Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers a potential drug delivery system

Synthetic ¹²⁵I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [¹²⁵I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.     

In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells

Isolation and separation of rat liver cells into endothelial, Kupffer, and parenchymal cell fractions were performed at different times after injection of human 125I-acetyl low density lipoproteins (LDL). In order to minimize degradation and redistribution of the injected lipoprotein during cell isolation, a low temperature (8 degrees C) procedure was applied. Ten min after injection, isolated endothelial cells contained 5 times more acetyl-LDL apoprotein per mg of cell protein than the Kupffer cells and 31 times more than the hepatocytes. A similar relative importance of the different cell types in the uptake of acetyl-LDL was observed 30 min after injection. For studies on the in vitro interaction of endothelial and Kupffer cells with acetyl-LDL, the cells were isolated with a collagenase perfusion at 37 degrees C. Pure endothelial (greater than 95%) and purified Kupffer cells (greater than 70%) were obtained by a two-step elutriation method. It is demonstrated that the rat liver endothelial cell possesses a high affinity receptor specific for the acetyl-LDL because a 35-fold excess of unlabeled acetyl-LDL inhibits association of the labeled compound for 70%, whereas unlabeled native human LDL is ineffective. Binding to the acetyl-LDL receptor is coupled to rapid uptake and degradation of the apolipoprotein. Addition of the lysosomotropic agents chloroquine (50 microM) or NH4Cl (10 mM) resulted in more than 90% inhibition of the high affinity degradation, indicating that this occurs in the lysosomes. With the purified Kupffer cell fraction, the cell association and degradation of acetyl-LDL was at least 4 times less per mg of cell protein than with the pure endothelial cells. Although cells isolated with the cold pronase technique are also still able to bind and degrade acetyl-LDL, it appeared that 40-60% of the receptors are destroyed or inactivated during the isolation procedure. It is concluded that the rat liver endothelial cell is the main cell type responsible for acetyl-LDL uptake.     

Nucleoside and deoxynucleoside phosphorylation in formamide solutions

Nucleosides or deoxynucleosides were converted to a number of phosphorylated nucleotide and deoxynucleotide derivatives by ammonium or alkali dihydrogen phosphates in formamide. Conversions were smaller and slower at room temperature and greater and faster at elevated temperatures. Nucleotides afforded product mixtures similar to those obtained for nucleosides under the same conditions, indicating the occurrence of transphosphorylation processes. Products of reaction at elevated temperatures were cyclic nucleotides, nucleoside monophosphates, nucleoside diphosphates and cyclic nucleotide phosphates. The relative amounts of products formed were quite temperature dependent. Cyclic nucleotides were found to be in greatest abudance for reactions run at 125° or above. Relative yields of 2, 3 and 5 nucleotides and 3 and 5 deoxynucleotides from several experiments are reported. 5-Monophosphates were generally found to be present in larger quantities than 2 or 3 monophosphates. 2-Deoxyadenosine showed a preference for phosphorylation at the 3 position. Conclusions reached from mechanistic studies are that the phosphorylations are a series of equilibrium reactions, with cyclic nucleotides being formed irreversibly.Presented in part at the 3rd Northwest and 5th Rocky Mountain Joint Regional ACS Meeting of the American Chemical Society, Salt Lake City, Utah, June, 1980.     

Transgelin Silencing Induces Different Processes in Different Breast Cancer Cell Lines

Transgelin is a protein reported to be a marker of several cancers. However, previous studies have shown both up‐ and down‐regulation of transgelin in tumors when compared with non‐tumor tissues and the mechanisms whereby transgelin may affect the development of cancer remain largely unknown. Transgelin is especially abundant in smooth muscle cells and is associated with actin stress fibers. These contractile structures participate in cell motility, adhesion, and the maintenance of cell morphology. Here, the role of transgelin in breast cancer is focused on. Initially, the effects of transgelin on cell migration of the breast cancer cell lines, BT 549 and PMC 42, is studied. Interestingly, transgelin silencing increased the migration of PMC 42 cells, but decreased the migration of BT 549 cells. To clarify these contradictory results, the changes in protein abundances after transgelin silencing in these two cell lines are analyzed using quantitative proteomics. The results confirmed the role of transgelin in the migration of BT 549 cells and suggest the involvement of transgelin in apoptosis and small molecule biochemistry in PMC 42 cells. The context‐dependent function of transgelin reflects the different molecular backgrounds of these cell lines, which differ in karyotypes, mutation statuses, and proteome profiles.     

Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis

WW domain binding protein 1‐like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6‐RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non‐haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4‐family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l‐deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.     

Activity of the tick Ixodes ricinus monitored in a suburban park in Brno,Czech Republic,in association with the evaluation of selected repellents

The ever‐increasing number of Lyme borreliosis patients led us to consider more effective procedures for disease prevention. The aim of our study was to monitor the annual activity and infectivity of Ixodes ricinus ticks in the Pisárky region, City of Brno, CR, and to test the responses of the locally‐captured ticks to selected repellents. The result of regular one‐hour‐perweek monitoring in 2011 was the collection of ticks that directly reflected the highest number of Lyme disease patients (4,835) detected throughout the period of recording in the Czech Republic. The ticks were examined for spirochaetes by dark field microscopy. The positive samples were identified by PCR analysis, confirming that 76% of these were infected with Borrelia burgdorferi sensu lato. Ticks were most abundant in May and June, with August having the highest risk for spirochaetal infection. Tick activity was statistically correlated with temperature. The moving‐object‐bioassay was used to study repellent efficiency on the Ixodes ricinus nymphs captured in the above‐mentioned suburban park. Five selected commercial repellents based on DEET (N, N‐diethyl3methylbenzamide) showed statistically different effects on the non‐repellent control group.     

