BBA - General Subjects (v.1860, #7)

Central role of betaine–homocysteine S-methyltransferase 3 in chondral ossification and evidence for sub-functionalization in neoteleost fish by Joana Rosa; Daniel M. Tiago; Cátia L. Marques; Parameswaran Vijayakumar; Luis Fonseca; M. Leonor Cancela; Vincent Laizé (1373-1387).
To better understand the complex mechanisms of bone formation it is fundamental that genes central to signaling/regulatory pathways and matrix formation are identified. Cell systems were used to analyze genes differentially expressed during extracellular matrix mineralization and bhmt3, coding for a betaine–homocysteine S-methyltransferase, was shown to be down-regulated in mineralizing gilthead seabream cells.Levels and sites of bhmt3 expression were determined by qPCR and in situ hybridization throughout seabream development and in adult tissues. Transcriptional regulation of bhmt3 was assessed from the activity of promoter constructs controlling luciferase gene expression. Molecular phylogeny of vertebrate BHMT was determined from maximum likelihood analysis of available sequences. bhmt3 transcript is abundant in calcified tissues and localized in cartilaginous structures undergoing endo/perichondral ossification. Promoter activity is regulated by transcription factors involved in bone and cartilage development, further demonstrating the central role of Bhmt3 in chondrogenesis and/or osteogenesis. Molecular phylogeny revealed the explosive diversity of bhmt genes in neoteleost fish, while tissue distribution of bhmt genes in seabream suggested that neoteleostean Bhmt may have undergone several steps of sub-functionalization.Data on bhmt3 gene expression and promoter activity evidences a novel function for betaine–homocysteine S-methyltransferase in bone and cartilage development, while phylogenetic analysis provides new insights into the evolution of vertebrate BHMTs and suggests that multiple gene duplication events occurred in neoteleost fish lineage.High and specific expression of Bhmt3 in gilthead seabream calcified tissues suggests that bone-specific betaine–homocysteine S-methyltransferases could represent a suitable marker of chondral ossification.
Keywords: Gilthead seabream Sparus aurata; Betaine–homocysteine S-methyltransferase; Bone formation; In vitro mineralization; Molecular evolution; Gene expression; Transcriptional regulation; Taxonomic distribution;

New partners and phosphorylation sites of focal adhesion kinase identified by mass spectrometry by Maria del Mar Masdeu; Beatriz G. Armendáriz; Eduardo Soriano; Jesús Mariano Ureña; Ferran Burgaya (1388-1394).
The regulation of focal adhesion kinase (FAK) involves phosphorylation and multiple interactions with other signaling proteins. Some of these pathways are relevant for nervous system functions such as branching, axonal guidance, and plasticity. In this study, we screened mouse brain to identify FAK-interactive proteins and phosphorylatable residues as a first step to address the neuronal functions of this kinase. Using mass spectrometry analysis, we identified new phosphorylated sites (Thr 952, Thr 1048, and Ser 1049), which lie in the FAT domain; and putative new partners for FAK, which include cytoskeletal proteins such as drebrin and MAP 6, adhesion regulators such as neurabin-2 and plakophilin 1, and synapse-associated proteins such as SynGAP and a NMDA receptor subunit. Our findings support the participation of brain-localized FAK in neuronal plasticity.
Keywords: Focal adhesion kinase; Mass spectrometry; Brain; Synapse;

Ordered chimerogenesis applied to CYP2B P450 enzymes by Thomas Lautier; Philippe Urban; Jacqueline Loeper; Laetitia Jezequel; Denis Pompon; Gilles Truan (1395-1403).
Structural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11.A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast.Most of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F′/G′ cassette role and substrate access channels.Some sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity.The result provides a new basis for determining the structural elements that control functions in complex enzymes.Display Omitted
Keywords: P450; CYP2B6; CYP2B11; Chimera; Cyclophosphamide; Channel;

