BBA - General Subjects (v.1840, #6)

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies by Yuki Kawakami; Shiori Hirano; Mai Kinoshita; Akemi Otsuki; Toshiko Suzuki-Yamamoto; Makiko Suzuki; Masumi Kimoto; Sae Sasabe; Mitsuo Fukushima; Koji Kishimoto; Takashi Izumi; Toru Oga; Shuh Narumiya; Mitsuaki Sugahara; Masashi Miyano; Shozo Yamamoto; Yoshitaka Takahashi (1625-1633).
Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC4 (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC4 and LTD4 by competing their binding to their receptors.mAbLTC and scFvLTC inhibited their binding of LTC4 or LTD4 to CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R) overexpressed in Chinese hamster ovary cells. The induction by LTD4 of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT1R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC4- and LTD4-induced aggregation of mouse platelets expressing CysLT2R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT2R antagonists but not to CysLT1R antagonists.These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT1R and CysLT2R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.mAbLTC can be used in the treatment of inflammatory diseases such as asthma.
Keywords: Arachidonic acid; CysLT1 receptor; CysLT2 receptor; Cysteinyl leukotriene; Monoclonal antibody; Single-chain variable fragment;

Cancer cells have extremely active metabolism, which supports high proliferation rates. Metabolic profiles of human colon cancer cells have been extensively studied, but comparison with non-tumour counterparts has been neglected.Here we compared the metabolic flux redistribution in human colon adenocarcinoma cells (HT29) and the human colon healthy cell line NCM460 in order to identify the main pathways involved in metabolic reprogramming. Moreover, we explore if induction of differentiation in HT29 by trichostatin A (TSA) reverts the metabolic reprogramming to that of NCM460. Cells were incubated with [1,2-13C2]-d-glucose as a tracer, and Mass Isotopomer Distribution Analysis was applied to characterize the changes in the metabolic flux distribution profile of the central carbon metabolism.We demonstrate that glycolytic rate and pentose phosphate synthesis are 25% lower in NCM460 with respect to HT29 cells. In contrast, Krebs cycle activity in the former was twice that recorded in the latter. Moreover, we show that TSA-induced HT29 cell differentiation reverts the metabolic phenotype to that of healthy NCM460 cells whereas TSA does not affect the metabolism of NCM460 cells.We conclude that pentose phosphate pathway, glycolysis, and Krebs cycle are key players of colon adenocarcinoma cellular metabolic remodeling and that NCM460 is an appropriate model to evaluate the results of new therapeutic strategies aiming to selectively target metabolic reprogramming.Our findings suggest that strategies to counteract robust metabolic adaptation in cancer cells might open up new avenues to design multiple hit and targeted therapies.
Keywords: Colon cancer; Glycolysis; Metabolic adaptation; Metabolic profile; Tumour metabolism; Trichostatin-A;

Glutamate transporter type 3 regulates mouse hippocampal GluR1 trafficking by Jiangbei Cao; Hongying Tan; Weidong Mi; Zhiyi Zuo (1640-1645).
Rapid trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) to the plasma membrane is considered a fundamental biological process for learning and memory. GluR1 is an AMPAR subunit. We have shown that mice with knockout of excitatory amino acid transporter type 3 (EAAT3), a neuronal glutamate transporter, have impaired learning and memory. The mechanisms for this impairment are not known and may be via regulation of AMPAR trafficking.Freshly prepared 300 μm coronal hippocampal slices from wild-type or EAAT3 knockout mice were incubated with or without 25 mM tetraethylammonium for 10 min. The trafficking of GluR1, an AMPAR subunit, to the plasma membrane and its phosphorylation were measured.Tetraethylammonium increased the trafficking of GluR1 and EAAT3 to the plasma membrane in the wild-type mouse hippocampal slices but did not cause GluR1 trafficking in the EAAT3 knockout mice. Tetraethylammonium also increased the phosphorylation of GluR1 at S845, a protein kinase A (PKA) site, in the wild-type mice but not in the EAAT3 knockout mice. The PKA antagonist KT5720 attenuated tetraethylammonium-induced GluR1 phosphorylation and trafficking in the wild-type mice. The PKA agonist 6-BNz-cAMP caused GluR1 trafficking to the plasma membrane in the EAAT3 knockout mice. In addition, EAAT3 was co-immunoprecipitated with PKA.These results suggest that EAAT3 is upstream of PKA in a pathway to regulate GluR1 trafficking.Our results provide initial evidence for the involvement of EAAT3 in the biochemical cascade of learning and memory.
Keywords: α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid receptor; GluR1; Glutamate transporters type 3; Trafficking; Mice;

Regulation of the yeast trehalose–synthase complex by cyclic AMP-dependent phosphorylation by Eduardo T.V. Trevisol; Anita D. Panek; Joelma F. De Mesquita; Elis C.A. Eleutherio (1646-1650).
Trehalose is an important protectant in several microorganisms. In Saccharomyces cerevisiae, it is synthesized by a large complex comprising the enzymes Tps1 and Tps2 and the subunits Tps3 and Tsl1, showing an intricate metabolic control.To investigate how the trehalose biosynthesis pathway is regulated, we analyzed Tps1 and Tps2 activities as well as trehalose and trehalose-6-phosphate (T6P) contents by mass spectrometry.Tsl1 deficiency totally abolished the increase in Tps1 activity and accumulation of trehalose in response to a heat stress, whereas absence of Tps3 only reduced Tps1 activity and trehalose synthesis. In extracts of heat stressed cells, Tps1 was inhibited by T6P and by ATP. Mg2 + in the presence of cAMP. In contrast, cAMP-dependent phosphorylation did not inhibit Tps1 in tps3 cells, which accumulated a higher proportion of T6P after stress. Tps2 activity was not induced in a tps3 mutant.Taken together these results suggest that Tsl1 is a decisive subunit for activity of the TPS complex since in its absence no trehalose synthesis occurred. On the other hand, Tps3 seems to be an activator of Tps2. To perform this task, Tps3 must be non-phosphorylated. To readily stop trehalose synthesis during stress recovery, Tps3 must be phosphorylated by cAMP-dependent protein kinase, decreasing Tps2 activity and, consequently, increasing the concentration of T6P which would inhibit Tps1.A better understanding of TPS complex regulation is essential for understanding how yeast deals with stress situations and how it is able to recover when the stress is over.
Keywords: Trehalose; Saccharomyces cerevisiae; Stress response; Phosphorylation; Ras/cAPK pathway;

Comparison of quantum dot-binding protein tags: Affinity determination by ultracentrifugation and FRET by Mara Werwie; Niklas Fehr; Xiangxing Xu; Thomas Basché; Harald Paulsen (1651-1656).
Hybrid complexes of proteins and colloidal semiconductor nanocrystals (quantum dots, QDs) are of increasing interest in various fields of biochemistry and biomedicine, for instance for biolabeling or drug transport. The usefulness of protein–QD complexes for such applications is dependent on the binding specificity and strength of the components. Often the binding properties of these components are difficult and time consuming to assess.In this work we characterized the interaction between recombinant light harvesting chlorophyll a/b complex (LHCII) and CdTe/CdSe/ZnS QDs by using ultracentrifugation and fluorescence resonance energy transfer (FRET) assay experiments. Ultracentrifugation was employed as a fast method to compare the binding strength between different protein tags and the QDs. Furthermore the LHCII:QD stoichiometry was determined by separating the protein–QD hybrid complexes from unbound LHCII via ultracentrifugation through a sucrose cushion.One trimeric LHCII was found to be bound per QD. Binding constants were evaluated by FRET assays of protein derivatives carrying different affinity tags. A new tetra-cysteine motif interacted more strongly (Ka  = 4.9 ± 1.9 nM− 1) with the nanoparticles as compared to a hexahistidine tag (His6 tag) (Ka  ~ 1 nM− 1).Relative binding affinities and binding stoichiometries of hybrid complexes from LHCII and quantum dots were identified via fast ultracentrifugation, and binding constants were determined via FRET assays.The combination of rapid centrifugation and fluorescence-based titration will be useful to assess the binding strength between different types of nanoparticles and a broad range of proteins.
Keywords: Nanoparticle; Protein; Stoichiometry; Ultracentrifugation; Fluorescence resonance energy transfer (FRET); Light-harvesting complex II (LHCII);

Type 1 ribotoxin-curcin conjugated biogenic gold nanoparticles for a multimodal therapeutic approach towards brain cancer by M. Sheikh Mohamed; Srivani Veeranarayanan; Aby Cheruvathoor Poulose; Yutaka Nagaoka; Hiroaki Minegishi; Yasuhiko Yoshida; Toru Maekawa; D. Sakthi Kumar (1657-1669).
Gliomas have been termed recurrent cancers due to their highly aggressive nature. Their tendency to infiltrate and metastasize has posed significant roadblocks to in attaining fool proof treatment solutions. An initiative to curb such a scenario was successfully demonstrated in vitro, utilizing a multi-conceptual gold nanoparticle based photo-thermal and drug combination therapy.Gold nanoparticles (Au NPs) were synthesized with a highly environmentally benign process. The Au NPs were PEGylated and conjugated with folate and transferrin antibody to achieve a dual targeted nano-formulation directed towards gliomas. Curcin, a type 1 ribosome inactivating protein, was attached to the Au NPs as the drug candidate, and its multifarious toxic aspects analyzed in vitro. NIR photo-thermal properties of the Au nano-conjugates were studied to selectively ablate the glioma cancer colonies.Highly cyto-compatible, 10–15 nm Au NP conjugates were synthesized with pronounced specificity towards gliomas. Curcin was successfully conjugated to the Au NPs with pH responsive drug release. Prominent toxic aspects of curcin, such as ROS generation, mitochondrial and cytoskeletal destabilization were witnessed. Excellent photo-thermal ablation properties of gold nanoparticles were utilized to completely disrupt the cancer colonies with significant precision.The multifunctional nanoconjugate projects its competence in imparting complete arrest of the future proliferation or migration of the cancer mass.With multifunctionality the essence of nanomedicine in recent years, the present nanoconjugate highlights itself as a viable option for a multimodal treatment option for brain cancers and the like.Multifunctional, dual targeted Au NP based cancer cell destruction by the synergistic action of photo-thermal and pH-responsive drug effects.Display Omitted
Keywords: Gold nanoparticle; Curcin; Glioma; Photo-thermal ablation; Dual targeting;

