Lex glycosphingolipids-mediated cell aggregation

Glycoconjugates bearing oligosaccharide Lex, Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->3R, are found on the surface of several cell types. Although recent studies have indicated that Lexon both glycosphingolipids (GSL) and polylactosaminoglycans can mediate under certain experimental conditions Lex-Lexinteractions, cell-cell interactions based exclusively on LexGSLs have not been demonstrated. In this study we show that preincubation of nonaggregating rat basophilic leukemia (RBL) cells with purified LexGSLs resulted in incorporation of the GSLs into plasma membrane, as determined by immunostaining, and formation of aggregates in the presence of Ca2+; no aggregates were formed after preincubation of the cells with globoside or sphingomyelin. Lex-mediated aggregation was inhibited by removal of Ca2+or by addition of lactofucopentaose III but not by lactose or lacto- N-fucopentaose II. In a mixture of Lex-positive and Lex-negative RBL cells most of the aggregates were composed exclusively of Lex-positive cells. The combined data suggest that interactions between LexGSL on opposite cell surfaces are strong enough to allow formation of stable cell-cell contacts.      

Cannabinoid Effects on β Amyloid Fibril and Aggregate Formation,Neuronal and Microglial-Activated Neurotoxicity In Vitro

Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands against β amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter β amyloid (Aβ) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aβ_1–42 directly or microglial (BV-2 cells) conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ⁹-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55 ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aβ_1–42. Aβ_1–42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aβ-evoked SH-SY5Y cell viability, whereas JWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aβ fibrils and aggregates, there was no clear correlation between effects on Aβ morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.     

UV-B radiation and selenium affected energy availability in green alga <Emphasis Type="Italic">Zygnema</Emphasis>

Green alga Zygnema was exposed to three concentrations of selenium and two levels of UV-B radiation. The combined effects of both treatments on energy availability; photochemical quantum yield and respiratory potential were studied. Our findings show that traces of selenium enhance metabolic process connected with photochemical quantum yield and mitochondrial respiration. Surprisingly, selenium does not diminish the effects of UV-B radiation; on the contrary, the combined action of UV-B radiation and traces of selenium leads to pronounced negative effects on photochemical quantum yield and the respiratory potential. Selenium is involved in the activation of energy resources in green alga Zygnema. The importance of selenium for activity of the mitochondria is possibly an evolutionary recollection from an endosymbiotic bacterium.     

Temperature-Sensitive Microemulsion Gel: An Effective Topical Delivery System for Simultaneous Delivery of Vitamins C and E

Microemulsions (ME)—nanostructured systems composed of water, oil, and surfactants—have frequently been used in attempts to increase cutaneous drug delivery. The primary objective addressed in this work has been the development of temperature-sensitive microemulsion gel (called gel-like ME), as an effective and safe delivery system suitable for simultaneous topical application of a hydrophilic vitamin C and a lipophilic vitamin E. By changing water content of liquid o/w ME (o/w ME), a gel-like ME with temperature-sensitive rheological properties was formed. The temperature-driven changes in its microstructure were confirmed by rotational rheometry, viscosity measurements, and droplet size determination. The release studies have shown that the vitamins’ release at skin temperature from gel-like ME were comparable to those from o/w ME and were much faster and more complete than from o/w ME conventionally thickened with polymer (o/w ME carbomer). According to effectiveness in skin delivery of both vitamins, o/w ME was found the most appropriate, followed by gel-like ME and by o/w ME carbomer, indicating that no simple correlation between vitamins release and skin absorption could be found. The cytotoxicity studies revealed good cell viability after exposure to ME and confirmed all tested microemulsions as nonirritant. This work was supported by a grant of Slovenian Research Agency.     

Degradation of the Adriatic medusa <Emphasis Type="Italic">Aurelia</Emphasis> sp. by ambient bacteria

The decomposition of jellyfish after major bloom events results in the release of large amounts of nutrients, which can significantly alter nutrient and oxygen dynamics in the surrounding environment. The response of the ambient bacterial community to decomposing jellyfish biomass was evaluated in two marine ecosystems, the Gulf of Trieste (northern Adriatic Sea) and Big Lake (Mljet Island, southern Adriatic Sea). The major difference between these two ecosystems is that Aurelia sp. medusae occur throughout the year in the oligotrophic Big Lake, whereas in the mesotrophic Gulf of Trieste, they occur only seasonally and often as blooms. Addition of homogenized jellyfish to enclosed bottles containing ambient water from each of these systems triggered considerable changes in the bacterial community dynamics and in the nutrient regime. The high concentrations of protein, dissolved organic phosphorous (DOP), and PO₄ ³⁻ immediately after homogenate addition stimulated increase in bacterial abundance and production rate, coupled with NH₄ ⁺ accumulation in both ecosystems. Our preliminary results of the bacterial community structure, as determined with denaturing gradient gel electrophoresis, indicated differences in the bacterial community response between the two ecosystems. Despite divergence in the bacterial community responses to jellyfish homogenate, increased bacterial biomass and growth rates in both distinctive marine systems indicate potentially significant effects of decaying jellyfish blooms on microbial plankton.     