Ionization basis for activation of enzymes soluble in ionic liquids by Guangnan Ou; Biyan He; Peter Halling (1404-1408).
The complex interactions between electrolytes and proteins have been studied for more than a century. However, understanding is not yet complete and does not provide a basis for predicting the activity of enzymes in ionic media. The use of ionic liquids (ILs) as reaction medium has opened up new opportunities for better understanding of the mechanism of enzymatic catalysis. Although a number of properties of ILs have been correlated with enzyme function, these relationships are not completely understood at a molecular level.We propose that ILs must be able to promote ionization of protein ionizable groups in order to dissolve active enzymes. The biocompatible IL need to possess a functional group with large donor number and acceptor number in both cationic and anionic units, each of which is based on a high dielectric constant lead structure. We designed and synthesized two series of ILs and determined their ionizing–dissociating abilities and activities of lipases soluble in these new ILs.The results showed that the ionizing–dissociating abilities of ILs paralleled the catalytic activity trend of lipases dissolved in the ILs. The activities of lipases soluble in the newly designed ILs were comparable to those in water.We can conclude that ionizing–dissociating abilities of an IL can be used as a basis for predicting the activity of enzymes soluble in the IL.General significanceIonization basis for activation of enzymes gives a deeper understanding of the behavior of enzymes in non-aqueous media at a molecular level.
Keywords: Ionization; Activation; Enzyme; Ionic liquid;

Metmyoglobin (MbFeIII) reaction with H2O2 has been a subject of study over many years. H2O2 alone promotes heme destruction frequently denoted “suicide inactivation,” yet the mechanism underlying H2O2 dismutation associated with MbFeIII inactivation remains obscure.MbFeIII reaction with excess H2O2 in the absence and presence of the nitroxide was studied at pH 5.3–8.1 and 25 °C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H2O2 depletion, O2 evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy.The rates of both H2O2 dismutation and heme inactivation processes depend on [MbFeIII], [H2O2] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFeIII molecule catalyzes the dismutation of 50 ± 10 H2O2 molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFeIII while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFeIV =O by H2O2 and by nitroxide, respectively.The nitroxide effects on H2O2 dismutation catalyzed by MbFeIII demonstrate that MbFeIV =O reduction by H2O2 is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I–H2O2 adduct, which decomposes to a MbFeIV =O and an inactivated heme at a ratio of 25:1.The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.Display Omitted
Keywords: Heme protein; Suicide inactivation; Compound II; Nitroxide; Kinetics; Mechanism;

Rhamnetin induces sensitization of hepatocellular carcinoma cells to a small molecular kinase inhibitor or chemotherapeutic agents by Hui Jia; Qian Yang; Tao Wang; Yu Cao; Qi-yu Jiang; Hong-da Ma; Hui-wei Sun; Ming-xiao Hou; Yong-ping Yang; Fan Feng (1417-1430).
The rapid development of multi-drug resistance (MDR) process has hindered the effectiveness of advanced hepatocellular carcinoma (HCC) treatments. Notch-1 pathway, which mediates the stress-response, promotes cell survival, EMT (epithelial–mesenchymal transition) process and induces anti-apoptosis in cancer cells, would be a potential target for overcoming MDR process. This study investigated the potential application of rhamnetin, a specific inhibitor of Notch-1 pathway, in anti-tumor drug sensitization of HCC treatment.The expression of miR-34a, proteins belonging to Notch-1 signaling pathway or MDR-related proteins was detected by quantitative polymerase chain reaction (qPCR) and western blot assay. To identify whether rhamnetin induces the chemotherapeutic sensitization in HCC cells, the MTT-assays, flow cytometry, soft agar, trans-well and nude mice assays were performed.The endogenous expression of miR-34a was significantly increased and the expression of Notch-1 and Survivin was downregulated after rhamnetin treatment. Treatment of rhamnetin also reduced the expression of MDR related proteins P-GP (P-glycoprotein) and BCRP (breast cancer resistance protein). Rhamnetin increased the susceptibility of HCC cells and especially HepG2/ADR, a MDR HCC cell line, to a small molecular kinase inhibitor sorafenib or chemotherapeutic drugs etoposide and paclitaxel. The IC 50 value of those drugs correspondingly decreased.Together, our findings suggest that rhamnetin treatment may attenuate the MDR process in HCC cells. These findings may contribute to more effective strategies for HCC therapy.Rhamnetin acts as a promising sensitizer to chemotherapy and may be a novel approach to overcome the MDR process of HCC.
Keywords: HCC; MDR; Rhamnetin; MiR-34a; Notch-1 pathway; MDR-related proteins;