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid by Hussein Kaddour; Jacques Vergne; Guy Herve; Marie-Christine Maurel (1670-1675).
Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication. In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors.Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with Ki of 17 ± 5 μM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with Ki of 32 ± 5 μM and IC50 of 177 ± 5 nM, respectively.This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups.This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.
Keywords: RNA; Viroid; Ribozyme; Polyamine; Chrysanthemum;

OsRPK1, a novel leucine-rich repeat receptor-like kinase, negatively regulates polar auxin transport and root development in rice by Yu Zou; Xiaoyu Liu; Qing Wang; Yu Chen; Cheng Liu; Yang Qiu; Wei Zhang (1676-1685).
Leucine-rich-repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of putative RLKs in plants. Although several members in this subfamily have been identified, the studies about the relationships between LRR-RLKs and root development are still few. We previously identified a novel LRR-RLK in rice roots, and named it OsRPK1.In this study, we first detected OsRPK1 kinase activity in vitro, and assessed its expression profile. We then investigated its biological function using transgenic rice plants over- and under-expressing OsRPK1.The OsRPK1 gene, which encodes a Ca2 +-independent Ser/Thr kinase, was predominantly expressed in root tips, leaf blades, and undifferentiated suspension cells, and was markedly induced by treatment with auxin or ABA. Knockdown of OsRPK1 promoted the growth of transgenic rice plants, and increased plant height and tiller numbers. In contrast, over-expressing plants showed undeveloped adventitious roots, lateral roots, and a reduced root apical meristem. OsRPK1 over-expression also inhibited the expression of most auxin efflux carrier OsPIN genes, which was accompanied by changes in PAT and endogenous free IAA distribution in the leaves and roots.The data indicated that OsRPK1, a novel leucine-rich-repeat receptor-like kinase, affects the root system architecture by negatively regulating polar auxin transport in rice.This study demonstrated a common regulatory pathway of root system development in higher plants, which might be initiated by external stimuli via upstream receptor-like kinases and downstream carriers for polar auxin transport.
Keywords: Leucine-rich-repeat receptor-like kinase; Oryza sativa L; PIN-FORMED family; Polar auxin transport; Lateral roots; Adventitious roots;

PEP-1-PEA-15 protects against toxin-induced neuronal damage in a mouse model of Parkinson's disease by Eun Hee Ahn; Dae Won Kim; Min Jea Shin; Hye Ri Kim; So Mi Kim; Su Jung Woo; Seon Ae Eom; Hyo Sang Jo; Duk-Soo Kim; Sung-Woo Cho; Jinseu Park; Won Sik Eum; Soo Young Choi (1686-1700).
PEA-15 is abundantly expressed in both neurons and astrocytes throughout the brain. It is a multifunctional protein with the ability to increase cell survival via anti-apoptotic and anti-proliferative properties. However, the function of PEA-15 in neuronal diseases such as Parkinson's disease (PD) remains unclear. In this study, we investigated the protective effects of PEA-15 on neuronal damage induced by MPP+ in neuroblastoma SH-SY5Y and BV2 microglia cells and in a MPTP-induced PD mouse model using cell-permeable PEP-1-PEA-15.PEP-1-PEA-15 was purified using affinity chromatography. Cell viability and DNA fragmentation were examined by MTT assay and TUNEL staining. Dopaminergic neuronal cell death in the animal model was examined by immunohistochemistry.PEP-1-PEA-15 transduced into the SH-SY5Y and BV2 cells in a time- and dose-dependent manner. Transduced PEP-1-PEA-15 protected against MPP+-induced toxicity by inhibiting intracellular ROS levels and DNA fragmentation. Further, it enhanced the expression levels of Bcl-2 and caspase-3 while reducing the expression levels of Bax and cleaved caspase-3. We found that PEP-1-PEA-15 transduced into the substantia nigra and prevented dopaminergic neuronal cell death in a MPTP-induced PD mouse. Also, we showed the neuroprotective effects in the model by demonstrating that treatment with PEP-1-PEA-15 ameliorated MPTP-induced behavioral dysfunctions and increased dopamine levels in the striatum.PEP-1-PEA-15 can efficiently transduce into cells and protects against neurotoxin-induced neuronal cell death in vitro and in vivo.These results demonstrate the potential for PEP-1-PEA-15 to provide a new strategy for protein therapy treatment of a variety of neurodegenerative diseases including PD.
Keywords: Oxidative stress; Neurotoxin; Parkinson's disease; PEP-1-Phosphoprotein enriched in astrocytes 15; Protein therapy;

Cloning, characterization, and expression of glucose transporter 2 in the freeze-tolerant wood frog, Rana sylvatica by Andrew J. Rosendale; Benjamin N. Philip; Richard E. Lee; Jon P. Costanzo (1701-1711).
The essential role of glucose transporter 2 (GLUT2) in glucose homeostasis has been extensively studied in mammals; however, little is known about this important protein in lower vertebrates. The freeze-tolerant wood frog (Rana sylvatica), which copiously mobilizes glucose in response to freezing, represents an excellent system for the study of glucose transport in amphibians.GLUT2 was sequenced from northern and southern phenotypes of R. sylvatica, as well as the freeze-intolerant Rana pipiens. These proteins were expressed and functionally characterized in Xenopus oocytes. Abundance of GLUT2 in tissues was analyzed using immunoblotting techniques.GLUT2s cloned from these anurans encoded proteins with high sequence homologies to known vertebrate GLUT2s and had similar transport properties, although, notably, transport of the glucose analog 3-O-methyl-d-glucose (3-OMG) was strongly inhibited by 150 mM urea. Proteins from all study subjects had similar affinity constants (~ 12 mM) and other kinetic properties; however, GLUT2 abundance in liver was 3.5-fold greater in northern R. sylvatica than in the southern conspecific and R. pipiens.Our results indicate that amphibian GLUT2s are structurally and functionally similar to their homologs in other vertebrates, attesting to the conserved nature of this transport protein. The greater abundance of this protein in the northern phenotype of R. sylvatica suggests that these transporters contribute importantly to freezing survival.This study provides the first functional characterization of any GLUT isoform from an anuran amphibian and novel insights into the role of these proteins in glucose homeostasis and cryoprotectant mobilization in freeze-tolerant vertebrates.
Keywords: Anuran; Cryoprotectant; Ecogeography freeze tolerance; Glucose transport;

Deciphering metal-induced oxidative damages on glycated albumin structure and function by Jennifer Baraka-Vidot; Giovanna Navarra; Maurizio Leone; Emmanuel Bourdon; Valeria Militello; Philippe Rondeau (1712-1724).
Metal ions such as copper or zinc are involved in the development of neurodegenerative pathologies and metabolic diseases such as diabetes mellitus. Albumin structure and functions are impaired following metal- and glucose-mediated oxidative alterations. The aim of this study was to elucidate effects of Cu(II) and Zn(II) ions on glucose-induced modifications in albumin by focusing on glycation, aggregation, oxidation and functional aspects.Aggregation and conformational changes in albumin were monitored by spectroscopy, fluorescence and microscopy techniques. Biochemical assays such as carbonyl, thiol groups, albumin-bound Cu, fructosamine and amine group measurements were used. Cellular assays were used to gain functional information concerning antioxidant activity of oxidized albumins.Both metals promoted inhibition of albumin glycation associated with an enhanced aggregation and oxidation process. Metal ions gave rise to the formation of β-amyloid type aggregates in albumin exhibiting impaired antioxidant properties and toxic activity to murine microglia cells (BV2). The differential efficiency of both metal ions to inhibit albumin glycation, to promote aggregation and to affect cellular physiology is compared.Considering the key role of oxidized protein in pathology complications, glycation-mediated and metal ion-induced impairment of albumin properties might be important parameters to be followed and fought.
Keywords: Glycation; Albumin; Protein aggregation; Spectroscopy; Light scattering; Metal;

