Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

The second messenger NAADP triggers Ca²⁺ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2^−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca²⁺ responses as assessed by single-cell Ca²⁺ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P₂ were only partially dependent on TPCs. In Tpcn1/2^−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca²⁺-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca²⁺ release. High-affinity [³²P]NAADP binding still occurs in Tpcn1/2^−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca²⁺-permeable channels indispensable for NAADP signalling.     

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

Artefacts induced on <Emphasis Type="Italic">c</Emphasis>-type haem proteins by electrode surfaces

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met–His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c ₃ from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.     

Evidence for branching in cryptococcal capsular polysaccharides and consequences on its biological activity

The encapsulated fungus Cryptococcus neoformans is a common cause of life-threatening disease in immunocompromised individuals. Its major virulence determinant is the polysaccharide (PS) capsule. An unsolved problem in cryptococcal biology is whether the PSs composing the capsule are linear or complex branched polymers, as well as the implications of this structural composition in pathogenesis. In this study we approached the problem by combining static and dynamic light scattering, viscosity analysis, and high-resolution microscopy and correlated the findings with biological properties. Analysis of the dependence of capsular PS molecular mass and the radius of gyration provided strong evidence against a simple linear PS configuration. Shape factors calculated from light scattering measurements in solution revealed values consistent with polymer branching. Furthermore, viscosity measurements provided complementary evidence for structural branching. Electron microscopy showed PS spherical-like structures similar to other branched PS. Finally, we show that the capacity of capsular PS to interfere in complement-mediated phagocytosis, inhibit nitric oxide production by macrophage-like cells, protect against reactive oxygen species, antibody reactivity and half-life in serum were influenced by the degree of branching, providing evidence for the notion that PS branching is an important parameter in determining the biological activity of C. neoformans PS.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l(-1) caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

Enhancement of antibody synthesis in rats by feeding cis-9,trans-11 conjugated linoleic acid during early life

Previous studies have demonstrated that the intake of a 1% conjugated linoleic acid (CLA) diet in an 80:20 mixture of cis-9,trans-11 and trans-10,cis-12 exerts age-specific effects on the immune system: immunoglobulin enhancement and proliferative down-modulation in neonatal and adult rats, respectively. The present study evaluates the influence of the same diet on antibody synthesis of early infant Wistar rats during suckling and/or after weaning. Dietary supplementation was performed during suckling and early infancy (4 weeks), only during suckling (3 weeks), or only in early infancy (1 week). CLA content in plasma and serum immunoglobulin (Ig) G, IgM and IgA concentration were determined. Proliferation, cytokines and Ig production were evaluated on isolated splenocytes. Cis-9,trans-11- and trans-10,cis-12-CLA isomers were detected in the plasma of all CLA-supplemented animals, and the highest content was quantified in those rats supplemented over the longest period. These rats also exhibited higher concentrations of serum IgG, IgM and IgA. Moreover, splenocytes from CLA-supplemented rats showed the highest IgM and IgG synthesis and interleukin (IL)-6 production, whereas their proliferative ability was lower. In summary, in infant rats, we observed both the enhance antibody synthesis previously reported in neonates, and the reduced lymphoproliferation previously reported in adults.     

Synthesis of sugars embodying conjugated carbonyl systems and related triazole derivatives from carboxymethyl glycoside lactones. Evaluation of their antimicrobial activity and toxicity

The synthesis of a series of pyranoid derivatives comprising a conjugated carbonyl function and related triazole derivatives, structurally suitable for bioactivity evaluation, was achieved in few steps starting from readily available carboxymethyl glycoside lactones (CMGL). 3-Enopyranosid-2-uloses were generated by oxidation/elimination of tri-O-acylated 2-hydroxy pyranosides. Subsequent Wittig olefination provided stereoselectively 2-C-branched-chain conjugated dienepyranosides with (E)-configuration around the exocyclic double bond. A heterogeneous CuI/Amberlyst-catalyzed ‘click’ chemistry protocol was used to convert glycosides bearing a propargyl moiety into the corresponding 1,2,3-triazoles. These new molecules were screened for their in vitro antibacterial and antifungal activities and those containing conjugated carbonyl systems demonstrated the best efficacy. (N-Dodecylcarbamoyl)methyl enone glycerosides were the most active ones among the enones tested. The ??-anomer displayed very strong activities against Bacillus cereus and Bacillus subtilis and strong activity toward Enterococcus faecalis and the fungal pathogen Penicillium aurantiogriseum. The corresponding ??-anomer presented a very strong inhibitory effect against two fungal species (Aspergillus niger and P. aurantiogriseum). (N-Dodecyl-/N-propargyl/or N-benzylcarbamoyl)methyl dienepyranosides exhibited selectively a strong activity toward E. faecalis. Further acute toxicity evaluation indicated low toxic effect of the (N-dodecylcarbamoyl)methyl enone glyceroside ??-anomer and of the carbamoylmethyl dienepyranosides N-protected with propargyl or benzyl groups.     

In situ forming chitosan hydrogels prepared via ionic/covalent co-cross-linking

In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.