Uroguanylin modulates (Na+  + K+)ATPase in a proximal tubule cell line: Interactions among the cGMP/protein kinase G, cAMP/protein kinase A, and mTOR pathways by Francisco J. Arnaud-Batista; Diogo B. Peruchetti; Thiago P. Abreu; Nilberto R.F. do Nascimento; Gerhard Malnic; Manasses C. Fonteles; Celso Caruso-Neves (1431-1438).
The natriuretic effect of uroguanylin (UGN) involves reduction of proximal tubule (PT) sodium reabsorption. However, the target sodium transporters as well as the molecular mechanisms involved in these processes remain poorly understood.To address the effects of UGN on PT (Na+  + K+)ATPase and the signal transduction pathways involved in this effect, we used LLC-PK1 cells. The effects of UGN were determined through ouabain-sensitive ATP hydrolysis and immunoblotting assays during different experimental conditions.We observed that UGN triggers cGMP/PKG and cAMP/PKA pathways in a sequential way. The activation of PKA leads to the inhibition of mTORC2 activity, PKB phosphorylation at S473, PKB activity and, consequently, a decrease in the mTORC1/S6K pathway. The final effects are decreased expression of the α1 subunit of (Na+  + K+)ATPase and inhibition of enzyme activity.These results suggest that the molecular mechanism of action of UGN on sodium reabsorption in PT cells is more complex than previously thought. We propose that PKG-dependent activation of PKA leads to the inhibition of the mTORC2/PKB/mTORC1/S6K pathway, an important signaling pathway involved in the maintenance of the PT sodium pump expression and activity.The current results expand our understanding of the signal transduction pathways involved in the overall effect of UGN on renal sodium excretion.
Keywords: Uroguanylin; Proximal tubule; Sodium reabsorption; Sodium pump; Cell signaling; Kidney;

The cytotoxicity of garlic-related disulphides and thiosulfonates in WHCO1 oesophageal cancer cells is dependent on S-thiolation and not production of ROS by Muneerah Smith; Roger Hunter; Nashia Stellenboom; Daniel A. Kusza; M. Iqbal Parker; Ahmed N.H. Hammouda; Graham Jackson; Catherine H. Kaschula (1439-1449).
Garlic has been used for centuries in folk medicine for its health promoting and cancer preventative properties. The bioactive principles in crushed garlic are allyl sulphur compounds which are proposed to chemically react through (i) protein S-thiolation and (ii) production of ROS.A collection of R-propyl disulphide and R-thiosulfonate compounds were synthesised to probe the importance of thiolysis and ROS generation in the cytotoxicity of garlic-related compounds in WHCO1 oesophageal cancer cells.A significant correlation (R2  = 0.78, Fcrit (7,1) α = 0.005) was found between the cytotoxicity IC50 and the leaving group pKa of the R-propyl disulphides and thiosulfonates, supporting a mechanism that relies on the thermodynamics of a mixed disulphide exchange reaction. Disulphide (1) and thiosulfonate (11) were further evaluated mechanistically and found to induce G2/M cell-cycle arrest and apoptosis, inhibit cell proliferation, and generate ROS. When the ROS produced by 1 and 11 were quenched with Trolox, ascorbic acid or N-acetyl cysteine (NAC), only NAC was found to counter the cytotoxicity of both compounds. However, NAC was found to chemically react with 11 through mixed disulphide formation, providing an explanation for this apparent inhibitory result.Cellular S-thiolation by garlic related disulphides appears to be the cause of cytotoxicity in WHCO1 cells. Generation of ROS appears to only play a secondary role.Our findings do not support ROS production causing the cytotoxicity of garlic-related disulphides in WHCO1 cells. Importantly, it was found that the popular ROS inhibitor NAC interferes with the assay.