DNase antibodies can play an important role in the pathogenesis of different autoimmune pathologies.An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with systemic lupus erythematosus (SLE) was used. The small pools of phage particles displaying DNA binding light chains with different for DNA were isolated by affinity chromatography on DNA-cellulose and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Forty-five of 451 individual colonies were randomly chosen for a study of MLChs with DNase activity. The clones were expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography followed by gel filtration, and studied in detail.Fifteen of 45 MLChs efficiently hydrolyzed DNA, and fourteen of them demonstrated various optimal concentrations of KCl or NaCl in a 1–100 mM range and showed one or two pH optima in a 4.8–9.1 range. All MLChs were dependent on divalent metal cations: the ratio of relative DNase activity in the presence of Mn2 +, Ca2 +, Mg2 +, Ni2 +, Zn2 +, Cu2 +, and Co2 + was individual for each MLCh preparation. Fourteen MLChs demonstrated a comparable affinity for DNA (260–320 nM), but different k cat values (0.02–0.7 min− 1).These observations suggest an extreme diversity of DNase abzymes from SLE patients.SLE light chain repertoire can serve as a source of new types of DNases.An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Fourteen individual DNase monoclonal light chains (MLChs) were purified by metal chelating chromatography followed by gel filtration. The MLChs demonstrated 1 or 2 pH optima and were inactive after dialysis against EDTA, but could be activated by several externally added metal ions; the ratio of relative activity in the presence of different metal ions; Mg2, mN2 +, and cO2 + being among the best activators of their DNase activity. The DNase activity of some MLChs did not require K+ or Na+, while the others could be activated by KCl or NaCl at optimal concentrations from 1 to 100 mM.Display Omitted
Keywords: Systemic lupus erythematosus; Extreme diversity; Recombinant monoclonal light chain; DNase activity;

Antarease-like Zn-metalloproteases are ubiquitous in the venom of different scorpion genera by Ernesto Ortiz; Martha Rendón-Anaya; Solange Cristina Rego; Elisabeth Ferroni Schwartz; Lourival Domingos Possani (1738-1746).
The venoms of several scorpion species have long been associated with pancreatitis in animal models and humans. Antarease, a Zn-metalloprotease from Tityus serrulatus, is able to penetrate intact pancreatic tissue and disrupts the normal vesicular traffic necessary for secretion, so it could play a relevant role in the onset of acute pancreatitis.The cDNA libraries from five different scorpion species were screened for antarease homologs with specific primers. The amplified PCR products were cloned and sequenced. A structural model was constructed to assess the functionality of the putative metalloproteases. A phylogenetic analysis was performed to identify clustering patterns of these venom components.Antarease-like sequences were amplified from all the screened cDNA libraries. The complete sequence of the antarease from T. serrulatus was obtained. The structural model of the putative antarease from Tityus trivittatus shows that it may adopt a catalytically active conformation, sharing relevant structural elements with previously reported metalloproteases of the ADAM family. The phylogenetic analysis reveals that the reported sequences cluster in groups that correlate with the geographical localization of the respective species.Antareases are ubiquitous to a broad range of scorpion species, where they could be catalytically active enzymes. These molecules can be used to describe the evolution of scorpion venoms under different ecogeographic constrains.For the first time the complete sequence of the antareases is reported. It is demonstrated that antareases are common in the venom of different scorpion species. They are now proposed as targets for antivenom therapies.
Keywords: Antarease; cDNA library; Pancreatitis; Scorpion; Zn-metalloprotease;

Nrf2 regulates the expression of the peptide transporter PEPT1 in the human colon carcinoma cell line Caco-2 by Kerstin E. Geillinger; Anna P. Kipp; Kristin Schink; Pia V. Röder; Britta Spanier; Hannelore Daniel (1747-1754).
PEPT1 is a rheogenic transport protein in the apical membrane of intestinal epithelial cells capable of transporting essentially all possible di- and tripeptides that are generated from the luminal protein breakdown. In addition, several anticancer, antimicrobial and antiviral drugs are taken up from the intestinal lumen via PEPT1 and therefore PEPT1 is a target for efficient drug delivery via prodrug approaches. Thus, understanding PEPT1 gene regulation is not only of importance for dietary adaptation but also for drug treatment.In silico analysis of the Pept1 promoter was performed using MatInspector. Pept1 promoter constructs were generated and cotransfected with an Nrf2 expression plasmid. Caco-2 cells were stimulated with Nrf2 inducers followed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Biological relevance was investigated using western blot analysis and transport activity assays.Reporter gene assays showed transcriptional activation of the Pept1 promoter in response to Nrf2 overexpression. EMSA as well as ChIP analysis validated Nrf2 binding to the ARE located closest to the start codon (Pept1-ARE1). Induction of the Nrf2 pathway resulted in increased endogenous PEPT1 protein abundance as well as transport activity. Moreover, we demonstrate that also the induction of autophagy by MG132 resulted in elevated Nrf2 binding to Pept1-ARE1 and increased PEPT1 protein expression.In summary, we identified a biologically active Nrf2 binding site within the Pept1 promoter which links Pept1 to the cellular defense program activated by Nrf2.This study identifies Pept1 as an inducible target gene of the Nrf2 pathway.Display Omitted
Keywords: MG132; ARE; Transcription; Resveratrol; Sulforaphane; PEPT1;

Glycyrrhizin inhibits lipopolysaccharide-induced inflammatory response by reducing TLR4 recruitment into lipid rafts in RAW264.7 cells by Yunhe Fu; Ershun Zhou; Zhengkai Wei; Xiaojing Song; Zhicheng Liu; Tiancheng Wang; Wei Wang; Naisheng Zhang; Guowen Liu; Zhengtao Yang (1755-1764).
The aim of this study was to investigate the effect of glycyrrhizin on LPS-induced endotoxemia in mice and clarify the possible mechanism.An LPS-induced endotoxemia mouse model was used to confirm the anti-inflammatory activity of glycyrrhizin in vivo. In vitro, RAW264.7 cells were stimulated with LPS in the presence or absence of glycyrrhizin. The expression of cytokines was determined by ELISA. Toll-like receptor 4 (TLR4) was determined by Western blot analysis. Nuclear factor-kB (NF-κB) and Interferon regulatory factor 3 (IRF3) activation were detected by Western blotting and luciferase assay. Lipid raft staining was detected by immunocytochemistry.In vivo, the results showed that glycyrrhizin can improve survival during lethal endotoxemia. In vitro, glycyrrhizin dose-dependently inhibited the expression of TNF-α, IL-6, IL-1β and RANTES in LPS-stimulated RAW264.7 cells. Western blot analysis showed that glycyrrhizin suppressed LPS-induced NF-κB and IRF3 activation. However, glycyrrhizin did not inhibit NF-κB and IRF3 activation induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. Moreover, glycyrrhizin did not affect the expression of TLR4 and CD14 induced by LPS. Significantly, we found that glycyrrhizin decreased the levels of cholesterol of lipid rafts and inhibited translocation of TLR4 to lipid rafts. Moreover, glycyrrhizin activated ABCA1, which could induce cholesterol efflux from lipid rafts.Glycyrrhizin exerts an anti-inflammatory property by disrupting lipid rafts and inhibiting translocation of TLR4 to lipid rafts, thereby attenuating LPS-mediated inflammatory response.Learning the anti-inflammatory mechanism of glycyrrhizin is crucial for the anti-inflammatory drug development.
Keywords: Cytokine; Glycyrrhizin; Nuclear factor κB; Interferon regulatory factor 3; Toll-like Receptor 4; Lipid raft;

Cyclic AMP and alkaline pH downregulate carbonic anhydrase 2 in mouse fibroblasts by Pablo Mardones; Jung Chin Chang; Ronald P.J. Oude Elferink (1765-1770).
The hydration of CO2 catalyzed by the ubiquitous carbonic anhydrase 2 (Ca2) is central for bicarbonate transport, bone metabolism and acid–base homeostasis in metazoans. There is evidence that in some tissues Ca2 expression can be acutely induced by cAMP, whereas in other cell types it is unresponsive to cAMP-mediated transcriptional activation.We isolated fibroblasts from wild type and mice lacking the ubiquitous chloride/bicarbonate exchanger (Ae2a,b −/− mice). In these cells the regulation of carbonic anhydrase 2 by cAMP was studied.We show that Ca2 expression is strongly inhibited by chronic incubation with dibutyryl-cAMP, forskolin or alkaline pH in cultured mouse fibroblasts. Furthermore, fibroblasts obtained from anion exchanger 2 deficient (Ae2a,b −/−) mice, which display intracellular alkalosis and increased cAMP production, express less than 10% of control Ca2 mRNA and protein. Surprisingly, inhibition of the bicarbonate-sensitive soluble adenylyl cyclase (sAC) was found to reduce CA2 expression instead of increasing it.CA2 expression is strongly regulated by intracellular pH and by cAMP, suggesting a role for soluble adenylyl cyclase. Regulation occurs in opposite directions which may be explained by an incoherent feedforward loop consisting of activation by pCREB and repression by ICER.
Keywords: Carbonic anhydrase; Intracellular pH; Soluble adenylyl cyclase; Chloride/bicarbonate exchanger; Bicarbonate; Cyclic AMP;

Iron uptake and transfer from ceruloplasmin to transferrin by Chantal Eid; Miryana Hémadi; Nguyêt-Thanh Ha-Duong; Jean-Michel El Hage Chahine (1771-1781).
Dietary and recycled iron are in the Fe2 + oxidation state. However, the metal is transported in serum by transferrin as Fe3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe2 + and transported Fe3 +.This study uses the techniques of chemical relaxation and spectrophotometric detection.Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe2 + oxidation by Cu2 + D6. Fe3 + is then transferred from the binding site to the holding site. Cu+ D6 is then re-oxidized by a Cu2 + of the trinuclear cluster in about 200 ms. The second Fe2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.Fe3 + is transferred after Fe2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.Ceruloplasmin is a go-between dietary or recycled Fe2 + and transferrin transported Fe3 +.Display Omitted
Keywords: Iron-metabolism; Multi-copper oxidase; Metal-transport; Aceruloplasminemia; Protein–protein adduct;

