Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC₅₀ values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC₅₀ values.     

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. We have recently shown that cholesterol regulates representative members of the Kir family and that in the majority of the cases, cholesterol suppresses channel function. Furthermore, recent data indicate that cholesterol regulates Kir channels by specific sterol-protein interactions, yet the location of the cholesterol binding site in Kir channels is unknown. Using a combined computational-experimental approach, we show that cholesterol may bind to two nonanular hydrophobic regions in the transmembrane domain of Kir2.1 located between adjacent subunits of the channel. The location of the binding regions suggests that cholesterol modulates channel function by affecting the hinging motion at the center of the pore-lining transmembrane helix that underlies channel gating either directly or through the interface between the N and C termini of the channel.     

Light Suppresses Sporulation and Epidemics of Peronospora belbahrii

Peronospora belbahrii is a biotrophic oomycete attacking sweet basil. It propagates asexually by producing spores on dichotomously branched sporophores emerging from leaf stomata. Sporulation occurs when infected plants are incubated for at least 7.5h in the dark in moisture-saturated atmosphere at 10-27°C. Exposure to light suppresses spore formation but allows sporophores to emerge from stomata. Incandescent or CW fluorescent light of 3.5 or 6 µmoles.m².s^-1 respectively, caused 100% inhibition of spore formation on lower leaf surface even when only the upper leaf surface was exposed to light. The inhibitory effect of light failed to translocate from an illuminated part of a leaf to a shaded part of the same leaf. Inhibition of sporulation by light was temperature-dependent. Light was fully inhibitory at 15-27°C but not at 10°C, suggesting that enzyme(s) activity and/or photoreceptor protein re-arrangement induced by light occur at ≥15°C. DCMU or paraquat could not abolish light inhibition, indicating that photosystem I and photosystem II are not involved. Narrow band led illumination showed that red light (λmax 625 nm) was most inhibitory and blue light (λmax 440 nm) was least inhibitory, suggesting that inhibition in P. belbahrii, unlike other oomycetes, operates via a red light photoreceptor. Nocturnal illumination of basil in the field (4-10 µmoles.m².s^-1 from 7pm to 7am) suppressed sporulation of P. belbahrii and reduced epidemics of downy mildew, thus reducing the need for fungicide applications. This is the first report on red light inhibition of sporulation in oomycetes and on the practical application of light for disease control in the field.     

Association of sugar content QTL and PQL with physiological traits relevant to frost damage resistance in pea under field and controlled conditions

To increase yield in pea (Pisum sativum L.), autumn sowing would be preferable. Hence, frost tolerance of pea became a major trait of interest for breeders. In order to better understand the cold acclimation in pea, Champagne a frost tolerant line and Terese, a frost sensitive line, and their recombinant inbred lines (RIL) were studied. RIL frost tolerance was evaluated by a frost damage scale under field as well as controlled conditions. A quantitative trait loci (QTL) approach was used to identify chromosomal regions linked to frost tolerance. The detected QTL explained from 6.5 to 46.5% of the phenotypic variance. Amongst them, those located on linkage groups 5 and 6 were consistent with over all experiments, in field as well as in controlled environments. In order to improve the understanding of the frost tolerance mechanisms, several cold acclimation key characters such as concentration of sugars, electrolyte leakage, osmotic pressure, and activity of RuBisCO were assessed. Some of these physiological QTL colocalised with QTL for frost damage, in particular two raffinose QTL on LG5 and LG6 and one RuBisCO activity QTL on LG6, explaining 8.8 to 27.0% of the phenotypic variance. In addition, protein quantitative loci were mapped; some of them colocalised with frost damage and physiological QTL on LG5 and LG6, explaining 16.0–43.6% of the phenotypic variance. Raffinose metabolism and RuBisCO activity and its effect on photosynthesis might play a major role in cold acclimation of pea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Seed Transmission of Pseudoperonospora cubensis