The spermatozoa undergo a series of changes in the epididymis to mature after their release from the testis and subsequently in the female reproductive tract after ejaculation to get capacitated and achieve fertilization potential. Despite having a silenced protein synthesis machinery, the dynamic change in protein profile of the spermatozoa is attributed either to acquisition of new proteins via vescicular transport or to several post-translational modifications (PTMs) occurring on the already expressed protein complement.In this review emphasis is given on the PTMs already reported on the human sperm proteins under normal and pathologic conditions with particular reference to sperm function such as motility and fertilization. An attempt has been made to summarize different protocols and methods used for analysis of PTMs on sperm proteins and the newer trends those were emerging.Deciphering the differential occurrence of PTM on protein at ultrastructural level would give us a better insight of structure-function relationship of the particular protein. Protein with multiple PTMs could be used to generate the complex interaction network involved in a physiological function of a sperm. It can be speculated that crosstalk between different PTMs occurring either on same/ other proteins actually regulate the protein stability and activity both in physiological and pathological states.The analytical prospective of various PTMs reported in human spermatozoa and their relevance to sperm function particularly in various pathophysiological states, would pave way for development of biomarkers for diagnosis, prognosis and therapeutic intervention of male infertility.Display Omitted
Keywords: Spermatozoa; Proteome; Post-translational modifications; Infertility; Male factor;

Smyd3 open & closed lock mechanism for substrate recruitment: The hinge motion of C-terminal domain inferred from μ-second molecular dynamics simulations by Balasubramanian Chandramouli; Valentina Silvestri; Marco Scarno; Laura Ottini; Giovanni Chillemi (1466-1474).
The human lysine methyltransferase Smyd3, a member of the SET and MYND domain containing protein family, harbors methylation activity on both histone and non-histone targets in a tightly regulated manner. The mechanism of how Smyd3 dynamically regulates substrate recognition is still not fully unveiled.Here, we employed molecular dynamics simulations on full length human Smyd3, performed to a total of 1.2 μ-second, in the presence (holo) and absence (apo) of the S-Adenosyl methionine (AdoMet) cofactor. The dynamical features of Smyd3 in apo and holo states have been examined and compared via examining geometrical and electrostatic properties.The results show a distinct dynamics of the C-terminal domain (CTD) in the two states. In the apo state, the CTD undergoes a large hinge like motion and samples more opened configurations, thus acting like a loosened clamp and resulting in expanded substrate binding crevice. In the holo state, the CTD exhibits a restricted motion while the overall structure remains compact, mimicking a closed clamp. This leads to a localized increase in the negative potential at the substrate binding cleft. Further, solvent accessibility of critical residues at the target lysine access channel, important for methylation activity, is increased.We postulate that AdoMet cofactor acts like a key and locks Smyd3 in a closed conformation. In effect, the cofactor binding restricts the elasticity of the CTD, presenting a compact substrate binding cleft with high negative potential, which may have implications on substrate recruitment via long range electrostatics.The deletion of the CTD from Smyd3 has been shown to abolish the basal histone methylation activity. Our study highlights the importance of the CTD elasticity in shaping the substrate binding site for recognition and supports the previously proposed role of the CTD in stabilizing the active site for methylation activity.Display Omitted
Keywords: Lysine methyltransferase; Smyd3; SET and MYND domain; CTD hinge motion; Histone methylation; AdoMet;

‘Traffic light rules’: Chromatin states direct miRNA-mediated network motifs running by integrating epigenome and regulatome by Hongying Zhao; Guanxiong Zhang; Lin Pang; Yujia Lan; Li Wang; Fulong Yu; Jing Hu; Feng Li; Tingting Zhao; Yun Xiao; Xia Li (1475-1488).
Epigenetic marks can cooperatively regulate chromatin accessibility and in turn facilitate or impede the binding of regulatory factors to various elements, suggesting their important roles in regulatory circuits. However, it remains elusive as to how epigenetic marks cooperate in the operations of regulatory network.Here, we systematically characterized chromatin states of 26 epigenetic marks on different elements of protein-coding genes and miRNAs. We comprehensively analyzed, by using an integrative regulatory network, how cooperation among epigenetic, transcriptional, and post-transcriptional regulations came about.We observed extensive cooperation of epigenetic marks on local functional elements and complex epigenetic patterns corresponding to different biological functions. By identifying the significantly epigenetic state-modified motifs, we found that multiple combinations of epigenetic states were associated with a specific type of motif. Interestingly, miRNA-mediated motifs were linked to stable epigenetic states of downstream targets. Changes in epigenetic states of downstream targets in miRNA-mediated motifs can buffer the effects of upstream regulator on target genes, suggesting that miRNA-mediated motifs require the cooperation of epigenetic marks.Overall, epigenetic marks are involved in the running of regulatory motifs in the way traffic lights control traffic flows and hence should be part of the architecture of complex regulatory circuits.We demonstrated a detailed analysis of the cooperation of multiple epigenetic marks and how epigenetic regulation was organized into a human regulatory network. The findings form a basis for further understanding of the complicated roles of epigenetic marks on regulatory circuits.
Keywords: miRNA; Chromatin states; miRNA-mediated network motifs; Epigenetic regulation; Transcriptional regulation; Post-transcriptional regulation;;

