Acridine Orange/exosomes increase the delivery and the effectiveness of Acridine Orange in human melanoma cells: A new prototype for theranostics of tumors

Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466?nm. However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration. The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules. In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models. The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO. This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.     

Microphytobenthos activity and fluxes at the sediment-water interface: interactions and spatial variability

In this study oxygen and nutrient fluxes and denitrification rates across the sediment-water interface were measured via intact core incubations with a twofold aim: show whether microphytobenthos activity affects these processes and analyse the dispersion of replicate measurements. Eighteen intact sediment cores (i.d. 8 cm) were randomly sampled from a shallow microtidal brackish pond at Tjarno, on the west coast of Sweden, and were incubated in light and in darkness simulating in situ conditions. During incubation O₂, inorganic N and SiO₂ fluxes and denitrification rates (isotope pairing) were measured. Assuming mean values of 18 cores as best estimate of true average (BEA), the accuracy of O₂, NH₄ ⁺, NO₃ ^- and SiO₂ fluxes calculated for an increasing number of subsamples was tested. At the investigated site, microalgae strongly influenced benthic O₂, inorganic N and SiO₂ fluxes and coupled (D_n) and uncoupled (D_w) denitrification through their photosynthetic activity. In the shift between dark and light conditions NH₄ ⁺ and SiO₂ effluxes (60 and 110 µmol m^-2h^-1) and D_n (5 µmol m^-2 h^-1) dropped to zero, NO₃ ^- uptake (70 µmol m^-2 h^-1) showed a 30% increase, while D_w (20 µmol m^-2 h^-1) showed an 80% decrease. For O₂ and NO₃ ^- dark fluxes, 4 core replicates were sufficient to obtain averages within 5-10% of the best estimated mean, while 10-20% accuracy was obtained with 4-12 replicates for SiO₂ and >10 replicates for NH₄ ⁺ dark fluxes. Mean accuracy was considerably lower for all light incubations, probably due to the patchy distribution of the benthic microalgal community.     

Local adaptation and ecological genetics of host-plant specialization in a leaf beetle

The tendency of insect species to evolve specialization to one or a few plant species is probably a major reason for the remarkable diversity of herbivorous insects. The suggested explanations for this general trend toward specialization include a range of evolutionary mechanisms, whose relative importance is debated. Here we address two potentially important mechanisms: (i) how variation in the geographic distribution of host use may lead to the evolution of local adaptation and specialization; (ii) how selection for specialization may lead to the evolution of trade‐offs in performance between different hosts. We performed a quantitative genetic experiment of larval performance in three different populations of the alpine leaf beetle Oreina elongata reared on two of its main host plants. Due to differences in host availability, each population represents a distinctly different selective regime in terms of host use including selection for specialization on one or the other host as well as selection for utilizing both hosts during the larval stage. The results suggest that selection for specialization has lead to some degree of local adaptations in host use: both single‐host population had higher larval growth rate on their respective native host plant genus, while there was no difference between plant treatments in the two‐host population. However, differences between host plant treatments within populations were generally small and the degree of local adaptation in performance traits seems to be relatively limited. Genetic correlations in performance traits between the hosts ranged from zero in the two‐host population to significantly positive in the single‐host populations. This suggests that selection for specialization in single host populations typically also increased performance on the alternative host that is not naturally encountered. Moreover, the lack of a positive genetic correlation in the two host‐population give support for the hypothesis that performance trade‐offs between two host plants may typically evolve when a population have adapted to both these plants. We conclude that although there is selection for specialization in larval performance traits it seems as if the genetic architecture of these traits have limited the divergence between populations in relative performance on the two hosts.     

Kinetic studies on the cis-trans isomerization of farnesol by liver alcohol dehydrogenase

The isomerization of 2-trans,6-trans farnesol to the 2-cis,6-trans isomer proceeds through the corresponding aldehydes. The rate of the reaction in the presence of liver alcohol dehydrogenase (LADH) and diaphorase has been compared with the rates of the isomerization of 2-trans,6-trans farnesal with different catalysts (OH^?, H⁺, -SH diaphorase alone, albumin). The results are in keeping with enzymatic catalysis by LADH.     