Antibiotic bacitracin induces hydrolytic degradation of nucleic acids by Jerzy Ciesiołka; Małgorzata Jeżowska-Bojczuk; Jan Wrzesiński; Kamila Stokowa-Sołtys; Justyna Nagaj; Aleksandra Kasprowicz; Leszek Błaszczyk; Wojciech Szczepanik (1782-1789).
Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA.In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5′ ends with 32P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined.Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg2 +, Mn2 + or Zn2 + are absolutely necessary.The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.
Keywords: Ribonucleic acid (RNA) degradation; Deoxyribonucleic acid (DNA) degradation; Nucleolytic property; Hydrolytic cleavage mechanism;

Galectin-3 binds to MUC1-N-terminal domain and triggers recruitment of β-catenin in MUC1-expressing mouse 3T3 cells by Shuhei Tanida; Yugo Mori; Akiko Ishida; Kaoru Akita; Hiroshi Nakada (1790-1797).
Galectin-3 is expressed in a variety of tumors and its expression level is related with tumor progression. Aberrant expression of MUC1 in various tumors is also associated with a poor prognosis. It has been reported that MUC1 is a natural ligand of galectin-3.A stable MUC1 transfectant was produced by introducing MUC1 cDNA into mouse 3T3 fibroblasts (MUC1/3T3 cells). MUC1 was prepared from MUC1/3T3 cells; MUC1-N-terminal domain (MUC1-ND) and -C-terminal domain (MUC1-CD) were separated by CsCl ultracentrifugation, and then the galectin-3-binding domain was determined by co-immuniprecipitation assay. After ligation of galectin-3 to 3T3/MUC1 cells, MUC1-CD was immunoprecipitated from the cell lysate. The immunoprecipitate was subjected to SDS-PAGE and Western blotting, followed by detection of co-immunoprecipitated β-catenin.Galectin-3 binds to the N-terminal domain of MUC1 but not to the C-terminal one. Galectin-3 present on the cell surface increased with the expression of MUC1 and is colocalized with MUC1. It should be noted that β-catenin was detected in the immunoprecipitate with anti-MUC1-CD Ab from a lysate of galectin-3-treated 3T3/MUC1 cells.Galectin-3 binds to MUC1-ND and triggers MUC1-mediated signaling in 3T3/MUC1 cells, leading to recruitment of β-catenin to MUC1-CD.This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated pathway.
Keywords: MUC1; Galectin-3; β-Catenin;

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: Binding of substrate induces a second class of site with lowered affinity and catalytic activity by Joel L. Asenjo; Heide C. Ludwig; Cristian A. Droppelmann; Juan G. Cárcamo; Ilona I. Concha; Alejandro J. Yáñez; María L. Cárdenas; Athel Cornish-Bowden; Juan C. Slebe (1798-1807).
Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P 2 and by high concentrations of its substrate Fru-1,6-P 2. The mechanism that produces substrate inhibition continues to be obscure.Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P 2 and Fru-1,6-P 2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244.The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P 2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P 2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a “stapler” that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition.Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits.Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.
Keywords: FBPase; Gluconeogenesis; Substrate inhibition; Fructose-1,6-bisphosphate; Affinity site; Subunit interaction;

A new tellurium-containing amphiphilic molecule induces apoptosis in HCT116 colon cancer cells by Peng Du; Nathaniel Edward Bennett Saidu; Johanna Intemann; Claus Jacob; Mathias Montenarh (1808-1816).
Chalcogen-based redox modulators over the years have attracted considerable attention as anti-cancer agents. New selenium- and tellurium-containing compounds with a polar head group and aryl-groups of various lengths have recently been reported as biologically active in several organisms. In the present study, we used the most active of the tellurium compound DP41, and its selenium counterpart DP31 to investigate their effects on the human cancer cell line HCT116.Cells were treated with DP41 or DP31 and the formation of superoxide radicals was determined using dihydroethidium. Cell cycle analysis and apoptosis was determined by cytofluorimetry. Proteins involved in ER signaling and apoptosis were determined by Western blot analysis and fluorescence microscopy.With 50 μM of DP41, we observed an increase in O2 formation. There was, however, no such increase in O2 after treatment with the corresponding selenium compound under the same conditions. In the case of DP41, the production of O2 radicals was followed by an up-regulation of Nrf2, HO-1, phospho-eIF2α and ATF4. CHOP was also induced and cells entered apoptosis. Unlike the cancer cells, normal retinal epithelial ARPE-19 cells did not produce elevated levels of O2 radicals nor did they induce the ER signaling pathway or apoptosis.The tellurium-containing compound DP41, in contrast to the corresponding selenium compound, induces O2 radical formation and oxidative and ER stress responses, including CHOP activation and finally apoptosis.These results indicate that DP41 is a redox modulating agent with promising anti-cancer potentials.Display Omitted
Keywords: Reactive oxygen species; Endoplasmic reticulum stress pathway; Apoptosis; Cancer cell; Non-cancer cell;

Hypobaric hypoxia induced arginase expression limits nitric oxide availability and signaling in rodent heart by Manjulata Singh; Gayatri Padhy; Praveen Vats; Kalpana Bhargava; Niroj Kumar Sethy (1817-1824).
This study was aimed to evaluate regulation of cardiac arginase expression during hypobaric hypoxia and subsequent effect on nitric oxide availability and signaling.Rats were exposed to hypobaric hypoxia (282 mm Hg for 3 h) and ARG1 expression was monitored. The expression levels of eNOS and eNOSSer1177 were determined by Western blotting, cGMP levels were measured by ELISA and amino acid concentrations were measured by HPLC analysis. Transcription regulation of arginase was monitored by chromatin immunoprecipitation (ChIP) assay with anti-c-Jun antibody for AP-1 consensus binding site on ARG1 promoter. Arginase activity was inhibited by intra-venous dose of N-(ω)-hydroxy-nor-l-arginine (nor-NOHA) prior to hypoxia exposure and subsequent effect on NO availability and oxidative stress were evaluated.Hypobaric hypoxia induced cardiac arginase expression by recruiting c-Jun to AP-1 binding site on ARG1 promoter. This increased expression redirected l-arginine towards arginase and resulted in limited endothelial nitric oxide synthase (eNOS) activity, nitric oxide (NO) availability and cGMP mediated signaling. Inhibition of arginase restored the eNOS activity, promoted cardiac NO availability and ameliorated peroxynitrite formation during hypoxia.Hypoxic induced arginase under transcription control of AP-1 reciprocally regulates eNOS activity and NO availability in the heart. This also results in cardiac oxidative stress.This study provides understanding of hypoxia-mediated transcriptional regulation of arginase expression in the heart and its subsequent effect on eNOS activity, NO availability and signaling as well as cardiac oxidative stress. This information will support the use of arginase inhibitors as therapeutics for pathological hypoxia.
Keywords: Arginase; Nitric oxide; Hypoxia; Heart; Oxidative stress;

Identification, modeling and ligand affinity of early deuterostome CYP51s, and functional characterization of recombinant zebrafish sterol 14α-demethylase by Ann Michelle Stanley Morrison; Jared V. Goldstone; David C. Lamb; Akira Kubota; Benjamin Lemaire; John J. Stegeman (1825-1836).
Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes.PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC-MS.Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2 nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26 μM for ketoconazole and 0.64 μM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51s. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s.Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s.The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications.
Keywords: Sterol 14α-demethylase; CYP51; Zebrafish; Deuterostome; Sterol biosynthesis; Azole inhibition;

Corrigendum to “Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes” [Biochim. Biophys. Acta — Gen. Subj. 1830 (10) (2013) 4692–4707] by María Florencia Leal Denis; J. Jeremías Incicco; María Victoria Espelt; Sandra V. Verstraeten; Omar P. Pignataro; Eduardo R. Lazarowski; Pablo J. Schwarzbaum (1837).

