Plasma membrane carries out multiple physiological functions that require its dynamic and tightly regulated organization into specialized domains of different size, stability, and lipid/protein ...composition. Sphingolipids are a group of lipids in which the plasma membrane is particularly enriched, thus being crucial for its structure and function. A specific type of sphingolipid‐enriched plasma membrane domains, where ergosterol is depleted and lipids are tightly packed in a rigid gel phase, has recently been found in several fungal species, including yeasts and moulds. After presenting the main biophysical features of gel domains and the experimental method for their detection in the fungal plasma membrane, we review these sphingolipid‐enriched gel domains and illustrate their importance to both unicellular and multicellular fungi. First, the biophysical properties of the fungal sphingolipid‐enriched domains will be analysed taking into consideration the plasma membrane sphingolipidome. Next, their possible biological roles will be summarized, including their relations with plasma membrane compartments and involvement in stress responses. Moreover, since the plasma membrane is a target for several antifungal compounds, a biophysical connection between sphingolipid‐enriched domains and antifungal action will be explored.
Piano-stool-{CpRu} complexes containing 1,3,5-triaza-7-phosphaadamantane (PTA), N-methyl-1,3,5-triaza-7-phosphaadamantane (mPTA), and 3,7-dimethyl-1,3,7-triaza-5-phosphabyciclo3.3.1nonane (dmoPTA) ...were evaluated as drugs against breast cancer. The evaluated compounds include two new examples of this family, the complexes RuCp(DMSO-κS)(HdmoPTA)(PPh3)(CF3SO3)2 (8) and RuCp(PPh3)2-µ-dmoPTA-1κP-2κ2N,N′-PdCl2(CF3SO3) (11), which have been synthesized and characterized by NMR, IR, and single-crystal X-ray diffraction. The cytotoxic activity of compounds was evaluated against MDA-MB-231 breast cancer cells, and the three most active complexes were further tested against the hormone-dependent MCF-7 breast cancer cell line. Their cell death mechanism and ruthenium uptake were also evaluated, as well as their binding ability to human serum albumin.
The steady rise in the cancer burden and grim statistics set a vital need for new therapeutic solutions. Given their high efficiency, metallodrugs are quite appealing in cancer chemotherapy. This ...work examined the anticancer activity of an anti-trypanosomal ruthenium-based compound bearing the 5-nitrofuryl pharmacophore, Ru
(dmso)
(5-nitro-2-furaldehyde semicarbazone) (abbreviated as RuNTF; dmso is the dimethyl sulfoxide ligand). The cytotoxicity of RuNTF was evaluated in vitro against ovarian adenocarcinoma, hormone-dependent breast adenocarcinoma, prostate carcinoma (grade IV) and V79 lung fibroblasts human cells. The activity of RuNTF was similar to the benchmark metallodrug cisplatin for the breast line and inactive against the prostate line and lung fibroblasts. Given the known role of serum protein binding in drug bioavailability and the distribution via blood plasma, this study assessed the interaction of RuNTF with human serum albumin (HSA) by circular dichroism (CD) and fluorescence spectroscopy. The fluorescence emission quenching from the HSA-Trp214 residue and the lifetime data upon RuNTF binding evidenced the formation of a 1:1 {RuNTF-albumin} adduct with log
sv = (4.58 ± 0.01) and log
= (4.55 ± 0.01). This is supported by CD data with an induced CD broad band observed at ~450 nm even after short incubation times. Importantly, the binding to either HSA or human apo-transferrin is beneficial to the cytotoxicity of the complex towards human cancer cells by enhancing the cytotoxic activity of RuNTF.
The relevance of mannosyldiinositolphosphorylceramide M(IP)
C synthesis, the terminal complex sphingolipid class in the yeast
, for the lateral organization of the plasma membrane, and in particular ...for sphingolipid-enriched gel-like domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H
symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (
) and
Δ cells, which are unable to synthesize M(IP)
C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that
deletion strongly affects the rigidity of gel-like domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterolenriched domains. Moreover, in these cells lacking M(IP)
C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the
plasma membrane and suggests that M(IP)
C is important for a proper hydrophobic chain packing of sphingolipids in the gel-like domains of
cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.
