Normal Cell Membrane Research Articles

Correlation between the secondary structure and surface activity of β-sheet forming cationic amphiphilic peptides and their anticancer activity

Cancer is one of the main causes of death worldwide. The current cancer treatment strategies often lack selectivity for cancer cells resulting in dose-limiting adverse effects and reduced quality of life. Recently, anticancer peptides (ACPs) have emerged as an alternative treatment with higher selectivity, less adverse effects, and lower propensity for drug resistance. However, most of the current studies on the ACPs are focused on α-helical ACPs and there is lack of systematic studies on β-sheet forming ACPs. Herein we report the development of a new series of rationally designed short cationic amphiphilic β-sheet forming ACPs and their structure activity relationship. The peptides had the general formula (XY1XY2)3, with X representing hydrophobic amino acids (isoleucine (I) or leucine (L)), Y1 and Y2 representing cationic amino acids (arginine (R) or lysine (K)). The cytotoxicity of the designed ACPs in HCT 116 colorectal cancer, HeLa cervical cancer and human dermal fibroblast (HDF) cells was assessed by MTT test. The physicochemical properties of the peptides were characterized by various techniques including RP-HPLC, LC-MS, and Circular Dichroism (CD) spectroscopy. The surface activity of the peptides at the air-water interface and their interaction with the lipid monolayers as models for cell membranes were studied by Langmuir trough. The peptides consisting of I with R and K had selective anticancer activity while the combination of L and R diminished the anticancer activity of the peptides but rendered them more toxic to HDFs. The anticancer activity of the peptides was directed by their surface activity (amphiphilicity) and their secondary structure in hydrophobic surfaces including cancer cell membranes. The selectivity of the peptides for cancer cells was a result of their higher penetration into cancer cell membranes compared to normal cell membranes. The peptides exerted their anticancer activity by disrupting the mitochondrial membranes and eventually apoptosis. The results presented in this study provide an insight into the structure-activity relationship of this class of ACPs which can be employed as guidance to design new ACPs with improved anticancer activity and lower toxicity against normal cells.

Expression and subcellular localization of Discoidin Domain Receptor 1 (DDR1) define prostate cancer aggressiveness

BackgroundThe Discoidin Domain Receptor 1 (DDR1) is one of the two members of a unique family of receptor tyrosine kinase receptors that signal in response to collagen, which has been implicated in cancer progression. Here, we examined the expression of DDR1 in prostate cancer (PCa), and assessed its potential value as a prognostic marker, as a function of grade, stage and other clinicopathologic parameters.MethodsWe investigated the association between the expression level and subcellular localization of DDR1 protein and PCa aggressiveness by immunohistochemistry, using tissue microarrays (TMAs) encompassing 200 cases of PCa with various Gleason scores (GS) and pathologic stages with matched normal tissue, and a highly specific monoclonal antibody.ResultsDDR1 was found to be localized in the membrane, cytoplasm, and nuclear compartments of both normal and cancerous prostate epithelial cells. Analyses of DDR1 expression in low GS (≤ 7[3 + 4]) vs high GS (≥ 7[4 + 3]) tissues showed no differences in nuclear or cytoplasmic DDR1in either cancerous or adjacent normal tissue cores. However, relative to normal-matched tissue, the percentage of cases with higher membranous DDR1 expression was significantly lower in high vs. low GS cancers. Although nuclear localization of DDR1 was consistently detected in our tissue samples and also in cultured human PCa and normal prostate-derived cell lines, its presence in that site could not be associated with disease aggressiveness. No associations between DDR1 expression and overall survival or biochemical recurrence were found in this cohort of patients.ConclusionThe data obtained through multivariate logistic regression model analysis suggest that the level of membranous DDR1 expression status may represent a potential biomarker of utility for better determination of PCa aggressiveness.

Yippee like 4 (Ypel4) is essential for normal mouse red blood cell membrane integrity

The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.