Cigarette smoke-induced alveolar epithelial–mesenchymal transition is mediated by Rac1 activation by Hui-juan Shen; Yan-hong Sun; Shui-juan Zhang; Jun-xia Jiang; Xin-wei Dong; Yong-liang Jia; Jian Shen; Yan Guan; Lin-hui Zhang; Fen-fen Li; Xi-xi Lin; Xi-mei Wu; Qiang-min Xie; Xiao-feng Yan (1838-1849).
Epithelial–mesenchymal transition (EMT) is the major pathophysiological process in lung fibrosis observed in chronic obstructive pulmonary disease (COPD) and lung cancer. Smoking is a risk factor for developing EMT, yet the mechanism remains largely unknown. In this study, we investigated the role of Rac1 in cigarette smoke (CS) induced EMT.EMT was induced in mice and pulmonary epithelial cells by exposure of CS and cigarette smoke extract (CSE) respectively.Treatment of pulmonary epithelial cells with CSE elevated Rac1 expression associated with increased TGF-β1 release. Blocking TGF-β pathway restrained CSE-induced changes in EMT-related markers. Pharmacological inhibition or knockdown of Rac1 decreased the CSE exposure induced TGF-β1 release and ameliorated CSE-induced EMT. In CS-exposed mice, pharmacological inhibition of Rac1 reduced TGF-β1 release and prevented aberrations in expression of EMT markers, suggesting that Rac1 is a critical signaling molecule for induction of CS-stimulated EMT. Furthermore, Rac1 inhibition or knockdown abrogated CSE-induced Smad2 and Akt (PKB, protein kinase B) activation in pulmonary epithelial cells. Inhibition of Smad2, PI3K (phosphatidylinositol 3-kinase) or Akt suppressed CSE-induced changes in epithelial and mesenchymal marker expression.Altogether, these data suggest that CS initiates EMT through Rac1/Smad2 and Rac1/PI3K/Akt signaling pathway. Our data provide new insights into the fundamental basis of EMT and suggest a possible new course of therapy for COPD and lung cancer.
Keywords: Epithelial–mesenchymal transition; TGF-β1; Cigarette smoke extract; Rac1; Pulmonary fibrosis;

α-Melanocyte-stimulating hormone inhibits angiogenesis through attenuation of VEGF/VEGFR2 signaling pathway by Wen-Tsan Weng; Shih-Chung Huang; Yi-Ling Ma; Hoi-Hung Chan; Shih-Wei Lin; Jian-Ching Wu; Chang-Yi Wu; Zhi-Hong Wen; E-Ming Wang; Chao-Liang Wu; Ming-Hong Tai (1850-1860).
Gene therapy of proopiomelanocortin, the precursor of α-melanocyte-stimulating hormone (α-MSH), suppresses the neovascularization in tumors. However, the roles of α-MSH in angiogenesis remain unclear.The influence of α-MSH on angiogenesis was evaluated by ex vivo rat aorta and in vivo, including transgenic zebrafish and chicken chorioallantoic membrane (CAM) assays. The effect of α-MSH on proliferation, matrix metalloproteinase (MMP) secretion, migration and tube formation was examined using human umbilical vein endothelial cells (HUVECs). The expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) was investigated by quantitative RT-PCR, immunoblot and immunofluorescent analysis. Antibodies' neutralization was employed to dissect the receptor(s) transmitting α-MSH signaling.Application of α-MSH potently suppressed the microvessels sprouting in organotypic aortic rings. Besides, α-MSH perturbed the vessels development in zebrafish and chicken embryos. α-MSH (0.01–10 nM) inhibited the MMP-2 secretion, migration and tube formation of HUVECs without affecting proliferation. Mechanistic studies unveiled α-MSH decreased the VEGF expression and release in HUVECs. Besides, α-MSH downregulated the VEGFR2 expression at transcriptional and translational levels. Importantly, α-MSH attenuated the Akt phosphorylation, but enhanced the expression of PTEN, endogenous antagonist of PI3K/Akt signaling. Expression analysis and antibody neutralization revealed that MC1-R and MC2-R participated in α-MSH-induced blockage of migration and VEGF/VEGFR2/Akt signaling. However, VEGF supply failed to reverse the anti-angiogenic function of α-MSH.α-MSH inhibits the physiological angiogenesis by attenuating VEGF/VEGFR2/Akt signaling in endothelial cells.α-MSH is a potent angiogenesis inhibitor targeting at endothelial VEGF/VEGFR2 signaling, which may have potential for therapeutic application.Display Omitted
Keywords: Proopiomelanocortin; α-MSH; Angiogenesis; HUVEC; VEGF;

In Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of trehalose-6-phosphate synthase (TPS).The cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant.The purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14 kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30 °C and 7.0 respectively. The Km Vmax and Kcat against S-adenosyl-l-methionine (AdoMet) were 4.95 μM, 3.2 U/mg and 6.4 s− 1 respectively. Competitive inhibitor S-Adenosyl-l-homocysteine achieved a Ki as 10.9 μM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kanr exhibited almost 50% reduction in intracellular trehalose concentration.A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae.This is the first report about a cysteine methyltransferase which performs S methylation at cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.
Keywords: AdoMet; Trehalose; Methylation; Adox;

Role of glutamine-169 in the substrate recognition of human aminopeptidase B by Yuko Ogawa; Atsushi Ohnishi; Yoshikuni Goto; Yoshiki Sakuma; Jobu Watanabe; Akira Hattori; Masafumi Tsujimoto (1872-1881).
Aminopeptidase B (EC, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB.Site-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency.Substitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC50 values of inhibitors that interact with the catalytic pocket of APB. The EC50 value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion.Gln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APB via maintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters.
Keywords: Aminopeptidase; Catalytic pocket; Proteolytic processing; Site-directed mutagenesis; Substrate specificity;

Thiol oxidation is crucial in the desensitization of the mitochondrial F1FO-ATPase to oligomycin and other macrolide antibiotics by Salvatore Nesci; Vittoria Ventrella; Fabiana Trombetti; Maurizio Pirini; Alessandra Pagliarani (1882-1891).
The macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases.The inhibition mechanism of macrolides was explored on swine heart mitochondrial F1FO-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide–enzyme interaction.When individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I 1 and I 2 pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI 1 I 2), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT ≥ 1 μM. The TBT-driven enzyme desensitization was reversed by DTE.The macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of FO. The oxidation of highly conserved thiols in the ATP synthase c-ring of FO weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT.The findings, by elucidating the macrolide inhibitory mechanism on FO, indirectly cast light on the F1FO torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy.Display Omitted
Keywords: Mitochondrial F1FO-ATPase; Uncompetitive inhibition; Oligomycin; Macrolide binding region; Thiol oxidation; Tributyltin;

Direct cytocidal effect of galectin-9 localized on collagen matrices on human immune cell lines by Youko Fukata; Aiko Itoh; Yasuhiro Nonaka; Takashi Ogawa; Takanori Nakamura; Osamu Matsushita; Nozomu Nishi (1892-1901).
There is a continuous demand for new immunosuppressive agents for organ transplantation. Galectin-9, a member of the galactoside-binding animal lectin family, has been shown to suppress pathogenic T-cell responses in autoimmune disease models and experimental allograft transplantation. In this study, an attempt has been made to develop new collagen matrices, which can cause local, contact-dependent immune suppression, using galectin-9 and collagen-binding galectin-9 fusion proteins as active ingredients.Galectin-9 and galectin-9 fusion proteins having collagen-binding domains (CBDs) derived from bacterial collagenases and a collagen-binding peptide (CBP) were tested for their ability to bind to collagen matrices, and to induce Jurkat cell death in solution and in the collagen-bound state.Galectin-9-CBD fusion proteins exhibited collagen-binding activity comparable to or lower than that of the respective CBDs, while their cytocidal activity toward Jurkat cells in solution was 80 ~ 10% that of galectin-9. Galectin-9 itself exhibited oligosaccharide-dependent collagen-binding activity. The growth of Jurkat cells cultured on collagen membranes treated with galectin-9 was inhibited by ~ 90%. The effect was dependent on direct cell-to-membrane contact. Galectin-9-CBD/CBP fusion proteins bound to collagen membranes via CBD/CBP moieties showed a low or negligible effect on Jurkat cell growth.Among the proteins tested, galectin-9 exhibited the highest cytocidal effect on Jurkat cells in the collagen-bound state. The effect was not due to galectin-9 released into the culture medium but was dependent on direct cell-to-membrane contact.The study demonstrates the possible use of galectin-9-modified collagen matrices for local, contact-dependent immune suppression in transplantation.
Keywords: Galectin; Collagen; Collagen-binding domain; Immune suppression;

Omega-3 deficiency and neurodegeneration in the substantia nigra: Involvement of increased nitric oxide production and reduced BDNF expression by Henriqueta Dias Cardoso; Eraldo Fonseca dos Santos Junior; David Filipe de Santana; Catarina Gonçalves-Pimentel; Monara Kaélle Angelim; Alinny R. Isaac; Cláudia Jacques Lagranha; Rubem Carlos Araújo Guedes; Eduardo Isidoro Beltrão; Edgar Morya; Marcelo Cairrão Araújo Rodrigues; Belmira Lara da Silveira Andrade-da-Costa (1902-1912).
Our previous study demonstrated that essential fatty acid (EFA) dietary restriction over two generations induced midbrain dopaminergic cell loss and oxidative stress in the substantia nigra (SN) but not in the striatum of young rats. In the present study we hypothesized that omega-3 deficiency until adulthood would reduce striatum's resilience, increase nitric oxide (NO) levels and the number of BDNF-expressing neurons, both potential mechanisms involved in SN neurodegeneration.Second generation rats were raised from gestation on control or EFA-restricted diets until young or adulthood. Lipoperoxidation, NO content, total superoxide dismutase (t-SOD) and catalase enzymatic activities were assessed in the SN and striatum. The number of tyrosine hydroxylase (TH)- and BDNF-expressing neurons was analyzed in the SN.Increased NO levels were observed in the striatum of both young and adult EFA-deficient animals but not in the SN, despite a similar omega-3 depletion (~ 65%) in these regions. Increased lipoperoxidation and decreased catalase activity were found in both regions, while lower tSOD activity was observed only in the striatum. Fewer TH- (~ 40%) and BDNF-positive cells (~ 20%) were detected at the SN compared to the control.The present findings demonstrate a differential effect of omega-3 deficiency on NO production in the rat's nigrostriatal system. Prolonging omega-3 depletion until adulthood impaired striatum's anti-oxidant resources and BDNF distribution in the SN, worsening dopaminergic cell degeneration.Omega-3 deficiency can reduce the nigrostriatal system's ability to maintain homeostasis under oxidative conditions, which may enhance the risk of Parkinson's disease.
Keywords: Brain-derived neurotrophic factor; Lipoperoxidation; Docosahexaenoic acid; Dopamine; Oxidative stress; Striatum;

