Evidences for a Leaky Scanning Mechanism for the Synthesis of the Shorter M23 Protein Isoform of Aquaporin-4: IMPLICATION IN ORTHOGONAL ARRAY FORMATION AND NEUROMYELITIS OPTICA ANTIBODY INTERACTION*

Aquaporin-4 (AQP4) exists as two major isoforms that differ in the length of the N terminus, the shorter AQP4-M23 and the longer AQP4-M1. Both isoforms form tetramers, which can further aggregate in the plasma membrane to form typical orthogonal arrays of particles (OAPs) whose dimension depends on the ratio of the M1 and M23. In this study, we tested the hypothesis that the M23 isoform can be produced directly by the M1 mRNA. In cells transiently transfected with AQP4-M1 coding sequence we observed besides AQP4-M1 the additional presence of the AQP4-M23 isoform associated with the formation of typical OAPs observable by two-dimensional blue native/SDS-PAGE and total internal reflection microscopy. The mutation of the second in-frame methionine M23 in AQP4-M1 (AQP4-M1^M23I) prevented the expression of the M23 isoform and the formation of OAPs. We propose “leaky scanning” as a translational mechanism for the expression of AQP4-M23 protein isoform and that the formation of OAPs may occur even in the absence of AQP4-M23 mRNA. This mechanism can have important pathophysiological implications for the cell regulation of the M1/M23 ratio and thus OAP size. In this study we also provide evidence that AQP4-M1 is mobile in the plasma membrane, that it is inserted and not excluded into immobile OAPs, and that it is an important determinant of OAP structure and size.     

The effect of wrack composition and diversity on macrofaunal assemblages in intertidal marine sediments

Wrack (dead, washed-up seaweed and seagrass) buried in soft substrata may increase the organic content and alter the physical structure of sediments. These effects may influence the composition and structure of macrofaunal assemblages in the sediment. Such influences can be expected to vary according to the type and amount of wrack as well as the presence of invasive seaweeds in the wrack. In this study, we deliberately buried different amounts of the invasive species Sargassum muticum in isolation or mixed to the native species Ulva sp. and Fucus vesiculosus, in two intertidal sandflats to test some hypotheses about the response of macrofaunal assemblages. We tested whether (1) diversity of detritus (i.e. different mixtures), and (2) the amount of detritus of S. muticum influenced the composition and the relative abundance of macrofaunal assemblages. We also assessed whether the sediment organic carbon and the biomass of benthic microalgae varied depending on the diversity of detritus and the amount of detritus of S. muticum. Finally, we tested if these effects of wrack were consistent across sites. Results indicated that buried wrack affected the composition and structure of macrofaunal assemblages in short-term (i.e. 4 weeks), but there were no differences depending on detritus diversity or the amount of S. muticum. In addition, sediment organic matter and microalgal biomass were not affected by the addition of wrack. They instead varied greatly among small spatial scales (i.e. plots). Wrack composition or abundance of the invasive species S. muticum played thus a small role in shaping the structure of macrofaunal assemblages or the biomass of benthic microalgae in these intertidal sediments, probably because these sediments are frequently affected by various inputs of organic matter and benthic assemblages are already adapted to organically enriched sediments.     

Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle

Skeletal muscle fibers exhibit a high resting chloride conductance primarily determined by ClC-1 chloride channels that stabilize the resting membrane potential during repetitive stimulation. Although the importance of ClC-1 channel activity in maintaining normal muscle excitability is well appreciated, the subcellular location of this conductance remains highly controversial. Using a three-pronged multidisciplinary approach, we determined the location of functional ClC-1 channels in adult mouse skeletal muscle. First, formamide-induced detubulation of single flexor digitorum brevis (FDB) muscle fibers from 15-16-day-old mice did not significantly alter macroscopic ClC-1 current magnitude (at -140 mV; -39.0 +/- 4.5 and -42.3 +/- 5.0 nA, respectively), deactivation kinetics, or voltage dependence of channel activation (V(1/2) was -61.0 +/- 1.7 and -64.5 +/- 2.8 mV; k was 20.5 ± 0.8 and 22.8 +/- 1.2 mV, respectively), despite a 33% reduction in cell capacitance (from 465 +/- 36 to 312 +/- 23 pF). In paired whole cell voltage clamp experiments, where ClC-1 activity was measured before and after detubulation in the same fiber, no reduction in ClC-1 activity was observed, despite an approximately 40 and 60% reduction in membrane capacitance in FDB fibers from 15-16-day-old and adult mice, respectively. Second, using immunofluorescence and confocal microscopy, native ClC-1 channels in adult mouse FDB fibers were localized within the sarcolemma, 90 degrees out of phase with double rows of dihydropyridine receptor immunostaining of the T-tubule system. Third, adenoviral-mediated expression of green fluorescent protein-tagged ClC-1 channels in adult skeletal muscle of a mouse model of myotonic dystrophy type 1 resulted in a significant reduction in myotonia and localization of channels to the sarcolemma. Collectively, these results demonstrate that the majority of functional ClC-1 channels localize to the sarcolemma and provide essential insight into the basis of myofiber excitability in normal and diseased skeletal muscle.     

Inflammatory protein profile during systemic high dose interleukin-2 administration

Systemic interleukin-2 (IL-2) administration induces an assortment of downstream effects whose biological and therapeutic significance remains unexplored mostly because of the methodological inability to globally address their complexity. Protein array analysis of sera from patients with renal cell carcinoma obtained prior and during high-dose IL-2 therapy had previously revealed extensive alterations in expression of the soluble factors analyzed, whose discovery was limited by the number of capture antibodies selected for protein detection. Here, we expanded the analysis to SELDI-TOF-MS and quantitative protein analysis (nephelometry). All cytokines/chemokines detected by protein arrays were below the SELDI detection limit, while novel IL-2-specific changes in expression of acute-phase reactants and high-density lipoprotein metabolites could be identified. Serum amyloid protein A (SAA) and C-reactive protein expression were consistently up-regulated after four doses of IL-2, while other proteins were down-regulated. These findings were confirmed by SELDI immunoaffinity capture and nephelometry. Immunoaffinity capture revealed different, otherwise undetectable, isoforms of SAA. A linear correlation between peak area by SELDI and protein concentration by nephelometry was observed. Overall distinct yet complementary information was obtained using different platforms, which may better illustrate complex phenomena such as the systemic response to biological response modifiers.     

Human recombinant prolidase from eukaryotic and prokaryotic sources. Expression, purification, characterization and long-term stability studies

Prolidase is a Mn(2+)-dependent dipeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. In humans, a lack of prolidase activity causes prolidase deficiency, a rare autosomal recessive disease, characterized by a wide range of clinical outcomes, including severe skin lesions, mental retardation, and infections of the respiratory tract. In this study, recombinant prolidase was produced as a fusion protein with an N-terminal histidine tag in eukaryotic and prokaryotic hosts and purified in a single step using immobilized metal affinity chromatography. The enzyme was characterized in terms of activity against different substrates, in the presence of various bivalent ions, in the presence of the strong inhibitor Cbz-Pro, and at different temperatures and pHs. The recombinant enzyme with and without a tag showed properties mainly indistinguishable from those of the native prolidase from fibroblast lysate. The protein yield was higher from the prokaryotic source, and a detailed long-term stability study of this enzyme at 37 degrees C was therefore undertaken. For this analysis, an 'on-column' digestion of the N-terminal His tag by Factor Xa was performed. A positive effect of Mn(2+) and GSH in the incubation mixture and high stability of the untagged enzyme are reported. Poly(ethylene glycol) and glycerol had a stabilizing effect, the latter being the more effective. In addition, no significant degradation was detected after up to 6 days of incubation with cellular lysate. Generation of the prolidase in Escherichia coli, because of its high yield, stability, and similarity to native prolidase, appears to be the best approach for future structural studies and enzyme replacement therapy.     