Discovery of 1’-acetoxychavicol acetate (ACA) as a promising antibacterial compound from galangal (Alpinia galanga (Linn.) Willd)

Galangal (Alpinia galanga (Linn.) Willd) is a perennial aromatic rhizomatous plant with great industrial importance. In this study, four major antibacterial compounds were isolated from galangal and further identified as hydroxycinnamaldehyde, cinnamaldehyde, coumaryl alcohol, and 1′-acetoxychavicol acetate (ACA). Among them, ACA was a less investigated antibacterial compound in galangal, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) 0.313 and 0.625 mg/mL, respectively, against multidrug-resistant Staphylococcus aureus SJTUF 20758. The mechanism study found that ACA significantly influenced the bacterial morphology and damaged the bacterial cell membrane integrity. Furthermore, quantitative proteomic analysis indicated that ACA at sub-MIC, on one hand, inhibited the expression of proteins associated with cell wall and membrane synthesis, osmotic regulation, and bacterial adhesion and invasion. On the other hand, it increased the expression of proteins pertinent to oxidative stress response, respiratory chain, and ATP synthesis. Therefore, ACA might initially target cell membrane by changing the expression of key membrane proteins involved in maintaining normal cell membrane functions, leading to the damage of cell membrane integrity, and also might trigger the survival response of bacteria. In general, to the best of our knowledge, it is for the first time to clarify the antibacterial mechanism of ACA. In addition, galangal is a promising spice rich in antibacterial compounds, especially ACA, which may be used as natural preservatives in the food industry.

Acid Microenvironment in Bone Sarcomas.

Simple SummaryAlthough rare, malignant bone sarcomas have devastating clinical implications for the health and survival of young adults and children. To date, efforts to identify the molecular drivers and targets have focused on cancer cells or on the interplay between cancer cells and stromal cells in the tumour microenvironment. On the contrary, in the current literature, the role of the chemical-physical conditions of the tumour microenvironment that may be implicated in sarcoma aggressiveness and progression are poorly reported and discussed. Among these, extracellular acidosis is a well-recognized hallmark of bone sarcomas and promotes cancer growth and dissemination but data presented on this topic are fragmented. Hence, we intended to provide a general and comprehensive overview of the causes and implications of acidosis in bone sarcoma.In bone sarcomas, extracellular proton accumulation is an intrinsic driver of malignancy. Extracellular acidosis increases stemness, invasion, angiogenesis, metastasis, and resistance to therapy of cancer cells. It reprograms tumour-associated stroma into a protumour phenotype through the release of inflammatory cytokines. It affects bone homeostasis, as extracellular proton accumulation is perceived by acid-sensing ion channels located at the cell membrane of normal bone cells. In bone, acidosis results from the altered glycolytic metabolism of bone cancer cells and the resorption activity of tumour-induced osteoclasts that share the same ecosystem. Proton extrusion activity is mediated by extruders and transporters located at the cell membrane of normal and transformed cells, including vacuolar ATPase and carbonic anhydrase IX, or by the release of highly acidic lysosomes by exocytosis. To date, a number of investigations have focused on the effects of acidosis and its inhibition in bone sarcomas, including studies evaluating the use of photodynamic therapy. In this review, we will discuss the current status of all findings on extracellular acidosis in bone sarcomas, with a specific focus on the characteristics of the bone microenvironment and the acid-targeting therapeutic approaches that are currently being evaluated.

Biomarker Discovery: Novel Insights into Red Blood Cell Metabolomics in Patients with Sickle Cell Disease at Risk for Early Mortality