Synthesis and characterization of curcumin loaded polymer/lipid based nanoparticles and evaluation of their antitumor effects on MCF-7 cells by Sathish Sundar Dhilip Kumar; Ayyavu Mahesh; Surianarayanan Mahadevan; Asit Baran Mandal (1913-1922).
Hybrid materials are synthesized using hydrophilic polymer and lipids which ensure their long term systemic circulation through intravenous administration and enhance loading of hydrophobic drugs. The purpose of this study is to prepare, characterize and evaluate the in vitro efficacy of curcumin loaded poly-hydroxyethyl methacrylate/stearic acid nanoparticles in MCF-7.C-PSA-NPs, prepared using the emulsification–solvent evaporation method were characterized by dynamic laser scattering, SEM, AFM, FT-IR, X-ray diffraction, and TGA. The in vitro release behavior was observed in PBS pH 7.4, the anticancer potential was analyzed by MTT assay, cell cycle and apoptosis studies were performed through flow cytometry. C-PSA-NPs drug localization and cancer cell morphological changes were analyzed in MCF-7 cell line.C-PSA-NPs exhibited the mean particle size in the range of 184 nm with no aggregation. The surface charge of the material was around − 29.3 mV. Thermal studies (TGA) and surface chemistry studies (FT-IR, XRD) showed the existence of drug curcumin in C-PSA-NPs. The MTT assay indicated higher anticancer properties and flow cytometry studies revealed that there were better apoptotic activity and maximum localization of C-PSA-NPs than curcumin.Polymer lipid based drug delivery appeared as one of the advancements in drug delivery systems. Through the present study, a novel polymer lipid based nanocarrier delivery system loaded with curcumin was demonstrated as an effective and potential alternative method for tumor treatment in MCF-7 cell line.C-PSA-NPs exhibited potent anticancer activity in MCF-7 cell line and it indicates that C-PSA-NPs are a suitable carrier for curcumin.Display Omitted
Keywords: Polyhydroxyethyl methacrylate; Stearic acid; Curcumin; Drug delivery system; Polymeric lipid nanoparticle;

Nitric oxide (NO) is an important presynaptic modulator of synaptic transmission. Here, we aimed to correlate the release of the major inhibitory neurotransmitter GABA with intracellular events occurring in rat brain axon terminals during their exposure to NO in the range of nanomolar–low micromolar concentrations.Using [3H]GABA and fluorescent dyes (Fluo 4-AM, acridine orange and rhodamine 6G), the following parameters were evaluated: vesicular and cytosolic GABA pools, intracellular calcium concentration, synaptic vesicle acidification, and mitochondrial membrane potential. Diethylamine NONOate (DEA/NO) and S-nitroso-N-acetylpenicillamine (SNAP) were used as NO donors.DEA/NO and SNAP (in the presence of dithiothreitol (DTT)) stimulated external Ca2 +-independent [3H]GABA release, which was not attributed to a rise in intracellular calcium concentration. [3H]GABA release coincided with increasing GABA level in cytosol and decreasing the vesicular GABA content available for exocytotic release. There was a strong temporal correlation between NO-induced increase in cytosolic [GABA] and dissipation of both synaptic vesicle proton gradient and mitochondrial membrane potential. Dissipation was reversible, and recovery of both parameters correlated in time with re-accumulation of [3H]GABA into synaptic vesicles. The molar ratio of DTT to SNAP determined the rate and duration of the recovery processes.We suggest that NO can stimulate GABA release via GABA transporter reversal resulting from increased GABA levels in cytosol. The latter is reversible and appears to be due to S-nitrosylation of key proteins, which affect the energy status of the pre-synapse.Our findings provide new insight into molecular mechanism(s) underlying the presynaptic action of nitric oxide on inhibitory neurotransmission.
Keywords: Nitric oxide; [3H]GABA release; Synaptic vesicle acidification; Mitochondrial membrane potential; S-nitrosylation; Exocytosis;

Bundles of unipolar actin filaments (F-actin), cross-linked via the actin-binding protein fascin, are important in filopodia of motile cells and stereocilia of inner ear sensory cells. However, such bundles are also useful as shuttles in myosin-driven nanotechnological applications. Therefore, and for elucidating aspects of biological function, we investigate if the bundle tendency to follow straight paths (quantified by path persistence length) when propelled by myosin motors is directly determined by material properties quantified by persistence length of thermally fluctuating bundles.Fluorescent bundles, labeled with rhodamine-phalloidin, were studied at fascin:actin molar ratios: 0:1 (F-actin), 1:7, 1:4 and 1:2. Persistence lengths (Lp) were obtained by fitting the cosine correlation function (CCF) to a single exponential function: < cos(θ(0) − θ(s)) > = exp(−s  / (2Lp)) where θ(s) is tangent angle; s: path or contour lengths. < > denotes averaging over filaments.Bundle-Lp (bundles < 15 μm long) increased from ~ 10 to 150 μm with increased fascin:actin ratio. The increase was similar for path-Lp (path < 15 μm), with highly linear correlation. For longer bundle paths, the CCF-decay deviated from a single exponential, consistent with superimposition of the random path with a circular path as suggested by theoretical analysis.Fascin–actin bundles have similar path-Lp and bundle-Lp, both increasing with fascin:actin ratio. Path-Lp is determined by the flexural rigidity of the bundle.The findings give general insight into mechanics of cytoskeletal polymers that interact with molecular motors, aid rational development of nanotechnological applications and have implications for structure and in vivo functions of fascin–actin bundles.
Keywords: Persistence length; Fascin; Actin; Myosin; Motility assay; Monte-Carlo simulation;

Metformin has been shown to have a strong anti-proliferative effect in many breast cancer cell lines, mainly due to the activation of the energy sensing kinase, AMP-activated protein kinase (AMPK). MDA-MB-231 cells are aggressive and invasive breast cancer cells that are known to be resistant to several anti-cancer agents as well as to the anti-proliferative effect of metformin. As metformin is a glucose lowering drug, we hypothesized that normoglycemia will sensitize MDA-MB-231 cells to the anti-proliferative effect of metformin.MDA-MB-231 cells were treated with increasing metformin concentrations in hyperglycemic or normoglycemic conditions. The growth inhibitory effect of metformin was assessed by MTT assay. The expression of several proteins involved in cell proliferation was measured by Western blotting.In agreement with previous studies, treatment with metformin did not inhibit the growth of MDA-MB-231 cells cultured in hyperglycemic conditions. However, metformin significantly inhibited MDA-MB-231 growth when the cells were cultured in normoglycemic conditions. In addition, we show that metformin-treatment of MDA-MB-231 cells cultured in normoglycemic conditions and not in hyperglycemic conditions caused a striking activation of AMPK, and an AMPK-dependent inhibition of multiple molecular signaling pathways known to control protein synthesis and cell proliferation.Our data show that normoglycemia sensitizes the triple negative MDA-MB-231 breast cancer cells to the anti-proliferative effect of metformin through an AMPK-dependent mechanism.These findings suggest that tight normoglycemic control may enhance the anti-proliferative effect of metformin in diabetic cancer patients.
Keywords: MDA-MB-231; Breast cancer; Metformin; Diabetes; AMP-activated protein kinase;

Interaction of mutant p53 with p73: A Surface Plasmon Resonance and Atomic Force Spectroscopy study by Simona Santini; Silvia Di Agostino; Emilia Coppari; Anna Rita Bizzarri; Giovanni Blandino; Salvatore Cannistraro (1958-1964).
TP53 tumor suppressor gene is mutated in more than 50% of human tumors. Mutated p53 proteins could sequestrate and inactivate p73 reducing the apoptotic and anti-proliferative effects of the transcription factor, and yielding cancer cells more aggressive and chemoresistant. The possibility of using drugs to prevent the mutant p53/p73 complex formation preserving the p73 function, calls for a deeper insight into the molecular and biochemical mechanisms of mutant p53/p73 protein interaction.The kinetics of the mutant p53R175H/p73 complex was investigated with innovative and complementary techniques, operating in real time, in near physiological conditions and without any labeling. Specifically, Atomic Force Spectroscopy and Surface Plasmon Resonance working at single-molecule level and in bulk condition, respectively, were used.The two techniques revealed that a stable complex is formed between mutant p53R175H and p73 proteins; the complex being characterized by a high interaction force and a dissociation equilibrium constant in the order of 10− 7  M, as expected for specific interactions. No binding was instead observed between p73 and wild type p53.Mutant p53R175H protein, unlike wild type p53, can form a stable complex with p73. The mutant p53R175H/p73 protein complex could be a target for innovative pharmaceutical drugs that, by dissociating it or preventing biomolecule interaction thus preserving the p73 function, could enhance the response of cancerous cells carrying mutant p53R175H protein to common chemotherapeutic agents.The kinetic information obtained in vitro may help to design specific pharmaceutical drugs directed against cancerous cells carrying mutant p53 proteins.Display Omitted
Keywords: Mutated p53; p73; Atomic Force Spectroscopy; Surface Plasmon Resonance;