Pseudoperonospora cubensis, an obligate biotrophic oomycete causing devastating foliar disease in species of the Cucurbitaceae family, was never reported in seeds or transmitted by seeds. We now show that P. cubensis occurs in fruits and seeds of downy mildew-infected plants but not in fruits or seeds of healthy plants. About 6.7% of the fruits collected during 2012–2014 have developed downy mildew when homogenized and inoculated onto detached leaves and 0.9% of the seeds collected developed downy mildew when grown to the seedling stage. This is the first report showing that P. cubensis has become seed-transmitted in cucurbits. Species-specific PCR assays showed that P. cubensis occurs in ovaries, fruit seed cavity and seed embryos of cucurbits. We propose that international trade of fruits or seeds of cucurbits might be associated with the recent global change in the population structure of P. cubensis.     

New horizons in schizophrenia treatment: autophagy protection is coupled with behavioral improvements in a mouse model of schizophrenia

Autophagy plays a key role in the pathophysiology of schizophrenia as manifested by a 40% decrease in BECN1/Beclin 1 mRNA in postmortem hippocampal tissues relative to controls. This decrease was coupled with the deregulation of the essential ADNP (activity-dependent neuroprotector homeobox), a binding partner of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) another major constituent of autophagy. The drug candidate NAP (davunetide), a peptide fragment from ADNP, enhanced the ADNP-LC3B interaction. Parallel genetic studies have linked allelic variation in the gene encoding MAP6/STOP (microtubule-associated protein 6) to schizophrenia, along with altered MAP6/STOP protein expression in the schizophrenic brain and schizophrenic-like behaviors in Map6-deficient mice. In this study, for the first time, we reveal significant decreases in hippocampal Becn1 mRNA and reversal by NAP but not by the antipsychotic clozapine (CLZ) in Map6-deficient (Map6^+/−) mice. Normalization of Becn1 expression by NAP was coupled with behavioral protection against hyperlocomotion and cognitive deficits measured in the object recognition test. CLZ reduced hyperlocomotion below control levels and did not significantly affect object recognition. The combination of CLZ and NAP resulted in normalized outcome behaviors. Phase II clinical studies have shown NAP-dependent augmentation of functional activities of daily living coupled with brain protection. The current studies provide a new mechanistic pathway and a novel avenue for drug development.     

Proximity of cytoplasmic and periplasmic loops in NhaA Na+/H+ antiporter of Escherichia coli as determined by site-directed thiol cross-linking

The unique trypsin cleavable site of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, was exploited to detect a change in mobility of cross-linked products of NhaA by polyacrylamide gel electrophoresis. Double-Cys replacements were introduced into loops, one on each side of the trypsin cleavage site (Lys 249). The proximity of paired Cys residues was assessed by disulfide cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis(maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM). The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomly. In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H225C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C, N177C/S286C, and H225C/S286C. In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C; (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C. The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformation with pH are in close proximity within the NhaA protein. The data also suggest close proximity between N-terminal and C-terminal helices at both the cytoplasmic and the periplasmic face of NhaA.     

Structure-based functional study reveals multiple roles of transmembrane segment IX and loop VIII-IX in NhaA Na+/H+ antiporter of Escherichia coli at physiological pH

The three-dimensional crystal structure of Escherichia coli NhaA determined at pH 4 provided the first structural insights into the mechanism of antiport and pH regulation of a Na(+)/H(+) antiporter. However, because NhaA is activated at physiological pH (pH 6.5-8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII-IX. Here we studied this NhaA segment (Glu(241)-Phe(267)) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the "pH sensor." Furthermore mutation F267C reduced the H(+)/Na(+) stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na(+), suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu(255) and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10-18 A) implying a flexible interface. L255C formed intermolecular S-S bonds, cross-linked only with a 5-A cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent K(m) for Na(+) of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation.     