Background : In adults with sickle cell disease (SCD), an elevated tricuspid regurgitant jet velocity (TRV) by Doppler echocardiography is associated with increased morbidity and mortality. A TRV>2.9 m/s occurs in approximately 10% of patients with SCD, has a 67-75% positive predictive value for pulmonary hypertension identified by right heart cardiac catheterization, and is the most robust non-invasive biomarker of early mortality risk for patients with SCD identified to date. Metabolomics is an integrated biosystems approach to molecular fingerprinting that may be able to differentiate individual metabolic characteristics of patients at high vs. low risk of mortality based on TRV elevation.Objective: To compare the metabolic differences between patients with SCD at high risk for early mortality defined by a TRV>2.9 m/s on Doppler echocardiogram compared to a low risk group with a TRV<2.5 m/s.Methods: A liquid chromatography-high resolution mass spectrometry method was used to determine metabolites in sickle erythrocytes from 90 case-control selected participants from the walk-PHaSST screening cohort, matched for sickle genotype (Hb-SS), age, gender, hydroxyurea use and alpha-thalassemia trait, divided equally into patients with a TRV>2.9 m/s compared to those with a TRV<2.5 m/s. Statistical and bioinformatics analyses used one-way analysis of variance (ANOVA) and partial least-squares discriminant analysis (PLS-DA) to identify groupwise discriminatory factors contributing to 95% separation of high vs. normal TRV. Significant features with one-way ANOVA p-value<0.05 were used to perform pathway analysis.Results: Patient characteristics are summarized in Table 1. Differential expression analysis showed that 145 accurate mass m/z features extracted from C18 column with positive ionization mode were significantly different between the high TRV and normal TRV subjects based on one-way ANOVA with p<0.005 (FDR<0.2) and PLS-DA with VIP>2 but no significant metabolism pathways were identified. With less stringent threshold p<0.05, one-way ANOVA revealed 1702 significant features which can discriminate the samples according to TRV status. Pathway enrichment analysis using Mummichog and the 1702 significant features with p-value<0.05 revealed 9 significant pathways (Figure 1).Conclusions: Metabolic profiles in sickle erythrocytes differentiate SCD patients with a normal TRV and low mortality risk from those at high risk for early mortality with a TRV>2.9 m/s through unsupervised pathway enrichment analysis. The pathways identified are intriguing and warrant further study. Identification of the porphyrin metabolic pathway may be related to higher hemolytic rates, while carnitine shuttle holds importance in mitochondrial function. Cholecalciferol (vitamin D3) metabolism is of interest given this common vitamin deficiency in SCD and studies linking vitamin D to vaso-occlusive pain, while omega 3 fatty acid metabolism is critical to normal cell membrane function. This preliminary work identifies metabolic pathways for future targeted analysis, and may lead to novel mechanistic insight and therapies that target these pathways. [Display omitted] DisclosuresMorris:Lifetrients: Patents & Royalties: I am inventor of IP owned by USCF-Benioff Children's Hospital Oakland, licensed to Lifetrients, generating royalties; Nestle: Consultancy; Calithera Biosciences: Consultancy; MAST Therapeutics: Research Funding; Pfizer: Consultancy. Brown:Calithera: Research Funding. Kato:Novartis: Consultancy; Global Blood Therapeutics: Consultancy; MAST Therapeutics: Research Funding; Bayer: Research Funding.

Toxicity and membrane perturbation properties of the ribotoxin-like protein Ageritin.

Ageritin is the prototype of a new ribotoxin-like protein family, which has been recently identified also in basidiomycetes. The protein exhibits specific RNase activity through the cleavage of a single phosphodiester bond located at sarcin/ricin loop of the large rRNA, thus inhibiting protein biosynthesis at early stages. Conversely to other ribotoxins, its activity requires the presence of divalent cations. In the present study, we report the activity of Ageritin on both prokaryotic and eukaryotic cells showing that the protein has a prominent effect on cancer cells viability and no effects on eukaryotic and bacterial cells. In order to rationalize these findings, the ability of the protein to interact with various liposomes mimicking normal, cancer and bacterial cell membranes was explored. The collected results indicate that Ageritin can interact with DPPC/DPPS/Chol vesicles, used as a model of cancer cell membranes, and with DPPC/DPPG vesicles, used as a model of bacterial cell membranes, suggesting a selective interaction with anionic lipids. However, a different perturbation of the two model membranes, mediated by cholesterol redistribution, was observed and this might be at the basis of Ageritin selective toxicity towards cancer cells.