Myosin IIa activation is crucial in breast cancer derived galectin-1 mediated tolerogenic dendritic cell differentiation by Da-En Cheng; Jen-Yu Hung; Ming-Shyan Huang; Ya-Ling Hsu; Chi-Yu Lu; Eing-Mei Tsai; Ming-Feng Hou; Po-Lin Kuo (1965-1976).
Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed.Circulating CD14+CD16+ of breast cancer patients and induced CD14+CD16+ DCs were identified as tDCs by treating CD14+ monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo.The CD14+CD16+ tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c+ DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c+/Ly6G+/IL-10+ in the BALB/c mice.Galectin-1 can induce tDCs after internalizing into CD14+ monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy.Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development.
Keywords: Galectin-1; Breast cancer; Myosin IIa; Endocytosis; Dendritic cell;

Yap1 mediates tolerance to cobalt toxicity in the yeast Saccharomyces cerevisiae by Catarina Pimentel; Soraia M. Caetano; Regina Menezes; Inês Figueira; Claudia N. Santos; Ricardo B. Ferreira; Manuel A.S. Santos; Claudina Rodrigues-Pousada (1977-1986).
Cobalt has a rare occurrence in nature, but may accumulate in cells to toxic levels. In the present study, we have investigated how the transcription factor Yap1 mediates tolerance to cobalt toxicity.Fluorescence microscopy was used to address how cobalt activates Yap1. Using microarray analysis, we compared the transcriptional profile of a strain lacking Yap1 to that of its parental strain. To evaluate the extent of the oxidative damage caused by cobalt, GSH was quantified by HPLC and protein carbonylation levels were assessed.Cobalt activates Yap1 under aerobiosis and anaerobiosis growth conditions. This metal generates a severe oxidative damage in the absence of Yap1. However, when challenged with high concentrations of cobalt, yap1 mutant cells accumulate lower levels of this metal. Accordingly, microarray analysis revealed that the expression of the high affinity phosphate transporter, PHO84, a well-known cobalt transporter, is compromised in the yap1 mutant. Moreover, we show that Yap1 is a repressor of the low affinity iron transporter, FET4, which is also known to transport cobalt.Cobalt activates Yap1 that alleviates the oxidative damage caused by this metal. Yap1 partially controls cobalt cellular uptake via the regulation of PHO84. Although FET4 repression by Yap1 has no effect on cobalt uptake, it may be its first line of defense against other toxic metals.Our results emphasize the important role of Yap1 in mediating cobalt-induced oxidative damages and reveal new routes for cell protection provided by this regulator.
Keywords: Cobalt; Yap1; Yeast; Oxidative stress; Pho84; Fet4;

A vacuolar H+-pyrophosphatase differential activation and energy coupling integrate the responses of weeds and crops to drought stress by Josimara Barcelos Venancio; Michelle Guedes Catunda; Juarez Ogliari; Janaína Aparecida Hottz Rima; Anna Lvovna Okorokova-Facanha; Lev Alexandrovitich Okorokov; Arnoldo Rocha Facanha (1987-1992).
Cyperus rotundus L. is a C4 weed of large vegetative and reproductive vigor endowed with competitive advantages over most crop species mainly under adverse environmental conditions. Vacuole functions are critical for the mechanisms of drought resistance, and here the modulation of the primary system of vacuolar ion transport is investigated during a transient water stress imposed to this weed and to C4 crop species (Zea mays L.).The vacuolar H+ pumps, the H+-ATPase and H+-PPiase, expression, activities and the energy coupling were spectrophotometrically investigated as key elements in the differential drought-resistance mechanisms developed by weeds and crops.In C. rotundus tonoplasts, ATP hydrolysis was more sensitive to drought than its coupled H+ transport, which was in turn at least 3-folds faster than that mediated by the H+-PPiase. Its PPi hydrolysis was only slightly affected by severe water deficit, contrasting with the disruption induced in the PPi-dependent H+-gradient. This effect was antagonized by plant rehydration as the H+-PPiase activity was highly stimulated, reassuming a coupled PPi-driven H+ pumping. Maize tonoplasts exhibited 2–4 times lower hydrolytic activities than that of C. rotundus, but were able to overactivate specifically PPi-dependent H+ pumping in response to stress relief, resulting in an enhanced H+-pumps coupling efficiency.These results together with immunoanalysis revealed profiles consistent with pre- and post-translational changes occurring on the tonoplast H+-pumps, which differ between weeds and crops upon water deficit.The evidences highlight an unusual modulation of the H+-PPiase energy coupling as a key biochemical change related to environmental stresses adaptive capacity of plants.
Keywords: Membrane-bound pyrophosphatase; V-type ATPase; Cytoplasmic pyrophosphate; Metabolic energy; Electrochemical gradient; Phosphate pool dynamics;

Changes in glycosaminoglycan structure on differentiation of human embryonic stem cells towards mesoderm and endoderm lineages by Leyla Gasimli; Anne Marie Hickey; Bo Yang; Guoyun Li; Mitche dela Rosa; Alison V. Nairn; Michael J. Kulik; Jonathan S. Dordick; Kelley W. Moremen; Stephen Dalton; Robert J. Linhardt (1993-2003).
Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation.Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages, and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis, immunoblotting, immunofluorescence and disaccharide analysis.Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1, HS6ST2 and HS6ST3, three heparan sulfate biosynthetic enzymes, within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure.Differentiation of embryonic stem cells markedly changes the proteoglycanome.The glycosaminoglycan biosynthetic pathway is complex and highly regulated, and therefore, understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation.Display Omitted
Keywords: Glycosaminoglycan; Proteoglycans; Embryonic; Stem cells; Splanchnic mesoderm; Hepatocytes;

Curcusone D, a novel ubiquitin–proteasome pathway inhibitor via ROS-induced DUB inhibition, is synergistic with bortezomib against multiple myeloma cell growth by Mei-Na Cao; Yu-Bo Zhou; An-Hui Gao; Jia-Yi Cao; Li-Xin Gao; Li Sheng; Lei Xu; Ming-Bo Su; Xian-Chao Cao; Meng-meng Han; Ming-Kui Wang; Jia Li (2004-2013).
Ubiquitin–proteasome pathway (UPP) plays a very important role in the degradation of proteins. Finding novel UPP inhibitors is a promising strategy for treating multiple myeloma (MM).Ub-YFP reporter assays were used as cellular UPP models. MM cell growth, apoptosis and overall death were evaluated with the MTS assay, Annexin V/PI dual-staining flow cytometry, poly (ADP-ribose) polymerase (PARP) cleavage, and PI uptake, respectively. The mechanism of UPP inhibition was analyzed by western blotting for ubiquitin, in vitro and cellular proteasomal and deubiquitinases (DUBs) activity assays. Cellular reactive oxygen species (ROS) were measured with H2DCFDA.Curcusone D, identified as a novel UPP inhibitor, causes cell growth inhibition and apoptosis in MM cells. Curcusone D induced the accumulation of poly-ubiquitin-conjugated proteins but could not inhibit proteasomal activity in vitro or in cells. Interestingly, the mono-ubiquitin level and the total cellular DUB activity were significantly downregulated following curcusone D treatment. Furthermore, curcusone D could induce ROS, which were closely correlated with DUB inhibition that could be nearly completely reversed by NAC. Finally, curcusone D and the proteasomal inhibitor bortezomib showed a strong synergistic effect against MM cells.Curcusone D is novel UPP inhibitor that acts via the ROS-induced inhibition of DUBs to produce strong growth inhibition and apoptosis of MM cells and synergize with bortezomib.The anti-MM molecular mechanism study of curcusone D will promote combination therapies with different UPP inhibitors against MM and further support the concept of oxidative stress regulating the activity of DUBs.Display Omitted
Keywords: Ubiquitin–proteasome pathway; Deubiquitinase; ROS; Curcusone D; Bortezomib; Multiple myeloma;

Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease.We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging.Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90 nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional.We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells.The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomer distribution and properties, and the study of their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms of Parkinson's disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models.
Keywords: Fluorescence; Oligomers; Protein aggregation; Lysosomes; Parkinson's disease; Live imaging;

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes.We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64.Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide.Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis.Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.
Keywords: Saccharomyces cerevisiae; Parkinson's disease; LRRK2; Cellular dysfunction; Oxidative stress;

Malarial hemozoin: From target to tool by Lorena M. Coronado; Christopher T. Nadovich; Carmenza Spadafora (2032-2041).
Malaria is an extremely devastating disease that continues to affect millions of people each year. A distinctive attribute of malaria infected red blood cells is the presence of malarial pigment or the so-called hemozoin. Hemozoin is a biocrystal synthesized by Plasmodium and other blood-feeding parasites to avoid the toxicity of free heme derived from the digestion of hemoglobin during invasion of the erythrocytes.Hemozoin is involved in several aspects of the pathology of the disease as well as in important processes such as the immunogenicity elicited. It is known that the once best antimalarial drug, chloroquine, exerted its effect through interference with the process of hemozoin formation. In the present review we explore what is known about hemozoin, from hemoglobin digestion, to its final structural analysis, to its physicochemical properties, its role in the disease and notions of the possible mechanisms that could kill the parasite by disrupting the synthesis or integrity of this remarkable crystal.The importance and peculiarities of this biocrystal have given researchers a cause to consider it as a target for new antimalarials and to use it through unconventional approaches for diagnostics and therapeutics against the disease.Hemozoin plays an essential role in the biology of malarial disease. Innovative ideas could use all the existing data on the unique chemical and biophysical properties of this macromolecule to come up with new ways of combating malaria.
Keywords: Hemozoin synthesis; Hemoglobin degradation; Hemozoin structure; Hemozoin biophysical properties; Plasmodium falciparum;