Tumor necrosis factor reduces Plasmodium falciparum growth and activates calcium signaling in human malaria parasites by Laura N. Cruz; Yang Wu; Henning Ulrich; Alister G. Craig; Célia R.S. Garcia (1489-1497).
Plasmodium has a complex biology including the ability to interact with host signals modulating their function through cellular machinery. Tumor necrosis factor (TNF) elicits diverse cellular responses including effects in malarial pathology and increased infected erythrocyte cytoadherence. As TNF levels are raised during Plasmodium falciparum infection we have investigated whether it has an effect on the parasite asexual stage.Flow cytometry, spectrofluorimetric determinations, confocal microscopy and PCR real time quantifications were employed for characterizing TNF induced effects and membrane integrity verified by wheat germ agglutinin staining.TNF is able to decrease intracellular parasitemia, involving calcium as a second messenger of the pathway. Parasites incubated for 48 h with TNF showed reduced erythrocyte invasion. Thus, TNF induced rises in intracellular calcium concentration, which were blocked by prior addition of the purinergic receptor agonists KN62 and A438079, or interfering with intra- or extracellular calcium release by thapsigargin or EGTA (ethylene glycol tetraacetic acid). Importantly, expression of PfPCNA1 which encodes the Plasmodium falciparum Proliferating-Cell Nuclear Antigen 1, decreased after P. falciparum treatment of TNF (tumor necrosis factor) or 6-Bnz cAMP (N6-benzoyladenosine-3′,5′-cyclic monophosphate sodium salt).This is potentially interesting data showing the relevance of calcium in downregulating a gene involved in cellular proliferation, triggered by TNF.The data show that Plasmodium may subvert the immunological system and use TNF for the control of its proliferation within the vertebrate host.
Keywords: Malaria; Cytoadhesion; Tumor necrosis factor; Plasmodium falciparum; Calcium signaling; Proliferating cell nuclear antigen-1;

FRET reveals multiple interaction states between two component signalling system proteins of M. tuberculosis by Ruchi Agrawal; Prem Kumar V.; Harini Ramanan; Deepak Kumar Saini (1498-1507).
Two component signalling involves interaction between sensor kinase (SK) and response regulator (RR) proteins which depends on their phosphorylation status.In this study we report the development of an in vitro FRET assay for studying interaction between fluorescently tagged SK and RR proteins.Using TCS proteins of Mycobacterium tuberculosis, we demonstrate that phosphorylation status of SK affects the SK–RR interaction, which varies from one TCS to another. The observation was strengthened by recordings from mutant SK and RR proteins. The assay retained the specificity/crosstalk potential of the participating proteins and reflected the inherent phosphotransfer potentials.SK and RR proteins interact with each other in unphosphorylated state and the phosphorylation affects the interaction between SK and RR, which was reflected as reduction in FRET ratio.A non-radioactive, in vitro FRET based assay is reported, which can be utilized for studying genome-wide partner screening, identifying crosstalk or specificity in TCSs.Display Omitted
Keywords: Two component signalling; Sensor kinase; Response regulator; Crosstalk; FRET;