Effect of Natural Antimicrobial Substances with Packaging System on Improving Quality of ‘ETMANI’ Guava (Psidium guajava L.) Fruit during cold storage

The effect of postharvest treatments of thyme oil and chitosan under different concentrations and combined with the packaging on weight loss%, firmness, decay index, malondialdehyde(MDA) content, total chlorophyll, hue angle (ho), ripening index, ascorbic acid and pectin methylesterase enzyme(PME) on ‘Etmani’ guava (Psidium guajava L.) fruit, were investigated during 2017 and 2018 successive seasons. Obtained results indicate that weight loss %was diminished by (T7) thyme oil at 1000 μLL−1 + packaging and (T8) chitosan at 1% + packaging, as well as firmness and PME were reduced significantly (p˂0.05) when treated with chitosan at 1% and 2% (T3 and T 4) and thyme oil at 1000 μLL−1 ( T2) compared to control treatment (T0). Simultaneously, it indicated that the most effective treatment in decreasing MDA and decay index were the treatment T3 and T4. Therefore, Chitosan and Thyme oils treatments could maintain normal cell membrane structure and function through down-regulating MDA content and pectin methylesterase enzyme activity, which due to reducing softening, weight loss%, and decay index in guava cv. ‘Etmani’ fruits during storage periods in comparison to other treatments and control

Role of Serine 263 in the Human Choline Transporter 1 and Congenital Myasthenic Syndrome

Disruptions of the neuromuscular junction (NMJ) result in congenital myasthenic syndromes (CMS). CMS is characterized by a range of phenotypes including muscle weakness, apnoeas, akinesia or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). CHT1 re-uptake of choline is a requirement for acetylcholine synthesis and NMJ function. The 13 transmembrane domain CHT1 protein is also thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive variant c.C788>T in SLC5A7 (NM_021815.4), in a family with a history of CMS using whole exome sequencing. This variant encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether a change in phosphorylation and/or charge/size of position 263 results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetics p.S263D and p.S263E. Upon expression in HEK293 cells and the utilization of cell surface biotinylation experiments, we found that WT, p.S263F and p.S263A mutants were present at the plasma membrane. Radioactive transport assays indicated that the p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non-functional. Immobilized metal affinity electrophoresis suggests that wild-type CHT1 may be more phosphorylated than the p.S263F CHT1 mutant. These results support the essential role of the Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status as well as size/charge at this position.

Molecular mechanism underlying the selective attack of trehalose lipids on cancer cells as revealed by coarse-grained molecular dynamics simulations

The present study indicated that the mixed lipid bilayer of dimyristoylphosphatidylcholine (DMPC) and trehalosemonomyristate (TreC14) interacted strongly with the plasma membrane of cancer cells, and not that of normal cells, when the composition of TreC14 was 70%, as revealed by coarse-grained molecular dynamics simulations. These results were consistent with those of previous experimental studies, indicating that DMPC/TreC14 mixed liposomes (DMTreC14) with TreC14 composition at 70% exhibited a strong anti-cancer effect without affecting normal cells. The simulations also revealed that lipids with highly hydrophilic and bulky head groups, such as TreC14, phosphatidylinositol (PI), and phosphatidylserine (PS), showed the tendency to accumulate. This caused both the DMTreC14 and cancer cell membranes to bend into large positive curvatures, resulting in tight contact between them. In contrast, no apparent interaction between the DMTreC14 and normal cell membranes was observed because PI and PS did not exist in the extracellular monolayer of the normal cell membrane.

Carbon nanohorns as nanocontainers for cisplatin: insight into their interaction with the plasma membranes of normal and breast cancer cells.

Cisplatin (cddp)-based chemotherapy is one of the most effective therapeutic alternatives for breast cancer treatment, the most common form of cancer, despite the severe side effects related to the high toxicity and low selectivity of cddp. To circumvent these drawbacks, the encapsulation of cddp into oxidized carbon nanohorns (CNHoxs) has been shown as a promising formulation with biocompatibility and low toxicity. However, there is still a lack of studies regarding the behavior of this cddp@CNHox nanovector on the cell membranes. This study presents an in silico description of the interactions between cddp@CNHox and membrane models of cancer (C_memb) and normal (N_memb) cells referring to a typical human breast. The results revealed the interaction mechanism of the inclusion complex 3cddp@CNHox (three cddp molecules are included in the CNHox cavity) with these biomembranes, which is a multistep process including approach, landing, insertion, and penetration. The 3cddp@CNHox stability was monitored over time, and demonstrated the trapping of cddp molecules inside the CNHox cavity over all simulations. The van der Waals contribution played a primary role (∼74%) for the complex stability. Moreover, the binding free energy calculations indicated that the interaction of the 3cddp@CNHox complex with the C_memb model was slightly more favorable, on average, than with the N_memb model. Analysis of the hydrogen bonds (HBs) formed over simulations of 800 ns explains the selectivity for the C_memb model, since the total number of HBs established between the inclusion complex and the C_memb model was about three times greater than that with the N_memb model. By reinforcing the potentiality of oxidized CNHox as a nanovector of cddp, the results presented in this study may assist and drive new experimental studies with this nanomaterial, focusing on the development of less aggressive formulations for breast cancer treatment.