Growth of the seaweed Ulva rigida C. Agardh in relation to biomass densities, internal nutrient pools and external nutrient supply in the Sacca di Goro lagoon (Northern Italy)

Growth of the seaweed Ulva rigida C. Agardh was investigated in relation to biomass densities, internal nutrient pools and external nutrient supply. Research was carried out from 23 March to 5 July 1994 in the Sacca di Goro (Po Delta, Northern Italy), whose south-eastern part was covered by extensive mats of Ulva rigida. Two types of field experiments were conducted by incubating Ulva thalli inside large cages. In the first experiment, beginning on 23 March, 100 g of wet thalli were placed into the cages, allowed to grow for two weeks, then collected and replaced. This procedure was repeated 8 times over the study period. In the second experiment, Ulva thalli were left inside the cages and collected at selected time intervals (14, 27, 41, 64 and 76 days) in order to simulate the effects of increased density on growth and nutrient storage.We recorded specific growth rates (NGR) ranging from 0.025 to 0.081 d^–1 for a period up to two months in the repeated short-term experiments performed at relatively low initial algal densities (300–500 g AFDW m^–3). These NGR resulted significantly related to dissolved inorganic nitrogen (DIN) in the water column. Tissue concentrations of total Kjeldahl nitrogen (TN) were almost constant, while extractable nitrate decreased in a similar manner to DIN in the water column. Total phosphorus showed considerable variation, probably linked to pulsed freshwater inflow.In the long-term incubation experiment, NGR of Ulva was inversely related to density. Internal concentrations of both total P and TN reached maximum values after one month; thereafter P concentration remained almost constant, while TN decreased below 2% w/w (by dry weight). The TN decrease was also accompanied by an abrupt decrease in nitrate tissue concentration. The biomass incubated over the two month period suffered a progressive N limitation as shown by a decreasing NY ratio (49.4 to 14.6). The reciprocal control of Ulva against biogeochemical environment and viceversa is a key factor in explaining both resource competition and successional stages in primary producer communities dominated by Ulva. However, when the biomass exceeds a critical threshold level, approximately 1 kg AFDW m^–3, the macroalgal community switches from active production to rapid decomposition, probably as a result of selfshading, biomass density and development of anaerobic conditions within the macroalgal beds.     

Sulphide release from anoxic sediments in relation to iron availability and organic matter recalcitrance and its effects on inorganic phosphorus recycling

This research aims to analyse the sediment capacity to buffer free sulphide release in three coastal lagoons which differ in terms of eutrophication level, tide influence and primary producer communities. A preliminary estimate of soluble reactive phosphorus (SRP) regeneration coupled with sulphide fluxes is also made. Sediment profiles of ferrous and ferric iron and reduced sulphur pools were determined in three stations in the Bassin d'Arcachon (South West France), in one site in the Etang du Prévost lagoon (Southern France), and in three stations in the Sacca di Goro lagoon (Northern Italy). Laboratory experiments were also conducted by incubating sediment slurries. Slurries from the French lagoons were also enriched with about 2% d.w. of organic detritus obtained from the dominant macrophytes of each site, namely Zostera noltii and Ruppia cirrhosa (Bassin d'Arcachon), and Ulva rigida (Etang du Prévost). In the Sacca di Goro, slurry experiments were conducted at two sites with different salinity range, sediment composition and hydrodynamics.Field data showed that concentrations of available iron (Fe(II)+Fe(III)) ranged from a minimum of 28.5 µmol cm^–3 (Etang du Prévost) to a maximum of 275.7 µmol cm^–3 (Sacca di Goro). Moreover, in the French lagoons, acid volatile sulphide (AVS) accumulation in the superficial sediment was related to ferrous iron concentrations. Laboratory experiments showed that in spite of strong reducing conditions, sulphide and SRP release was weaker in iron-rich sediments and in those enriched with the most refractory organic matter. The highest fluxes were detected in sediment slurries from the Etang du Prévost, which had the lowest iron content, supplied by 2% of the labile detritus from Ulva rigida. In this case, SRP release was directly related to sulphide production.Two factors seem significant to evaluate the buffer capacity against free sulphide and SRP release from anoxic sediment: organic matter biodegradability, which forces sediment toward reducing conditions, and iron availability, which can affect sulphide mobility as well as the iron hydroxide-phosphate-sulphide system.     

Cytoskeletal Breakdown and Apoptosis Elicited by NO Donors in Cerebellar Granule Cells Require NMDA Receptor Activation

Abstract: We have recently demonstrated that nitric oxide (NO) donors can trigger either apoptosis or necrosis of neurons as a function of the intensity of the exposure. Here, we show that the apoptosis induced by the NO donors S -nitrosocysteine (SNOC) or S -nitroso- N -acetylpenicillamine (SNAP) in cultured cerebellar granule cells (CGCs) depends on NMDA receptor (NMDA-R) activation leading to intracellular Ca²⁺ overload. Early dissolution of actin filaments followed by breakdown of microtubules and nuclear lamins preceded the appearance of typical apoptotic features. NO donors induced tyrosine nitration in neurons, in a small population of contaminating astrocytes, and in cultures of cerebellar astroglial cells. However, astrocytes neither displayed cytoskeletal alterations nor underwent apoptosis. Competitive and uncompetitive NMDA receptor antagonists, such as d -aminophosphonovaleric acid and MK-801, did not influence tyrosine nitration but prevented the accumulation of intracellular Ca²⁺, cytoskeletal breakdown, and apoptosis induced by either SNOC or SNAP in CGCs. Taken together, these data strongly suggest that Ca²⁺ influx through NMDA-R-gated ion channels is a critical event in CGC apoptosis induced by NO donors.     

The Use of Crown Ethers in Peptide Chemistry – V. Solid-phase Synthesis of Peptides by the Fragment Condensation Approach using Crown Ethers as Non-covalent Protecting Groups

We have previously described the conditions by which peptide synthesis by the solid-phase fragment condensation approach can be carried out using crown ethers as non-covalent protection for the N^α -amino group. Here we demonstrate that the procedure can be extended to large, partially protected peptide fragments possessing free Lys and/or Arg residues. The first step was to ensure that complex formation on the side chain of amino acids was not detrimental to the methodology and exhibited the same solubility and coupling properties as N^α -complexed peptides. Thus, a model hexapeptide was synthesized using Fmoc chemistry containing Lys and Arg residues, which, when complexed with 18-Crown-6, was readily soluble in DCM and coupled quantitatively to a resin-bound tetrapeptide. Two tripeptides were then prepared, one containing a free Ser residue, the other free Tyr, to examine the possible occurrence of side reactions. After coupling using standard conditions only the former tripeptide exhibited the formation of the O-acylation by-product (5%). Another model hexapeptide containing Lys, Tyr, Ser and Asp protected with a TFA-stable adamantyl group was complexed with 18-Crown-6 and coupled to the resin-bound tetrapeptide with near quantative yield. Extending the length of the peptide to 21 and 40 residues, which represent sequences Gly⁵² to Leu⁷² (21-mer) and Pro³³ to Leu⁷² (40-mer) from Rattus norvegicus chaperonin 10 protein, respectively, resulted in partially protected fragments that were readily soluble in water, thus enabling purification by RP-HPLC. Complexation with 18-Crown-6 gave two highly soluble products that coupled to resin-board tetramer with 68% and 50% coupling efficiencies for the 21-mer and 40-mer, respectively. Treatment with 1% DIEA solutions followed by acidolytic cleavage and purification of the major product confirmed that the correct product had been formed, when analysed by amino acid analysis and ESI-MS. These results served to extend the methodology of non-covalent protection of large partially protected peptide fragments for the stepwise fragment condensation of polypeptides.     

Copper amine oxidases: Current state of knowledge

Summary A brief review on the structure nd functions of the copper-pyrroloquinoline quinone amine oxidases is presented. Data concerning the metal and organic cofactors are reported, giving special emphasis to their structural relationship with the protein molecule and mechanistic properties. Information on the functional role of these enzymes with respect to polyamine metabolism are also given.     

Concealment of epitope by reduction and alkylation in prion protein

Conversion of the cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)), the key molecular event in the pathogenesis of prion diseases, is accompanied by a conformational transition of alpha-helix into beta-sheet structures involving alpha-helix 1 (alpha1) domain from residues 144 to 154 of the protein. Reduction and alkylation of PrP(C) have been found to inhibit the conversion of PrP(C) into PrP(Sc) in vitro. Here we report that while antibody affinity of epitopes in the N- and C-terminal domains remained unchanged, reduction and alkylation of the PrP molecule induced complete concealment of an epitope in alpha1 for anti-PrP antibody 6H4 that is able to cure prion infection in the cell model. Mass spectrometric analysis of recombinant PrP showed that the alkylation reaction takes place at reduced cysteines but no modification was observed in this cryptic epitope. Our study suggests that reduction and alkylation result in local or global rearrangement of PrP tertiary structure that is maintained in both liquid and solid phases. The implications in the conversion of PrP(C) into PrP(Sc) and the therapeutics of prion diseases are discussed.