Land use intensification in grasslands: higher trophic levels are more negatively affected than lower trophic levels

Increasing land use intensity and human influence are leading to a reduction in plant and animal species diversity. However, little is known about how these changes may affect higher trophic levels, apart from simply reducing species numbers. Here we investigated, over 3 years, the influence of different land practices on a tritrophic system in grassland habitats. The system consisted of the host plant Plantago lanceolata L. (Plantaginaceae), two monophagous weevils, Mecinus labilis Herbst and Mecinus pascuorum Gyllenhal (Coleoptera: Curculionidae), and their parasitoid Mesopolobus incultus Walker (Hymenoptera: Pteromalidae). At over 70 sites across three geographic regions in Germany, we measured plant species diversity and vegetation structure, as well as abundance of P. lanceolata, the two weevils, and the parasitoid. Land use intensity (fertilization) and type (mowing vs. grazing) negatively affected not only plant species richness but also the occurrence of the two specialized herbivores and their parasitoid. In contrast, land use had a mostly positive effect on host plant size, vegetation structure, and parasitization rate. This study reveals that intensification of land use influences higher trophic organisms even without affecting the availability of the host plant. The observed relationships between land use, vegetation complexity, and the tritrophic system are not restricted locally; rather they are measureable along a broad range of environmental conditions and years throughout Germany. Our findings may have important implications for the conservation of insect species of nutrient‐poor grasslands.     

Generalization versus Specialization in Pollination Systems: Visitors,Thieves, and Pollinators of Hypoestes aristata (Acanthaceae)

Many recent studies have suggested that the majority of animal-pollinated plants have a higher diversity of pollinators than that expected according to their pollination syndrome. This broad generalization, often based on pollination web data, has been challenged by the fact that some floral visitors recorded in pollination webs are ineffective pollinators. To contribute to this debate, and to obtain a contrast between visitors and pollinators, we studied insect and bird visitors to virgin flowers of Hypoestes aristata in the Bamenda Highlands, Cameroon. We observed the flowers and their visitors for 2-h periods and measured the seed production as a metric of reproductive success. We determined the effects of individual visitors using 2 statistical models, single-visit data that were gathered for more frequent visitor species, and frequency data. This approach enabled us to determine the positive as well as neutral or negative impact of visitors on H. aristata’s reproductive success. We found that (i) this plant is not generalized but rather specialized; although we recorded 15 morphotaxa of visitors, only 3 large bee species seemed to be important pollinators; (ii) the carpenter bee Xylocopa cf. inconstans was both the most frequent and the most effective pollinator; (iii) the honey bee Apis mellifera acted as a nectar thief with apparent negative effects on the plant reproduction; and (iv) the close relationship between H. aristata and carpenter bees was in agreement with the large-bee pollination syndrome of this plant. Our results highlight the need for studies detecting the roles of individual visitors. We showed that such an approach is necessary to evaluate the pollination syndrome hypothesis and create relevant evolutionary and ecological hypotheses.     

Plant Trait Assembly Affects Superiority of Grazer's Foraging Strategies in Species-Rich Grasslands

Background

Current plant – herbivore interaction models and experiments with mammalian herbivores grazing plant monocultures show the superiority of a maximizing forage quality strategy (MFQ) over a maximizing intake strategy (MI). However, there is a lack of evidence whether grazers comply with the model predictions under field conditions.

Methodology/Findings

We assessed diet selection of sheep (Ovis aries) using plant functional traits in productive mesic vs. low-productivity dry species-rich grasslands dominated by resource-exploitative vs. resource-conservative species respectively. Each grassland type was studied in two replicates for two years. We investigated the first grazing cycle in a set of 288 plots with a diameter of 30 cm, i.e. the size of sheep feeding station. In mesic grasslands, high plot defoliation was associated with community weighted means of leaf traits referring to high forage quality, i.e. low leaf dry matter content (LDMC) and high specific leaf area (SLA), with a high proportion of legumes and the most with high community weighted mean of forage indicator value. In contrast in dry grasslands, high community weighted mean of canopy height, an estimate of forage quantity, was the best predictor of plot defoliation. Similar differences in selection on forage quality vs. quantity were detected within plots. Sheep selected plants with higher forage indicator values than the plot specific community weighted mean of forage indicator value in mesic grasslands whereas taller plants were selected in dry grasslands. However, at this scale sheep avoided legumes and plants with higher SLA, preferred plants with higher LDMC while grazing plants with higher forage indicator values in mesic grasslands.

Conclusions

Our findings indicate that MFQ appears superior over MI only in habitats with a predominance of resource-exploitative species. Furthermore, plant functional traits (LDMC, SLA, nitrogen fixer) seem to be helpful correlates of forage quality only at the community level.