscape features of the different patches of habitats themselves.This model aims to be a tool for decision support in sustainable landscape management. It is easy to use and to apply on other regions, although it should always be accompanied by a sensitivity analysis to reduce the subjectivity.     

Regional differences in mitotic activity due to injury in mouse skin

Summary Mitotic activity adjacent to a wound inflicted at different sites in the mouse skin was measured 24 h after injury. A regional difference in the epidermal mitotic activity due to injury was noted. Mitotic activity was high in the anterior parts of the body including the head, lower in the middle to posterior regions of the body and lowest in the posterior-most parts of the body. Regional differences in epidermal mitotic activity due to injury were demonstrated in both female and male mice. The existence of a cranio-caudal gradient in epidermal response to injury is suggested.     

A study of cell electrophoresis as a means of purifying growth hormone secreting cells

Growth hormone secreting cells of the rat anterior pituitary are heavily laden with granules of growth hormone and can be partially purified on the basis of their resulting high density. Two methods of preparative cell electrophoresis were investigated as methods of enhancing the purification of growth hormone producing cells: density gradient electrophoresis and continuous flows electrophoresis. Both methods provided a two- to four-fold enrichment in growth hormone production per cell relative to that achieved by previous methods. Measurements of electrophoretic mobilities by two analytical methods, microscopic electrophoresis and laser-tracking electrophoresis, revealed very little distinction between unpurified anterior pituitary cell suspensions and somatotroph-enriched cell suspensions. Predictions calculated on the basis of analytical electrophoretic data are consistent with the hypothesis that sedimentation plays a significant role in both types of preparative electrophoresis and the electrophoretic mobility of the growth hormone secreting subpopulation of cells remains unknown.     

Testing prediction accuracy in short-term ecological studies

Applied ecology is based on an assumption that a management action will result in a predicted outcome. Testing the prediction accuracy of ecological models is the most powerful way of evaluating the knowledge implicit in this cause-effect relationship, however, the prevalence of predictive modeling and prediction testing are spreading slowly in ecology. The challenge of prediction testing is particularly acute for small-scale studies, because withholding data for prediction testing (e.g., via k-fold cross validation) can reduce model precision. However, by necessity small-scale studies are common. We use one such study that explored small mammal abundance along an elevational gradient to test prediction accuracy of models with varying degrees of information content. For each of three small mammal species, we conducted 5000 iterations of the following process: (1) randomly selected 75 % of the data to develop generalized linear models of species abundance that used detailed site measurements as covariates, (2) used an information theoretic approach to compare the top model with detailed covariates to habitat type-only and null models constructed with the same data, (3) tested those models’ ability to predict the 25 % of the randomly withheld data, and (4) evaluated prediction accuracy with a quadratic loss function. Detailed models fit the model-evaluation data best but had greater expected prediction error when predicting out-of-sample data relative to the habitat type models. Relationships between species and detailed site variables may be evident only within the framework of explicitly hierarchical analyses. We show that even with a small but relatively typical dataset (n = 28 sampling locations across 125 km over two years), researchers can effectively compare models with different information content and measure models’ predictive power, thus evaluating their own ecological understanding and defining the limits of their inferences. Identifying the appropriate scope of inference through prediction testing is ecologically valuable and is attainable even with small datasets.