Effects of Carbon Ion Beam Irradiation on Butanol Tolerance and Production of Clostridium acetobutylicum.

Clostridium acetobutylicum (C. acetobutylicum) has considerable potential for use in bioenergy development. Owing to the repeated use of traditional mutagenesis methods, the strains have developed a certain tolerance. The rheology of the bioprocess and the downstream processing of the product heavily depend on the ability of C. acetobutylicum mutants to produce butanol. Carbon ion beam irradiation has advantages over traditional mutation methods for fermentative production because of its dose conformity and superb biological effectiveness. However, its effects on the specific productivity of the strains have not been clearly understood. In this study, we screened five mutants through carbon ion beam irradiation; mutant Y217 achieved a butanol-production level of 13.67 g/L, exceeding that of wild-type strain ATCC 824 (i.e., 9.77 g/L). In addition, we found that the mutant maintained normal cell membrane integrity under the stimulation of 15 g/L butanol, whereas the intracellular macromolecules of wild-type strain ATCC 824 leaked significantly. Subsequently, we used the response surface methodology (RSM) to determine if the mutant cell membrane integrity improved the butanol tolerance. We verified that with the addition of butanol, the mutant could be fermented to produce 8.35 g/L butanol, and the final butanol concentration in the fermentation broth could reach 16.15 g/L. In this study, we proved that under butanol stress, mutant Y217 features excellent butanol production and tolerance and cell membrane integrity and permeability; no prior studies have attempted to do so. This will serve as an interesting and important illustration of the complexity of genetic control of the irradiation mutation of C. acetobutylicum strains. It may also prove to be useful in the bioengineering of strains of the mutant for use in the predevelopment stage.

Identifying Synthetic Lectins from a Competitive Screen for the Detection of Prostate Cancer.

Herein, we report a dual dye competitive screening method for the identification of five boronic acid functionalized synthetic lectins (SLs) that are selective for prostate-associated targets with the goal of detecting and staging prostate cancer. This method uses differently labeled normal (RWEP-1) and diseased (PC3) cell membrane extracts in a competitive binding assay to identify SLs that bind either the cancerous or normal extracts but not both. Subsequent studies examined the efficacy of these new SL hits in an array format to discriminate six prostate cell lines. The SL array was able to (a) classify the prostate cell lines with 83% accuracy, (b) discriminate the same cell lines based on their metastatic potential (noncancerous/healthy, cancerous/lowly metastatic, and cancerous/metastatic) with 96% classification accuracy, and (c) exhibit enhanced selectivity for prostate-derived versus colon-derived cell lines. Further analysis delineated the contribution from each SL in these studies, providing a focused SL array having potential utility as a cancer diagnostic.

Desorpcija plazme pomoću masene spektrometrije kao brzi test za dijagnostiku leukemije goveda

Plasma-desorption mass spectrometry with ionization of 252Californium fission fragments (252Cf-PD MS) was used to develop an express diagnostic test for bovine leukaemia. The test is based on the mass spectrometric parameters of artificial lipid membranes formed on the gold substrate from the lipid fraction of blood cells (mainly lymphocytes), by the self-assembly technique according to the biomimetic principle. Mass spectrometric analysis of artificial lipid membranes obtained from blood plasma of control and leukemic cattle gave a reliable diagnosis of leukaemia. The basis for the diagnosis were the ionization parameters of the fragmentation of lipid molecules from normal and leukemic cell membranes. In leukemic cell membranes, separate dense areas (rafts) 200 – 300 nm in diameter, saturated with cholesterol and phospholipids, are formed. The number of rafts per unit of cell membrane of leukemic cells significantly exceeded the permitted parameters of normal cell membranes. The increased number of rafts in artificial membranes formed in the membranes of leukemic cells was reliably reflected in mass spectra, due to the 180 – 250% increase in intensity of peaks of cholesterol fragment ions in comparison to control cells.