Here, biophysical properties of membranes enriched in three metabolically related sterols are analyzed both
in vitro
and
in vivo
. Unlike cholesterol and ergosterol, the common metabolic precursor ...zymosterol is unable to induce the formation of a liquid ordered (
l
o
) phase in model lipid membranes and can easily accommodate in a gel phase. As a result, Zym has a marginal ability to modulate the passive membrane permeability of lipid vesicles with different compositions, contrary to cholesterol and ergosterol. Using fluorescence-lifetime imaging microscopy of an aminostyryl dye in living mammalian and yeast cells we established a close parallel between sterol-dependent membrane biophysical properties
in vivo
and
in vitro
. This approach unraveled fundamental differences in yeast and mammalian plasma membrane organization. It is often suggested that, in eukaryotes, areas that are sterol-enriched are also rich in sphingolipids, constituting highly ordered membrane regions. Our results support that while cholesterol is able to interact with saturated lipids, ergosterol seems to interact preferentially with monounsaturated phosphatidylcholines. Taken together, we show that different eukaryotic kingdoms developed unique solutions for the formation of a sterol-rich plasma membrane, a common evolutionary trait that accounts for sterol structural diversity.
Ketoconazole (Ke) is an important antifungal drug, and two of its diphenylphosphinemethyl derivatives (KeP: Ph2PCH2-Ke and KeOP: Ph2P(O)CH2-Ke) have shown improved antifungal activity, namely against ...a yeast strain lacking ergosterol, suggesting alternative modes of action for azole compounds. In this context, the interactions of these compounds with a model of the cell membrane were investigated, using POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) large unilamellar vesicles and taking advantage of the intrinsic fluorescence of Ke, KeP and KeOP. Steady-state fluorescence spectra and anisotropy, including partition and aggregation studies, as well as fluorescence lifetime measurements, were carried out. In addition, the ability of the compounds to increase membrane permeability was assessed through carboxyfluorescein leakage. The membrane/water mole fraction partition coefficients (Kp,x): (3.31 ± 0.36) x105, (8.31 ± 1.60) x105 and (4.66 ± 0.72) x106, for Ke, KeP and KeOP, respectively, show that all three compounds have moderate to high affinity for the lipid bilayer. Moreover, KeP, and particularly KeOP interact more efficiently with POPC bilayers than Ke, which correlates well with their in vitro antifungal activity. Furthermore, although the three compounds disturb the lipid bilayer, KeOP is the quickest and most efficient one. Hence, the higher affinity and ability to permeabilize the membrane of KeOP when compared to that of KeP, despite the higher lipophilicity of the latter, points to an important role of Ph2P(O)CH2- oxygen. Overall, this work suggests that membrane interactions are important for the antifungal activity of these azoles and should be considered in the design of new therapeutic agents.
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•Membrane partition of ketoconazole and diphenylphosphine derivatives was quantified.•Both diphenylphosphine derivatives have higher membrane affinity than ketoconazole.•All compounds distinctly and significantly increase the lipid bilayer permeability.•The oxide derivative has the strongest membrane effects and antifungal activity.•Drug-membrane interactions are relevant for azole antifungal activity.
The free amino acid profile of 18 samples of tronchuda cabbage (Brassica oleracea L. var. costata DC) leaves, harvested at three different months, was determined by HPLC/UV−vis. The tronchuda cabbage ...leaves total free amino acid content varied from 3.3 to 14.4 g/kg fresh weight. Generally, arginine was the major compound, followed by proline, threonine, glutamine, cysteine, and glutamic acid. This study indicates that free amino acids are not similarly distributed: in external leaves, proline and arginine were the major free amino acids, while in internal ones, arginine was the main free amino acid, followed by threonine, glutamine, and cysteine. Significant differences were observed for valine, proline, arginine, leucine, cysteine, lysine, histidine, and tyrosine contents. The levels of some free amino acids were significantly affected by the collection period. In external leaves, this occurred with glutamic acid, serine, valine, leucine, cysteine, and ornithine contents, while in internal leaves, it occurred with aspartic acid, arginine, and total contents.
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