Analysis of growth of Lactobacillus plantarum WCFS1 on a complex medium using a genome-scale metabolic model

A genome-scale metabolic model of the lactic acid bacterium Lactobacillus plantarum WCFS1 was constructed based on genomic content and experimental data. The complete model includes 721 genes, 643 reactions, and 531 metabolites. Different stoichiometric modeling techniques were used for interpretation of complex fermentation data, as L. plantarum is adapted to nutrient-rich environments and only grows in media supplemented with vitamins and amino acids. (i) Based on experimental input and output fluxes, maximal ATP production was estimated and related to growth rate. (ii) Optimization of ATP production further identified amino acid catabolic pathways that were not previously associated with free-energy metabolism. (iii) Genome-scale elementary flux mode analysis identified 28 potential futile cycles. (iv) Flux variability analysis supplemented the elementary mode analysis in identifying parallel pathways, e.g. pathways with identical end products but different co-factor usage. Strongly increased flexibility in the metabolic network was observed when strict coupling between catabolic ATP production and anabolic consumption was relaxed. These results illustrate how a genome-scale metabolic model and associated constraint-based modeling techniques can be used to analyze the physiology of growth on a complex medium rather than a minimal salts medium. However, optimization of biomass formation using the Flux Balance Analysis approach, reported to successfully predict growth rate and by product formation in Escherichia coli and Saccharomyces cerevisiae, predicted too high biomass yields that were incompatible with the observed lactate production. The reason is that this approach assumes optimal efficiency of substrate to biomass conversion, and can therefore not predict the metabolically inefficient lactate formation.     

Differential role of basal keratinocytes in UV-induced immunosuppression and skin cancer

Cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) comprise major UV-induced photolesions. If left unrepaired, these lesions can induce mutations and skin cancer, which is facilitated by UV-induced immunosuppression. Yet the contribution of lesion and cell type specificity to the harmful biological effects of UV exposure remains currently unclear. Using a series of photolyase-transgenic mice to ubiquitously remove either CPDs or 6-4PPs from all cells in the mouse skin or selectively from basal keratinocytes, we show that the majority of UV-induced acute effects to require the presence of CPDs in basal keratinocytes in the mouse skin. At the fundamental level of gene expression, CPDs induce the expression of genes associated with repair and recombinational processing of DNA damage, as well as apoptosis and a response to stress. At the organismal level, photolyase-mediated removal of CPDs, but not 6-4PPs, from the genome of only basal keratinocytes substantially diminishes the incidence of skin tumors; however, it does not affect the UVB-mediated immunosuppression. Taken together, these findings reveal a differential role of basal keratinocytes in these processes, providing novel insights into the skin's acute and chronic responses to UV in a lesion- and cell-type-specific manner.     

Electric-field controlled liposome formation with embedded superparamagnetic iron oxide nanoparticles

Liposomes are one of the most promising biomaterial carriers to deliver DNA,(1) proteins, drugs and medicine in human bodies. However, artificially formed liposomes have to satisfy some crucial functions such as: (i) to efficiently carry drugs to targeted systems, (ii) to be biologically stable until they are removed from human body, (iii) to be biodegradable, and (iv) to be sufficiently small in size for effective drug delivery. Here, we report an efficient and novel method to simultaneously manufacture and incorporate super-paramagnetic iron-oxide nanoparticles (efficient target finder in the presence of external magnetic field) into the liposome's interior and its bilayer. In this technique, we use electric field to control the formation of liposomes and the incorporation of iron oxide nanoparticles. Our preparation procedure does not require any chemical or ultrasound treatments. Apart from that, we also provide further experimental investigations on the role of electric fields on the formation of liposomes using XPS(2) and the magnetic-optical microscope.     

Thrombin Generation in the Glasgow Myocardial Infarction Study

Background

Thrombin is a key protease in coagulation also implicated in complex pathology including atherosclerosis. To address the role of thrombin in relation to myocardial infarction (MI) we explored thrombin generation analysis in plasma from patients and controls that had participated in the Glasgow MI Study (GLAMIS).

Methods

Thrombin generation at 1 and 2 pM TF and with and without thrombomodulin (TM) was performed on plasmas from 356 subjects (171 cases, 185 age and sex matched controls) from GLAMIS collected between 3 and 9 months after the MI event.

Results

Although thrombin generation was slightly delayed in cases (lag time increased from 3.3 to 3.6 min) at the highest trigger, the overall potential to generate thrombin was increased by 7% for the ETP and by 15% for the peak height (both at the 1 pM TF trigger) in cases. Addition of TM did not reveal differences. Furthermore, an increased thrombin generation was associated with MI [normalized ETP: adjusted OR for the highest percentile = 2.4 (95% CI 1.3–4.5) and normalized peak height: adjusted OR = 2.6 (1.3–5.0)] at the lowest trigger; normalized ETP and peak height being 2.1 (1.1–3.8) and 2.0 (1.0–4.1) at the higher 2 pM trigger.

Conclusion

In GLAMIS, patients with a previous MI had an increased thrombin generation compared to controls. The absence of a clear difference in TM reduction suggests an unaltered anticoagulant activity in these patients. Further research is needed in order to unravel the underlying mechanisms of enhanced thrombin generation after MI.