LRRC8 family proteins within lysosomes regulate cellular osmoregulation and enhance cell survival to multiple physiological stresses

LRRC8 family proteins on the plasma membrane play a critical role in cellular osmoregulation by forming volume-regulated anion channels (VRACs) necessary to prevent necrotic cell death. We demonstrate that intracellular LRRC8 proteins acting within lysosomes also play an essential role in cellular osmoregulation. LRRC8 proteins on lysosome membranes generate large lysosomal volume-regulated anion channel (Lyso-VRAC) currents in response to low cytoplasmic ionic strength conditions. When a double-leucine L706L707 motif at the C terminus of LRRC8A was mutated to alanines, normal plasma membrane VRAC currents were still observed, but Lyso-VRAC currents were absent. We used this targeting mutant, as well as pharmacological tools, to demonstrate that Lyso-VRAC currents are necessary for the formation of large lysosome-derived vacuoles, which store and then expel excess water to maintain cytosolic water homeostasis. Thus, Lyso-VRACs allow lysosomes of mammalian cells to act as the cell`s "bladder." When Lyso-VRAC current was selectively eliminated, the extent of necrotic cell death to sustained stress was greatly increased, not only in response to hypoosmotic stress, but also to hypoxic and hypothermic stresses. Thus Lyso-VRACs play an essential role in enabling cells to mount successful homeostatic responses to multiple stressors.

An inverted CAV1 (caveolin 1) topology defines novel autophagy-dependent exosome secretion from prostate cancer cells

ABSTRACT CAV1 (caveolin 1) expression and secretion is associated with prostate cancer (PCa) disease progression, but the mechanisms underpinning CAV1 release remain poorly understood. Numerous studies have shown CAV1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) CAV1 topology. Here we show that CAV1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive CAV1 secretion. Characterization of the isolated vesicles by electron microscopy, single-molecule fluorescence microscopy and proteomics reveals they represent a novel class of exosomes ~40 nm in diameter containing ~50-60 copies of CAV1 and, strikingly, are released via a non-canonical secretory macroautophagy/autophagy pathway. This study provides novel insights into a mechanism whereby CAV1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression. Abbreviations: 3-MA: 3-methyladenine; APEX: a modified soybean ascorbate peroxidase; ATG5: autophagy related 5; ATG9A: autophagy related 9A; ATG12: autophagy related 12; BHK: baby hamster kidney; C-exosomes: caveolin-exosomes; CAMKK2/CAMKKβ: calckum/calmodulin dependent protein kinase kinase 2; CAV1: caveolin 1; DAB: 3,3′-diaminobenzidine; DAPK: death associated protein kinase; EEA1: early endosome antigen 1; EM: electron microscopy; FCS: fluorescence correlation spectroscopy; GBP: GFP/YFP-binding peptide; GFP: green fluorescent protein; GOLGA2: golgin A2; ILVs: intralumenal vesicles; LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; MTORC1: mechanistic target of rapamycin kinase complex 1; MVBs: multivesicular bodies; PBS: phosphate-buffered saline; PCa: prostate cancer; PI3K: phosphoinositide 3-kinase; PM: plasma membrane; SFM: serum-free medium; TSG101: tumor susceptibility 101; WCL: whole cell lysates; WT: wild type; YFP: yellow fluorescent protein; βoG: β-octylglucoside

Different Polarity Extracts of Polygonum minus towards Cytotoxic Activities against Colon Cancer Cell Lines (HT-29, HCT-116, CT-26)