The first cyclomegastigmane rhododendroside A from Rhododendron brachycarpum alleviates HMGB1-induced sepsis by Wei Zhou; Joonseok Oh; Wonhwa Lee; Soyoung Kwak; Wei Li; Amar G. Chittiboyina; Daneel Ferreira; Mark T. Hamann; Seung Ho Lee; Jong-Sup Bae; MinKyun Na (2042-2049).
Endangered plant species are a vital resource for exploring novel drug prototypes. A Korean endangered plant Rhododendron brachycarpum G. Don is a broad-leaved shrub native to northern Korea and central Japan. The high mobility group box 1 protein (HMGB1) could be a specific target for the discovery of novel antiseptic agents.Gauge-invariant atomic orbital (GIAO) NMR chemical shift calculations were applied for investigation of stereochemical details with accuracy improved by application of DP4 analysis. In vitro antiseptic mechanisms were investigated utilizing immunofluorescence staining, ELISA and cell–cell adhesion assay. Cecal ligation and puncture (CLP) operation was employed to evaluate in vivo potential alleviating severe sepsis and septic shock.The first bicyclic megastigmane glucoside rhododendroside A (1) along with known megastigmane glucosides (25) were isolated from the leaves of R. brachycarpum. The structure of 1 was established by NMR analysis as well as comparison of the experimental chemical shifts with those of computed values employing DP4 application. In the CLP operation model that simulates severe sepsis, rhododendroside A (1) improved the survival rate up to 60%.Our results exhibit that R. brachycarpum may produce a unique scaffold that is developed into a drug lead mitigating HMGB1-induced vascular pro-inflammatory stimuli and thus alleviating severe sepsis and related manifestations.Discovery of new drug leads would warrant conservation efforts of endangered species.Display Omitted
Keywords: Rhododendron brachycarpum; Endangered species; Nuclear magnetic resonance chemical shift calculations; High mobility group box 1 protein; Sepsis;

G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection by Pasqualina Liana Scognamiglio; Concetta Di Natale; Marilisa Leone; Mattia Poletto; Luigi Vitagliano; Gianluca Tell; Daniela Marasco (2050-2059).
Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region.Through a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif.These results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability.These findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif.This study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease.
Keywords: Disordered protein region; Surface Plasmon Resonance; Helical stability;

GATA-4 induces changes in electrophysiological properties of rat mesenchymal stem cells by Hong-Xia Li; Ya-Feng Zhou; Bin Jiang; Xin Zhao; Ting-Bo Jiang; Xun Li; Xiang-Jun Yang; Wen-Ping Jiang (2060-2069).
Transplanted mesenchymal stem cells (MSC) can differentiate into cardiac cells that have the potential to contribute to heart repair following ischemic injury. Overexpression of GATA-4 can significantly increase differentiation of MSC into cardiomyocytes (CM). However, the specific impact of GATA-4 overexpression on the electrophysiological properties of MSC-derived CM has not been well documented.Adult rat bone marrow MSC were retrovirally transduced with GATA-4 (MSCGATA-4) and GFP (MSCNull) and subsequently co-cultured with neonatal rat ventricular cardiomyocytes (CM). Electrophysiological properties and mRNA levels of ion channels were assessed in MSC using patch-clamp technology and real-time PCR.MSCGATA-4 exhibited higher levels of the TTX-sensitive Na+ current (INa.TTX), L-type calcium current (ICa.L), transient outward K+ current (Ito), delayed rectifier K+ current (IKDR) and inwardly rectifying K+ current (IK1) channel activities reflective of electrophysiological characteristics of CM. Real-time PCR analyses showed that MSCGATA-4 exhibited upregulated mRNA levels of Kv1.2, Kv2.1, SCN2a1, CCHL2a, KV1.4 and Kir1.1 channels versus MSCNull. Interestingly, MSCGATA-4 treated with IGF-1 neutralizing antibodies resulted in a significant decrease in Kir1.1, Kv2.1, KV1.4, CCHL2a and SCN2a1 channel mRNA expression. Similarly, MSCGATA-4 treated with VEGF neutralizing antibodies also resulted in an attenuated expression of Kv2.1, Kv1.2, Kv1.4, Kir1.1, CCHL2a and SCN2a1 channel mRNAs.GATA-4 overexpression increases Ito, IKDR, IK1, INa.TTX and ICa.L currents in MSC. Cytokine (VGEF and IGF-1) release from GATA-4 overexpressing MSC can partially account for the upregulated ion channel mRNA expression.Our results highlight the ability of GATA4 to boost the cardiac electrophysiological potential of MSC.
Keywords: GATA-4; Mesenchymal stem cell; Ion channel; Transdifferentiation; Patch clamp; Electrophysiological property;

Post-transcriptional regulation of the creatine transporter gene: Functional relevance of alternative splicing by Joseph D.T. Ndika; Cristina Martinez-Munoz; Nandaja Anand; Silvy J.M. van Dooren; Warsha Kanhai; Desiree E.C. Smith; Cornelis Jakobs; Gajja S. Salomons (2070-2079).
Aberrations in about 10–15% of X-chromosome genes account for intellectual disability (ID); with a prevalence of 1–3% (Gécz et al., 2009 [1]). The SLC6A8 gene, mapped to Xq28, encodes the creatine transporter (CTR1). Mutations in SLC6A8, and the ensuing decrease in brain creatine, lead to co-occurrence of speech/language delay, autism-like behaviors and epilepsy with ID. A splice variant of SLC6A8SLC6A8C, containing intron 4 and exons 5–13, was identified. Herein, we report the identification of a novel variant — SLC6A8D, and functional relevance of these isoforms.Via (quantitative) RT-PCR, uptake assays, and confocal microscopy, we investigated their expression and function vis-à-vis creatine transport. SLC6A8D is homologous to SLC6A8C except for a deletion of exon 9 (without occurrence of a frame shift). Both contain an open reading frame encoding a truncated protein but otherwise identical to CTR1. Like SLC6A8, both variants are predominantly expressed in tissues with high energy requirement. Our experiments reveal that these truncated isoforms do not transport creatine. However, in SLC6A8 (CTR1)-overexpressing cells, a subsequent infection (transduction) with viral constructs encoding either the SLC6A8C (CTR4) or SLC6A8D (CTR5) isoform resulted in a significant increase in creatine accumulation compared to CTR1 cells re-infected with viral constructs containing the empty vector. Moreover, transient transfection of CTR4 or CTR5 into HEK293 cells resulted in significantly higher creatine uptake.CTR4 and CTR5 are possible regulators of the creatine transporter since their overexpression results in upregulated CTR1 protein and creatine uptake.Provides added insight into the mechanism(s) of creatine transport regulation.
Keywords: Na+/Cl cotransporter; Creatine transporter; Alternative splicing; Creatine uptake upregulation; Intellectual disability;

Tetracycline nanoparticles loaded calcium sulfate composite beads for periodontal management by N. Sindhura Reddy; S. Sowmya; Joel D. Bumgardner; K.P. Chennazhi; Raja Biswas; R. Jayakumar (2080-2090).
The objective of this study was to fabricate, characterize and evaluate in vitro, an injectable calcium sulfate bone cement beads loaded with an antibiotic nanoformulation, capable of delivering antibiotic locally for the treatment of periodontal disease.Tetracycline nanoparticles (Tet NPs) were prepared using an ionic gelation method and characterized using DLS, SEM, and FTIR to determine size, morphology, stability and chemical interaction of the drug with the polymer. Further, calcium sulfate (CaSO4) control and CaSO4-Tet NP composite beads were prepared and characterized using SEM, FTIR and XRD. The drug release pattern, material properties and antibacterial activity were evaluated. In addition, protein adsorption, cytocompatibility and alkaline phosphatase activity of the CaSO4-Tet NP composite beads in comparison to the CaSO4 control were analyzed.Tet NPs showed a size range of 130 ± 20 nm and the entrapment efficiency calculated was 89%. The composite beads showed sustained drug release pattern. Further the drug release data was fitted into various kinetic models wherein the Higuchi model showed higher correlation value (R2  = 0.9279) as compared to other kinetic models. The composite beads showed antibacterial activity against Staphylococcus aureus and Escherichia coli. The presence of Tet NPs in the composite bead didn't alter its cytocompatibility. In addition, the composite beads enhanced the ALP activity of hPDL cells.The antibacterial and cytocompatible CaSO4-Tet NP composite beads could be beneficial in periodontal management to reduce the bacterial load at the infection site.Tet NPs would deliver antibiotic locally at the infection site and the calcium sulfate cement, would itself facilitate tissue regeneration.Display Omitted
Keywords: Calcium sulfate; Tetracycline nanoparticles; Antibacterial; Cytocompatible; Periodontal regeneration;

Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa by Damien Keogh; Roisin Thompson; Ruth Larragy; Kenneth McMahon; Michael O'Connell; Brendan O'Connor; Paul Clarke (2091-2104).
Prokaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs.Specific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties.This study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated.The carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties.The novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification.
Keywords: LecA; N-acetyl-lactosamine; Galectins; Lectinology; Glycoprotein analysis; Glycoprotein purification;