Structural investigation and inhibitory response of halide on phosphoserine aminotransferase from Trichomonas vaginalis by Rohit Kumar Singh; Mohit Mazumder; Bhumika Sharma; Samudrala Gourinath (1508-1518).
Phosphoserine aminotransferase (PSAT) catalyses the second reversible step of the phosphoserine biosynthetic pathway in Trichomonas vaginalis, which is crucial for the synthesis of serine and cysteine.PSAT from T. vaginalis (TvPSAT) was analysed using X-ray crystallography, enzyme kinetics, and molecular dynamics simulations.The crystal structure of TvPSAT was determined to 2.15 Å resolution, and is the first protozoan PSAT structure to be reported. The active site of TvPSAT structure was found to be in a closed conformation, and at the active site PLP formed an internal aldimine linkage to Lys 202. In TvPSAT, Val 340 near the active site while it is Arg in most other members of the PSAT family, might be responsible in closing the active site. Kinetic studies yielded Km values of 54 μM and 202 μM for TvPSAT with OPLS and AKG, respectively. Only iodine inhibited the TvPSAT activity while smaller halides could not inhibit.Results from the structure, comparative molecular dynamics simulations, and the inhibition studies suggest that iodine is the only halide that can bind TvPSAT strongly and may thus inhibit the activity of TvPSAT.The long loop between β8 and α8 at the opening of the TvPSAT active site cleft compared to other PSATs, suggests that this loop may help control the access of substrates to the TvPSAT active site and thus influences the enzyme kinetics.Our structural and functional studies have improved our understanding of how PSAT helps this organism persists in the environment.
Keywords: Serine pathway; Phosphoserine aminotransferase; Structure; Enzyme kinetics; Inhibition by halides; Molecular dynamics simulation;

Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells by Ashutosh Kumar; Marilyn Ehrenshaft; Erik J. Tokar; Ronald P. Mason; Birandra K. Sinha (1519-1527).
Etoposide and doxorubicin, topoisomerase II poisons, are important drugs for the treatment of tumors in the clinic. Topoisomerases contain several free sulfhydryl groups which are important for their activity and are also potential targets for nitric oxide (NO)-induced nitrosation. NO, a physiological signaling molecule nitrosates many cellular proteins, causing altered protein and cellular functions.Here, we have evaluated the roles of NO/NO-derived species in the activity/stability of topo II both in vitro and in human tumor cells, and in the cytotoxicity of topo II-poisons, etoposide and doxorubicin.Treatment of purified topo IIα with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of both the catalytic and relaxation activity in vitro, and decreased etoposide-dependent cleavable complex formation in both human HT-29 colon and MCF-7 breast cancer cells. PPNO treatment also induced significant nitrosation of topo IIα protein in these human tumor cells. These events, taken together, caused a significant resistance to etoposide in both cell lines. However, PPNO had no effect on doxorubicin-induced cleavable complex formation, or doxorubicin cytotoxicity in these cell lines.Inhibition of topo II function by NO/NO-derived species induces significant resistance to etoposide, without affecting doxorubicin cytotoxicity in human tumor cells.As tumors express inducible nitric oxide synthase and generate significant amounts of NO, modulation of topo II functions by NO/NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.
Keywords: Nitric oxide; Topoisomerase II; VP-16; Doxorubicin; Cytotoxicity; Resistance;

Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity by Kadiyala Gopi; K. Anbarasu; Kadali Renu; S. Jayanthi; B.S. Vishwanath; Gurunathan Jayaraman (1528-1540).
The plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study. E. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC–MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico.The bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A2, hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to 107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom.For the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity.Exploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available.
Keywords: Euphorbia hirta; Snake venom; Naja naja; Quercetin-3-O-rhamnoside; PLA2;

An in vitro evaluation of epigallocatechin gallate (eGCG) as a biocompatible inhibitor of ricin toxin by Paul D.R. Dyer; Arun K. Kotha; Alex S. Gollings; Susan A. Shorter; Thomas R. Shepherd; Marie W. Pettit; Bruce D. Alexander; Giulia T.M. Getti; Samer El-Daher; Les Baillie; Simon C.W. Richardson (1541-1550).
The catechin, epigallocatechin gallate (eGCG), found in green tea, has inhibitory activity against a number of protein toxins and was investigated in relation to its impact upon ricin toxin (RT) in vitro. The IC50 for RT was 0.08 ± 0.004 ng/mL whereas the IC50 for RT + 100 μM eGCG was 3.02 ± 0.572 ng/mL, indicating that eGCG mediated a significant (p < 0.0001) reduction in ricin toxicity. This experiment was repeated in the human macrophage cell line THP-1 and IC50 values were obtained for RT (0.54 ± 0.024 ng/mL) and RT + 100 μM eGCG (0.68 ± 0.235 ng/mL) again using 100 μM eGCG and was significant (p = 0.0013). The documented reduction in ricin toxicity mediated by eGCG was found to be eGCG concentration dependent, with 80 and 100 μg/mL (i.e. 178 and 223 μM respectively) of eGCG mediating a significant (p = 0.0472 and 0.0232) reduction in ricin toxicity at 20 and 4 ng/ml of RT in Vero and THP-1 cells (respectively). When viability was measured in THP-1 cells by propidium iodide exclusion (as opposed to the MTT assays used previously) 10 ng/mL and 5 ng/mL of RT was used. The addition of 1000 μM and 100 μM eGCG mediated a significant (p = 0.0015 and < 0.0001 respectively) reduction in ricin toxicity relative to an identical concentration of ricin with 1 μg eGCG. Further, eGCG (100 μM) was found to reduce the binding of RT B chain to lactose-conjugated Sepharose as well as significantly (p = 0.0039) reduce the uptake of RT B chain in Vero cells. This data suggests that eGCG may provide a starting point to refine biocompatible substances that can reduce the lethality of ricin.
Keywords: Ricin toxin; Endocytosis; Polyphenol; Epigallocatechin gallate; eGCG; Tea;

Dermal fibroblasts activated by conductive polymer-mediated electrical stimulation (ES) have shown myofibroblast characteristics that favor wound healing. However, the signaling pathway related to this phenotype switch remains unclear, and the in vivo survival of the electrically activated cells has never been studied.Primary human skin fibroblasts were exposed to pulsed-ES mediated through polypyrrole (PPy) coated fabrics. The expression of α-smooth muscle actin (α-SMA) and the signaling pathways were investigated by ELISA, Western blot and specific inhibition test, and immunocytochemistry staining as well as qRT-PCR analysis. In vivo implantation was performed in a mouse model to clarify the cell fate or contractile phenotype maintenance following ES stimulation.We demonstrated the upregulation of TGFβ1 and phosph-ERK, and the NF-κB nuclear enrichment in the ES-activated cells. The ES-activated fibroblasts retained high level of α-smooth muscle actin expression even after prolonged subculture. Subcutaneous implantation for 15 days revealed more human myofibroblasts in the experimental groups.These findings demonstrate for the first time the involvement of the TGFβ1/ERK/NF-κB signaling pathway in ES-activated fibroblasts. The ES induced phenotype switch proves stable in subculture and in animal, pointing potential application in wound healing.Reveal of how ES activates cells and the implication of ES activated cells in wound healing.
Keywords: Wound healing; Fibroblasts; Electrical stimulation; Signaling; ERK1/2; Conductive polymer;

Neurofilament networks: Salt-responsive hydrogels with sidearm-dependent phase behavior by Joanna Deek; Peter J. Chung; Cyrus R. Safinya (1560-1569).
Neurofilaments (NFs) — the neuron-specific intermediate filament proteins — are assembled into 10 nm wide filaments in a tightly controlled ratio of three different monomer types: NF-Low (NF-L), NF-Medium (NF-M), and NF-High (NF-H). Previous work on reconstituted bovine NF hydrogels has shown the dependence of network properties, including filament alignment and spacing, on the subunit composition.We use polarized optical microscopy and SAXS to explore the full salt-dependent phase behavior of reconstituted bovine NF networks as a function of various binary and ternary subunit ratios.We observe three salt-induced liquid crystalline phases: the liquid-ordered BG and NG phases, and the disordered IG phase. We note the emergent sidearm roles, particularly that of NF-H in driving the parallel to cross-filament transition, and the counter-role of NF-M in suppressing the IG phase.In copolymers of NF-LH, NF-H shifts the IG to NG transition to nearer physiological salt concentrations, as compared to NF-M in copolymers of NF-LM. For ternary mixtures, the role of NF-H is modulated by the ratio of NF-M, where beneath 10 wt.% NF-M, NF-H drives the transition to the disordered phase, and above which NF-H increases interfilament spacing.Understanding the role of individual subunits in regulating the network structure will enable us to understand the mechanisms that drive the dysfunction of these networks, as observed in diseased conditions.Display Omitted
Keywords: Neurofilament; Network; Liquid crystal; Hydrogel; Sidearm-dependent; Salt-responsive;