Aims: To investigate the cytotoxic properties of different polarity solvents of Polygonum minus extracts towards colon cancer cell lines, HT-29, HCT-116 and CT-26.&#x0D; Study Design: Experimental study.&#x0D; Place and Duration of Study: Central Laboratory, Tissue Culture Laboratory, Universiti Sultan Zainal Abidin, Terengganu between September 2019 until December 2019.&#x0D; Methodology: The different polarity solvents of P. minus extracts had been led to tetrazolium salt reduction (MTT) assay and an inhibition concentration of 50 (IC50) value for their cytotoxic potential against colon cancer cells. Then, cell morphology observation and fluorescence double staining of treatment cells were determined using a light inverted microscope and acridine orange/propidium iodide staining.&#x0D; Results: The results indicated that an extraction yields aligned from 0.01% for acetone and ethyl acetate to 0.45% for aqueous solution with decreasing order of aqueous solution &gt; 70% aqueous ethanol &gt; 50% aqueous ethanol &gt; methanol &gt; ethanol &gt; acetone and ethyl acetate. Meanwhile, the ethyl acetate extract showed a higher cytotoxic effect at IC50 values of 7.00 ± 0.06 µg/mL and 7.00 ± 0.30 µg/mL towards the HCT-116 and CT-26 cells; and 50% aqueous ethanol towards HT-29 cells (24.00 ± 0.01 µg/mL). The different solvent extracts of P. minus induced cytotoxic effects on the treated cell lines by altering their normal cell morphology and cell membrane integrity (except for acetone extract).&#x0D; Conclusion: Therefore, the use of different polarity solvent extracts of P. minus as an anti-cancer agent is promising more on ethyl acetate and warrants further investigation.

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing.

Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (ΔΨm), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P<0.05), and RSV did not significantly increase the sperm motility (0.44 vs. 0.40, P>0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ΔΨm. RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 μmol/L vs. 0.25 μmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor- and mitochondria-mediated apoptotic pathways.

The role of melatonin in alleviating the postharvest browning of lotus seeds through energy metabolism and membrane lipid metabolism

Abstract To investigate the underlying mechanism of melatonin (MT) involvement in the browning of lotus pods and seeds, the samples were immersed in 100 μmol L−1 MT and 10 μmol L−1 luzindole, a nonselective MT1/MT2 receptor antagonist. The energy and membrane lipid metabolisms of lotus seeds during 8 d at 25 ± 1 °C were evaluated. The results indicated that MT treatment suppressed the browning of lotus seeds through enhancing the endogenous MT content, while luzindole demonstrated an opposite effect. Compared with the control, the MT treatment ensured the energy status of lotus seeds, as the ATP content and energy charge were promoted. The activities of cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), and ATPases were enhanced by the treatment, which might contributued to the ATP production. Additionally, the cell membrane integrity of lotus seeds was maintained by MT treatment through reducing the cell membrane permeability, O2 ¯ production rate, and malondialdehyde and H2O2 contents, as well as enhancing the unsaturated-to-saturated fatty acids ratio, which could be explained by inhibited lipoxygenase (LOX), lipase (LPS) and phospholipase D (PLD) activities. Thus, the sufficient energy supply and normal cell membrane function may be responsible for the attenuated browning of lotus pods and seeds by MT treatment.

Phosphorylation of Serine 263 in the Human Choline Transporter 1 alters the transporter’s function

Disruptions of the neuromuscular junction result in congenital myasthenic syndromes (CMS). CMS is characterized by akinesia, apneas or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). The 13 transmembrane domain CHT1 protein is thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive substitution c.C788&gt;T in SLC5A7 in a family with a history of CMS using whole exome sequencing. This mutation encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether 1) the p.S263F substitution alters CHT1 phosphorylation state, and 2) a change in phosphorylation results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetic p.S263D and p.S263E. Upon expression in HEK293 cells, we found that p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non‐functional, suggesting that S263 phosphorylation inhibits choline transport. Immobilized metal affinity electrophoresis supports that wild‐type CHT1 may be more phosphorylated than p.S263F CHT1 mutant. These results support the essential role of Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status.Support or Funding InformationWomen and Children’s Health Research Institute, Stollery Children’s Hospital Foundation, Canadian Institute of Health Research &amp; The University of Alberta