Direct Regulation of Prokaryotic Kir Channel by Cholesterol

Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by ⁸⁶Rb⁺ uptake. Our results show that ⁸⁶Rb⁺ flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses ⁸⁶Rb⁺ flux by >50%, and activity is completely inhibited at 12–15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated ⁸⁶Rb⁺ uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 μg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific.Inwardly rectifying potassium channels (Kir) are known to play critical roles in the regulation of multiple cellular functions including membrane excitability, heart rate, and vascular tone (1–3). Kir channels are classified into seven subfamilies (Kir1–7) identified by distinct biophysical properties and sensitivities to different regulators (2). Our earlier studies have shown that Kir2 channels, one of the major subfamilies of Kir that are responsible for maintaining membrane potential in a variety of cell types, are strongly suppressed by the elevation of membrane cholesterol (4, 5). Cholesterol-induced suppression of Kir2 was first observed in aortic endothelial cells (4), in which resting K⁺ conductance is dominated by Kir2.1 and Kir2.2 channels (6), and then when channels were heterologously expressed in Chinese hamster ovary cells (5, 7). Furthermore, the same effect was observed ex vivo in endothelial cells and bone marrow-derived progenitor cells isolated from hypercholesterolemic pigs (8, 9).In terms of the mechanism, the first insights came from comparing the effects of cholesterol and of its chiral analogue, epicholesterol. Although the two sterols are known to have almost identical effects on the biophysical properties of the lipid bilayer (10, 11), their impact on Kir activity is completely different; partial substitution of endogenous cholesterol with epicholesterol resulted in significant increase in Kir current in endothelial cells (4). These observations suggest that specific sterol-protein interactions may be involved in the cholesterol sensitivity of Kir2 channels. However, in the complex environment of the plasma membrane, cholesterol may interact not only with the channels themselves but also with other proteins, which in turn may regulate the activity of the channels. In the cellular environment, therefore, it is impossible to discriminate between direct channel-cholesterol interactions and indirect effects. Moreover, it is impossible to define the actual concentrations of cholesterol in any given membrane compartment. To quantitatively test direct cholesterol-protein interactions, it is necessary to examine sensitivity of pure Kir channels to membrane cholesterol in a membrane of defined lipid composition. To date, only the cytoplasmic domains of several mammalian Kir channels have been purified (Kir2.1, Kir3.1, and Kir3.2) (12–15). We therefore concentrate in this study on the effect of cholesterol on two bacterial K⁺ channels that differ in the level of their homology to mammalian Kir channels, KirBac1.1 and KcsA. KirBac channels have high sequence homology with mammalian Kirs (e.g. 52% homology between KirBac1.1 and Kir2.1; see Fig. 7A) and have now been extensively used as structural models of mammalian Kir channels (3, 16, 17). The sequence similarity between KcsA and mammalian K channels lies mainly in the transmembrane domain (18). The overall sequence homology of KcsA to mammalian Kir channels is relatively low (e.g. 22% homology between KcsA and Kir2.1; see Fig. 7A), with an entirely different cytoplasmic domain structure.Open in a separate windowFIGURE 7.Cholesterol has no effect on KcsA-mediated ⁸⁶Rb⁺ uptake. A, time courses of ⁸⁶Rb⁺ uptake into liposomes reconstituted with 50 μg of cholesterol/mg of PL and as compared with liposomes containing no cholesterol (control). Both batches of liposomes contained 5 μg of KcsA/mg of PL. Blank liposomes contain no protein. The points represent averages of three independent experiments (means ± S.D.). B, normalized time courses of ⁸⁶Rb⁺ uptake in liposomes incorporating 50, 150, and 250 μg of cholesterol/mg of PL. C, maximal uptake of ⁸⁶Rb⁺ after 240 s in liposomes containing 10, 25, 50, 100, 150, 200, and 250 μg of cholesterol/mg of PL normalized to control (means ± S.D. of 3–5 independent experiments; *, p < 0.05). DPM, disintegrations per minute.Here we show that, similarly to Kir2 channels, prokaryotic Kir channels incorporated into liposomes are strongly suppressed by an increase in membrane cholesterol. Furthermore, the sensitivity of prokaryotic Kir to cholesterol is stereo-selective to cholesterol optical analogues. In contrast, KscA channels are insensitive to membrane cholesterol. These observations suggest that cholesterol directly suppresses Kir channels.     

Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses

Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR^(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8⁺- and CD4⁺-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8⁺ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection.