ALBERT

Description: It has been hypothesised that positive associations between age and levels of oxidative stress-generated damage to DNA may be related to an age-dependent decline in DNA repair activity. The objective of this study was to investigate the association between age and repair activity of oxidatively damaged DNA in peripheral blood mononuclear cells (PBMCs). We isolated PBMCs from subjects aged 18–83 years, as part of a health survey of the Danish population that focussed on lifestyle factors. The level of DNA repair activity was measured as incisions on potassium bromate-damaged DNA by the comet assay. There was an inverse association between age and DNA repair activity with a 0.65% decline in activity per year from age 18 to 83 (95% confidence interval: 0.16–1.14% per year). Univariate regression analysis also indicated inverse associations between DNA repair activity and waist-hip ratio ( P 〈 0.05) and plasma concentrations of glycosylated hemoglobin ( P = 0.07). However, multivariate regression analysis only showed an inverse association between age and DNA repair activity ( P 〈 0.05), indicating that the decline in repair activity was not mediated by metabolic risk factors. In summary, the results show an inverse association between age and DNA repair activity of oxidatively damaged DNA.

Description: Exposure to traffic-related particulate matter (PM) has been associated with increased risk of lung disease, cancer and cardiovascular disease especially in elderly and overweight subjects. The proposed mechanisms involve intracellular production of reactive oxygen species (ROS), inflammation and oxidation-induced DNA damage studied mainly in young normal-weight subjects. We performed a controlled cross-over, randomised, single-blinded, repeated-measure study where 60 healthy subjects (25 males and 35 females) with age 55–83 years and body mass index above 25kg/m 2 were exposed for 5h to either particle-filtered or sham-filtered air from a busy street with number of concentrations and PM 2.5 levels of 1800/cm 3 versus 23 000/cm 3 and 3 µg/m 3 versus 24 µg/m 3 , respectively. Peripheral blood mononuclear cells (PBMCs) were collected and assayed for production of ROS with and without ex vivo exposure to nanosized carbon black as well as expression of genes related to inflammation ( chemokine (C-C motif) ligand 2 , interleukin-8 and tumour necrosis factor ), oxidative stress response ( heme oxygenase (decycling)-1 ) and DNA repair ( oxoguanine DNA glycosylase ). DNA strand breaks and oxidised purines were assayed by the alkaline comet assay. No statistically significant differences were found for any biomarker immediately after exposure to PM from urban street air although strand breaks and oxidised purines combined were significantly associated with the particle number concentration during exposure. In conclusion, 5h of controlled exposure to PM from urban traffic did not change the gene expression related to inflammation, oxidative stress or DNA repair, ROS production or oxidatively damaged DNA in PBMCs from elderly overweight human subjects.

Description: Ionising radiation causes free radical–mediated damage in cellular DNA. This damage is manifested as chromosomal aberrations and micronuclei (MN) in proliferating cells. Sesamol, present in sesame seeds, has the potential to scavenge free radicals; therefore, it can reduce radiation-induced cytogenetic damage in cells. The aim of this study was to investigate the radioprotective potential of sesamol in bone marrow cells of mice and related haematopoietic system against radiation-induced genotoxicity. A comparative study with melatonin was designed for assessing the radioprotective potential of sesamol. C57BL/6 mice were administered intraperitoneally with either sesamol or melatonin (10 and 20mg/kg body weight) 30min prior to 2-Gy whole-body irradiation (WBI) and sacrificed after 24h. Total chromosomal aberrations (TCA), MN and cell cycle analyses were performed using bone marrow cells. The comet assay was performed on bone marrow cells, splenocytes and lymphocytes. Blood was drawn to study haematological parameters. Prophylactic doses of sesamol (10 and 20mg/kg) in irradiated mice reduced TCA and micronucleated polychromatic erythrocyte frequency in bone marrow cells by 57% and 50%, respectively, in comparison with radiation-only groups. Sesamol-reduced radiation-induced apoptosis and facilitated cell proliferation. In the comet assay, sesamol (20mg/kg) treatment reduced radiation-induced comets (% DNA in tail) compared with radiation only ( P 〈 0.05). Sesamol also increased granulocyte populations in peripheral blood similar to melatonin. Overall, the radioprotective efficacy of sesamol was found to be similar to that of melatonin. Sesamol treatment also showed recovery of relative spleen weight at 24h of WBI. The results strongly suggest the radioprotective efficacy of sesamol in the haematopoietic system of mice.

Description: Ferritins and other cage proteins have been utilized as models to understand the fundamentals of protein folding and self-assembly. The bacterioferritin (BFR) from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for a mutagenesis study to investigate the role of electrostatic intermolecular interactions mediated through charged amino acids. Through structural and computational analyses, three charged amino acids R30, D56 and E60 which involved in an electrostatic interaction network were mutated to the opposite charge. Four mutants, R30D, D56R, E60H and D56R-E60H, were expressed, purified and characterized. All of the mutants fold into α-helical structures. Consistent with the computational prediction, they all show a lowered thermostability; double mutant D56R-E60H was found to be 16°C less stable than the wild type. Except for the mutant E60H, all the other mutations completely shut down the formation of protein cages to favour the dimer state in solution. The mutants, however, retain their ability to form cage-like nanostructures in the dried, surface immobilized conditions of transmission electron microscopy. Our findings confirm that even a single charge-inversion mutation at the 2-fold interface of BFR can affect the quaternary structure of its dimers and their ability to self-assemble into cage structures.

Description: Most of bacteria can swim by rotating flagella bidirectionally. The C ring, located at the bottom of the flagellum and in the cytoplasmic space, consists of FliG, FliM and FliN, and has an important function in flagellar protein secretion, torque generation and rotational switch of the motor. FliG is the most important part of the C ring that interacts directly with a stator subunit. Here, we introduced a three-amino acids in-frame deletion mutation (PSA) into FliG from Vibrio alginolyticus , whose corresponding mutation in Salmonella confers a switch-locked phenotype, and examined its phenotype. We found that this FliG mutant could not produce flagellar filaments in a fliG null strain but the FliG(PSA) protein could localize at the cell pole as does the wild-type protein. Unexpectedly, when this mutant was expressed in a wild-type strain, cells formed flagella efficiently but the motor could not rotate. We propose that this different phenotype in Vibrio and Salmonella might be due to distinct interactions between FliG mutant and FliM in the C ring between the bacterial species.

Description: Sulphation is known to be critically involved in the metabolism of acetaminophen in vivo . This study aimed to systematically identify the major human cytosolic sulfotransferase (SULT) enzyme(s) responsible for the sulphation of acetaminophen. A systematic analysis showed that three of the twelve human SULTs, SULT1A1, SULT1A3 and SULT1C4, displayed the strongest sulphating activity towards acetaminophen. The pH dependence of the sulphation of acetaminophen by each of these three SULTs was examined. Kinetic parameters of these three SULTs in catalysing acetaminophen sulphation were determined. Moreover, sulphation of acetaminophen was shown to occur in HepG2 human hepatoma cells and Caco-2 human intestinal epithelial cells under the metabolic setting. Of the four human organ samples tested, liver and intestine cytosols displayed considerably higher acetaminophen-sulphating activity than those of lung and kidney. Collectively, these results provided useful information concerning the biochemical basis underlying the metabolism of acetaminophen in vivo previously reported.

Description: In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and purified to homogeneity. The purified protein was shown to be a monomeric protein with a molecular mass of ~146 kDa on sodium dodecyl sulphate—polyacrylamide gel electrophoresis. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cys109 with Ser severely abrogated the Ub-releasing activity of USP47, indicating that USP47 is indeed a cysteine DUB. An assay using Ub dimer substrates showed that the enzyme cleaved a variety of isopeptide bonds between 2 Ub molecules, including the Lys48- and Lys63-linked isopeptide bonds. USP47 also released a Ub moiety from Lys48- and Lys63-linked polyUb chains. Of the inhibitors tested, N -ethylmaleimide, Zn ion and Ub aldehyde revealed a dose-dependent inhibition of USP47. In this study, clear differences in the enzymatic properties between USP47 and USP7 (the most closely related proteins among DUBs) were also found. Therefore, our results suggest that USP47 may play distinct roles in Ub-mediated cellular processes via DUB activity.

Description: P24 antigen is the main structural protein of HIV-1, its detection provide a means to aid the early diagnosis of HIV-1 infection. The aim of this study was to improve the selectivity and sensitivity of the HIV P24 diagnostic assay by developing a cohort of 9E8 affinity-matured antibodies through in vitro phage affinity maturation which was performed by complementarity determining region (CDR)-hot spot mutagenesis strategy. Antibody 9E8-491 had an affinity constant of 5.64 x 10 –11 M, which was 5.7-fold higher than that of the parent antibody (9E8). Furthermore, the affinity, sensitivity and specificity of 9E8-491 were higher than those of 9E8, which indicate that 9E8-491 is a good candidate detection antibody for HIV P24 assay. Structure analysis of matured variants revealed that most hydrogen bonds resided in HCDR3. Among the antibody–antigen predicted binding residues, Tyr 100A/100B was the original conserved residue that was commonly present in HCDR3 of 9E8 and variants. Arg 100 /Asp 100C was the major variant substitution that most likely influenced the binding differences among variants and 9E8 monoclonal antibody. Both efficient library panning and predicted structural data were in agreement that the binding residues were mostly located in HCDR3 and enabled identification of key residues that influence antibody affinity.

Description: Many skeletal diseases have common pathological phenotype of defective osteogenesis of bone marrow stromal cells (BMSCs), in which histone modifications play an important role. However, few studies have examined the dynamics of distinct histone modifications during osteogenesis. In this study, we examined the dynamics of H3K9/K14 and H4K12 acetylation; H3K4 mono-, di- and tri-methylation; H3K9 di-methylation and H3K27 tri-methylation in osteogenic genes, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase, bone sialoprotein and osteocalcin, during C3H10T1/2 osteogenesis. H3 and H4 acetylation and H3K4 di-methylation were elevated, and H3K9 di-methylation and H3K27 tri-methylation were reduced in osteogenic genes during C3H10T1/2 osteogenesis. C3H10T1/2 osteogenesis could be modulated by altering the patterns of H3 and H4 acetylation and H3K27 tri-methylation. In a glucocorticoid-induced osteoporosis mouse model, we observed the attenuation of osteogenic potential of osteoporotic BMSCs in parallel with H3 and H4 hypo-acetylation and H3K27 hyper-tri-methylation in Runx2 and Osx genes. When H3 and H4 acetylation was elevated, and H3K27 tri-methylation was reduced, the attenuated osteogenic potential of osteoporotic BMSCs was rescued effectively. These observations provide a deeper insight into the mechanisms of osteogenic differentiation and the pathophysiology of osteoporosis and can be used to design new drugs and develop new therapeutic methods to treat skeletal diseases.

Description: Dihydrouridine (D) is formed by tRNA dihydrouridine synthases (Dus). In mesophiles, multiple Dus enzymes bring about D modifications at several positions in tRNA. The extreme-thermophilic eubacterium Thermus thermophilus , in contrast, has only one dus gene in its genome and only two D modifications (D20 and D20a) in tRNA have been identified. Until now, an in vitro assay system for eubacterial Dus has not been reported. In this study, therefore, we constructed an in vitro assay system using purified Dus. Recombinant T. thermophilus Dus lacking bound tRNA was successfully purified. The in vitro assay revealed that no other factors in living cells were required for D formation. A dus gene disruptant ( dus ) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The dus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNA Phe transcript at 60°C, demonstrating that other modifications in tRNA are not essential for Dus activity. However, a comparison of the formation of D in native tRNA Phe purified from the dus strain and tRNA Phe transcript revealed that other tRNA modifications are required for D formation at high temperatures.

Description: Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae , with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc /LDH strains are expected to be of use for versatile analyses of human LDH.

Description: RelB is activated by the non-canonical NF-B pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb Venus / Venus mice developed without any abnormalities observed in the Relb –/– mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb Venus / Venus DC analyses revealed that DCs consist of RelB – , RelB low and RelB high populations. The RelB high population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB low population was in the cytoplasm. Although both the RelB low and RelB – populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB low than in the RelB – splenic conventional DCs. Taken together, our results identify the RelB low population as a possible novel intermediate maturation stage of cDCs and the Relb Venus / Venus mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-B pathway.

Description: Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea . Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

Description: The scaffolding protein Salvador (Sav) plays a key role in the Hippo (Hpo) signalling pathway, which controls tissue growth by inhibiting cell proliferation and promoting apoptosis. Dysregulation of the Hippo pathway contributes to cancer development. Since the identification of the first Sav gene in 2002, very little is known regarding the molecular basis of Sav-SARAH mediating interactions due to its insolubility. In this study, refolding of the first Sav (known as WW45)-SARAH provided insight into the biochemical and biophysical properties, indicating that WW45-SARAH exhibits properties of a disordered protein, when the domain was refolded at a neutral pH. Interestingly, WW45-SARAH shows folded and rigid conformations relative to the decrease in pH. Further, diffracting crystals were obtained from protein refolded under acidic pH, suggesting that the refolded WW45 protein at low pH has a homogeneous and stable conformation. A comparative analysis of molecular properties found that the acidic-stable fold of WW45-SARAH enhances a heterotypic interaction with Mst2-SARAH. In addition, using an Mst2 mutation that disrupts homotypic dimerization, we showed that the monomeric Mst2-SARAH domain could form a stable complex of 1:1 stoichiometric ratio with WW45 refolded under acidic pH.

Description: Hypercholesterolemia is one of the factors contributing to cardiovascular problems. Erythrocytes are known to contribute its cholesterol to atherosclerotic plaque. Our earlier study showed that erythrocytes overexpress chondroitin sulphate/dermatan sulphate (CS/DS), a linear co-polymer, during diabetes which resulted in increased cytoadherence to extracellular matrix (ECM) components. This study was carried out to determine whether diet-induced hypercholesterolemia had any effect on erythrocyte CS/DS and impacted cytoadherence to ECM components. Unlike in diabetes, diet-induced hypercholesterolemia did not show quantitative changes in erythrocyte CS/DS but showed difference in proportion of un-sulphated and 4- O -sulphated disaccharides. Erythrocytes from hypercholesterolemic rats showed increased adhesion to ECM components which was abrogated to various extents when subjected to chondroitinase ABC digestion. However, isolated CS/DS chains showed a different pattern of binding to ECM components indicating that orientation of CS/DS chains could be playing a role in binding.

Description: The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 x 10 7 from cDNA of peripheral blood mononuclear cells of an alpaca ( Lama pacos ) immunized with a fragment of IZUMO1 (IZUMO1 PFF ) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (〉93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.

Description: The autophosphorylation of specific tyrosine residues occurs in the cytoplasmic region of the insulin receptor (IR) upon insulin binding, and this in turn initiates signal transduction. The R3 subfamily (Ptprb, Ptprh, Ptprj and Ptpro) of receptor-like protein tyrosine phosphatases (RPTPs) is characterized by an extracellular region with 6–17 fibronectin type III-like repeats and a cytoplasmic region with a single phosphatase domain. We herein identified the IR as a substrate for R3 RPTPs by using the substrate-trapping mutants of R3 RPTPs. The co-expression of R3 RPTPs with the IR in HEK293T cells suppressed insulin-induced tyrosine phosphorylation of the IR. In vitro assays using synthetic phosphopeptides revealed that R3 RPTPs preferentially dephosphorylated a particular phosphorylation site of the IR: Y960 in the juxtamembrane region and Y1146 in the activation loop. Among four R3 members, only Ptprj was co-expressed with the IR in major insulin target tissues, such as the skeletal muscle, liver and adipose tissue. Importantly, the activation of IR and Akt by insulin was enhanced, and glucose and insulin tolerance was improved in Ptprj -deficient mice. These results demonstrated Ptprj as a physiological enzyme that attenuates insulin signalling in vivo , and indicate that an inhibitor of Ptprj may be an insulin-sensitizing agent.

Description: The diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 (A.7120) differentiates into specialized heterocyst cells that fix nitrogen under nitrogen starvation conditions. Although reducing equivalents are essential for nitrogen fixation, little is known about redox systems in heterocyst cells. In this study, we investigated thioredoxin (Trx) networks in Anabaena using TrxM, and identified 16 and 38 candidate target proteins in heterocysts and vegetative cells, respectively, by Trx affinity chromatography (Motohashi et al. (Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc Natl Acad Sci USA , 2001; 98 , 11224–11229)). Among these, the Fe–S cluster scaffold protein NifU that facilitates functional expression of nitrogenase in heterocysts was found to be a potential TrxM target. Subsequently, we observed that the scaffold activity of N-terminal catalytic domain of NifU is enhanced in the presence of Trx-system, suggesting that TrxM is involved in the Fe–S cluster biogenesis.

Description: In this study, we examined the role of aminopeptidases with reference to endoplasmic reticulum aminopeptidase 1 (ERAP1) in nitric oxide (NO) synthesis employing murine macrophage cell line RAW264.7 cells activated by lipopolysaccharide (LPS) and interferon (IFN)- and LPS-activated peritoneal macrophages derived from ERAP1 knockout mouse. When NO synthesis was measured in the presence of peptides having N-terminal Arg, comparative NO synthesis was seen with that measured in the presence of Arg. In the presence of an aminopeptidase inhibitor amastatin, NO synthesis in activated RAW264.7 cells was significantly decreased. These results suggest that aminopeptidases are involved in the NO synthesis in activated RAW264.7 cells. Subsequently, significant reduction of NO synthesis was observed in ERAP1 knockdown cells compared with wild-type cells. This reduction was rescued by exogenously added ERAP1. Furthermore, when peritoneal macrophages prepared from ERAP1 knockout mouse were employed, reduction of NO synthesis in knockout mouse macrophages was also attributable to ERAP1. In the presence of amastatin, further reduction was observed in knockout mouse-derived macrophages. Taken together, these results suggest that several aminopeptidases play important roles in the maximum synthesis of NO in activated macrophages in a substrate peptide-dependent manner and ERAP1 is one of the aminopeptidases involved in the NO synthesis.