Glutamine-binding protein from Escherichia coli specifically binds a wheat gliadin peptide. 2. Resonance energy transfer studies suggest a new sensing approach for an easy detection of wheat gliadin

In this work is presented the first attempt to develop a fluorescence assay for detection of traces of gluten in food by utilizing the recombinant glutamine-binding protein (GlnBP) from E. coli. We found that GlnBP specifically binds the sequence of amino acids present both in gliadin and other prolamines classified as toxic for celiac patients. Affinity chromatography experiments together with mass spectrometry experiments demonstrated that GlnBP can bind the following amino acid sequence XXQPQPQQQQQQQQQQQQL. Sequence alignment experiments pointed out that this sequence is exclusively representative of the gliadin and the other prolamines considered toxic for celiac patients. These findings suggest the development of a competitive resonance energy transfer (RET) assay for an easy and rapid detection of this sequence in raw and cooked food.     

Protein, carbohydrate, lipid concentrations and HSP 70–HSP 90 (stress protein) expression over an annual cycle: useful tools to detect feeding constraints in a benthic suspension feeder

In the present paper we suggest an effect of seasonal variations in food availability on two ecophysiological parameters in a warm temperate benthic suspension feeder: the tissue concentrations of proteins, carbohydrates and lipids on the one hand, and the expression of stress proteins (HSP 70 and 90, inducible and/or constitutive) on the other hand. The concentrations of biomacromolecules have already been used to describe bentho-pelagic and reproductive processes, but this is the first time that stress protein expression is suggested to be directly related with food constraints in marine organisms. Paramuricea clavata (Cnidaria: Gorgonacea) express HSP 70 and 90 (constitutive and/or inducible) throughout the seasonal cycle, and HSP 70 levels are twice as high as the levels of HSP 90. In summer and autumn, when seston availability to suspension feeders was low, P. clavata showed low levels of carbohydrates and lipids, but high levels of HSPs expression. The levels of HSP 70 and 90 expression fit with negative exponential functions of carbohydrate and lipid concentrations. We suggest a direct effect of food availability on the studied ecophysiological parameters while the effect of temperature may be rather indirect. HSP expression as well as the tissue concentrations of carbohydrate and lipids may be used as biomarkers of environmental changes and seston availability to benthic suspension feeders.     

A novel 10B-enriched carboranyl-containing phthalocyanine as a radio- and photo-sensitising agent for boron neutron capture therapy and photodynamic therapy of tumours: in vitro and in vivo studies.

The synthesis of a Zn(ii)-phthalocyanine derivative bearing four 10B-enriched o-carboranyl units (10B-ZnB4Pc) and its natural isotopic abundance analogue (ZnB4Pc) in the peripheral positions of the tetraazaisoindole macrocycle is presented. The photophysical properties of ZnB4Pc, as tested against model biological systems, were found to be similar with those typical of other photodynamically active porphyrin-type photosensitisers, including a singlet oxygen quantum yield of 0.67. The carboranyl-carrying phthalocyanine was efficiently accumulated by B16F1 melanotic melanoma cells in vitro, appeared to be partitioned in at least some subcellular organelles and, upon red light irradiation, induced extensive cell mortality. Moreover, ZnB4Pc, once i.v.-injected to C57BL/6 mice bearing a subcutaneously transplanted pigmented melanoma, photosensitised an important tumour response, provided that the irradiation at 600-700 nm was performed 3 h after the phthalocyanine administration, when appreciable concentrations of ZnB4Pc were still present in the serum. Analogously, irradiation of the 10B-ZnB4Pc-loaded pigmented melanoma with thermal neutrons 24 h after injection led to a 4 day delay in tumour growth as compared with control untreated mice. These results open the possibility to use one chemical compound as both a photosensitising and a radiosensitising agent for the treatment of tumours by the combined application of photodynamic therapy and boron neutron capture therapy.