Localized and transient changes in plasma membrane fluidity during in vitro myoblast fusion: an 1H-NMR study.

High resolution proton NMR was used to study the cell surface molecular events which take place during in vitro myoblast differentiation and fusion. The CH3 and (CH2)n spectral signals were followed throughout in vitro myogenic development. The results show that although both the T1 and T2 relaxation times of the CH3 and (CH2)n groups are sensitive to the fusion process, T1 is the most sensitive. Both T1 of CH3 and (CH2)n increased before fusion indicating a higher degree of molecular motion and then returned to their original values. These results demonstrate how mobile lipid domains observed with proton NMR can be used to study the changes taking place during myoblast differentiation, particularly myoblast membrane fusion.     

The signal transduction pathway involved in the migration induced by a monocyte chemotactic cytokine

Recombinant monocyte-chemotactic and activating factor (rMCAF; alternative acronyms MCP-1, TDCF, human JE) induced migration of human monocytes across polycarbonate or nitrocellulose filters. Maximal induction of migration was observed at a concentration of 10 ng/ml (10(-9) M). Checkerboard analysis revealed that rMCAF elicited true gradient-dependent chemotactic migration, although a gradient independent chemokinetic effect was observed at low concentrations (1-5 ng/ml). rMCAF caused a rapid (less than 5 s) and transient (approximately 1.5 min) increase of free cytosolic Ca2+ ions, as assessed by the fura-2 probe. No Ca2+ increase was detected in neutrophils or lymphocytes stimulated by rMCAF. Studies conducted in the absence of extracellular Ca2+ or in the presence of Ni2+ (an inhibitor of Ca2+ influx) suggested that the increase of intracellular Ca2+ induced by rMCAF is dependent on the influx of extracellular Ca2+ through plasma membrane channels. Bordetella pertussis toxin inhibited the intracellular Ca2+ elevation and chemotaxis caused by rMCAF. The possible involvement of Ca(2+)-dependent protein kinases in rMCAF signaling pathway(s) was explored using inhibitors. Inhibitors of GMP-dependent kinase and myosin L chain kinase had no effect on rMCAF-induced monocyte migration. In contrast, protein kinase C/cAMP-dependent kinase inhibitors (such as, C-I, H-7, HA-1004, KT5720, and Staurosporine) markedly decreased rMCAF induced chemotaxis suggesting the involvement of a serine/threonine protein kinase, possibly protein kinase C, in rMCAF signaling pathway.     

Porcine pancreatic lipase. Sequence of the first 234 amino acids of the peptide chain.

The single polypeptide chain of about 460 amino acids of porcine pancreatic lipase (EC 3.1.1.3) has been fragmented into five peptides by cyanogen bromide cleavage [Rovery, M., Bianchetta, J. & Guidoni, A. (1973) Biochim. Biophys. Acta, 328, 391--395]. The sequence of the first three cyanogen bromide peptides (CNI, CNII, CNIII), including a total of 234 amino acids, was fully elucidated. Automatic or manual Edman degradation was performed on the different peptides. Fragmentations of the CN peptides were accomplished by digestions with trypsin (after citraconylation or 1,2-cyclohexanedione treatment), chymotrypsin and Staphylococcus aureus external protease. Hydrolysis of unreduced material by pepsin and thermolysin, performed in order to determine the S-S bridge positions, provided useful overlapping peptides. The glycan moiety of lipase is bound to Asn-166. The non-essential tyrosine specifically blocked by diisopropylphosphorofluoridate is Tyr-49 in a cluster of asparagine and glutamine residues. The existence of a highly hydrophobic sequence (206--217) at the C terminus of the CNII fragment is noteworthy.     

Species specificity and intraspecific variation in the chemical profiles of Heliconius butterflies across a large geographic range

In many animals, mate choice is important for the maintenance of reproductive isolation between species. Traits important for mate choice and behavioral isolation are predicted to be under strong stabilizing selection within species; however, such traits can also exhibit variation at the population level driven by neutral and adaptive evolutionary processes. Here, we describe patterns of divergence among androconial and genital chemical profiles at inter‐ and intraspecific levels in mimetic Heliconius butterflies. Most variation in chemical bouquets was found between species, but there were also quantitative differences at the population level. We found a strong correlation between interspecific chemical and genetic divergence, but this correlation varied in intraspecific comparisons. We identified “indicator” compounds characteristic of particular species that included compounds already known to elicit a behavioral response, suggesting an approach for identification of candidate compounds for future behavioral studies in novel systems. Overall, the strong signal of species identity suggests a role for these compounds in species recognition, but with additional potentially neutral variation at the population level.     

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg · L⁻¹ of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment. Received: 12 June 1997 / Accepted: 24 October 1997     

Synthetic antimicrobial β‐peptide in dual‐treatment with fluconazole or ketoconazole enhances the in vitro inhibition of planktonic and biofilm Candida albicans

Fungal infections are a pressing concern for human health worldwide, particularly for immunocompromised individuals. Current challenges such as the elevated toxicity of common antifungal drugs and the emerging resistance towards these could be overcome by multidrug therapy. Natural antimicrobial peptides, AMPs, in combination with other antifungal agents are a promising avenue to address the prevailing challenges. However, they possess limited biostability and susceptibility to proteases, which has significantly hampered their development as antifungal therapies. β‐peptides are synthetic materials designed to mimic AMPs while allowing high tunability and increased biostability. In this work, we report for the first time the inhibition achieved in Candida albicans when treated with a mixture of a β‐peptide model and fluconazole or ketoconazole. This combination treatment enhanced the biological activity of these azoles in planktonic and biofilm Candida, and also in a fluconazole‐resistant strain. Furthermore, the in vitro cytotoxicity of the dual treatment was evaluated towards the human hepatoma cell line, HepG2, a widely used model derived from liver tissue, which is primarily affected by azoles. Analyses based on the LA‐based method and the mass‐action law principle, using a microtiter checkerboard approach, revealed synergism of the combination treatment in the inhibition of planktonic C. albicans. The dual treatment proved to be fungicidal at 48 and 72 h. Interestingly, it was also found that the viability of HepG2 was not significantly affected by the dual treatments. Finally, a remarkable enhancement in the inhibition of the highly azole‐resistant biofilms and fluconazole resistant C. albicans strain was obtained. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.     

EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP⁺). We investigated the effects of EGb761 pretreatment on MPP⁺ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP⁺, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP⁺ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP⁺ administration. MPP⁺-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP⁺ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP⁺-induced oxidative stress.