Description: Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits. Each subunit has two distinct maltooligosaccharide binding sites: a storage site and a catalytic site. Our characterization of the properties of these sites suggested that GP activity consists of two activities: (i) binding to the glycogen molecule and (ii) phosphorolysis of the non-reducing-end glucose residues. Activity (i) is mainly due to the activities of the two storage sites, which depended on the ionic strength of the medium and were directly inhibited by cyclodextrins (CDs). Activity (i) is of benefit to GP because a high concentration of non-reducing-end glucose residues is localized on the surface of the glycogen molecule. Activity (ii), the total activity of the two catalytic sites, exhibited relatively little ionic strength dependence. Because the combined activity of (i) and (ii) is deduced using glycogen as an assay substrate, the sole activity of (ii) must be measured using small maltooligosyl-substrates. By using a very low concentration of pyridylaminated maltohexaose, we demonstrated that the GP catalytic sites are active even in the presence of CDs, and that the actions of the catalytic site and the storage site are independent of each other.

Description: O -GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O -GlcNAc transferase (OGT) and O -GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O -GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O -GlcNAcylated protein production in E. coli . The approach was validated using the CKII and p53 protein as control. This compatible dual plasmid system contains an arabinose-inducible OGT expression vector with a pUC origin and an isopropyl β - d -thiogalactopyranoside-inducible OGT target substrate expression vector bearing a p15A origin. The dual plasmid system produces recombinant proteins with varying O -GlcNAcylation levels by altering the inducer concentration. More importantly, the O -GlcNAcylation efficiency was much higher than the previously reported system. Altogether, we established an adjustable compatible dual plasmid system that can effectively yield O -GlcNAcylated proteins in E. coli .

Description: Active equi-paritioning of the F plasmid is achieved by its sopABC gene. SopA binds to the sopAB promoter region and SopB binds to sopC . SopA also polymerizes in the presence of ATP and Mg(II), which is stimulated by SopB. Non-specific DNA is known to inhibit SopA polymerization and disassemble SopA polymer. This study followed kinetics of polymerization and de-polymerization of SopA by turbidity measurement and found new effects by DNA and SopB. Plasmid DNA, at low concentrations, shortened the lag (nucleation) phase of SopA polymerization and also caused an initial ‘burst’ of turbidity. Results with two non-specific 20-bp DNAs indicated sequence/length dependence of these effects. sopAB operator DNA only showed inhibition of SopA polymerization. Results of turbidity decrease of pre-formed SopA polymer in the presence of ethylenediaminetetraacetic acid showed that SopB also accelerates disassembly of the SopA polymer. The steady-state level of turbidity in the presence of SopB and plasmid DNA indicated synergy between SopB and DNA in the disassembly. SopB protein showed no effect on SopA polymerization, when SopB was specifically bound to DNA. This result and others with truncation mutants of SopB suggested that a proper configuration of the domains of SopB is important for SopA-SopB interactions.

Description: Influenza A virus (IAV) has been raising public health and safety concerns worldwide. Cyanovirin-N (CVN) is a prominent anti-IAV candidate, but both cytotoxicity and immunogenicity have hindered the development of this protein as a viable therapy. In this article, linker-CVN (LCVN) with a flexible and hydrophilic polypeptide at the N-terminus was efficiently produced from the cytoplasm of Escherichia coli at a 〉15-l scale. PEGylation at the N-terminal α-amine of LCVN was also reformed as 20 kDa PEGylated linkered Cyanovirin-N (PEG 20k –LCVN). The 50% effective concentrations of PEG 20k –LCVN were 0.43 ± 0.11 µM for influenza A/HK/8/68 (H3N2) and 0.04 ± 0.02 µM for A/Swan/Hokkaido/51/96 (H5N3), dramatically lower than that of the positive control, Ribavirin (2.88 ± 0.66 x 10 3 µM and 1.79 ± 0.62 x 10 3 µM, respectively). A total of 12.5 µM PEG 20k –LCVN effectively inactivate the propagation of H3N2 in chicken embryos. About 2.0 mg/kg/day PEG 20k –LCVN increased double the survival rate (66.67%, P = 0.0378) of H3N2 infected mice, prolonged the median survival period, downregulated the mRNA level of viral nuclear protein and decreased (attenuated) the pathology lesion in mice lung. A novel PEGylated CVN derivative, PEG 20k –LCVN, exhibited potent and strain-dependent anti-IAV activity in nanomolar concentrations in vitro, as well as in micromolar concentration in vivo .

Description: The semi-filamentous multicellular cyanobacterium Limnothrix / Pseudanabaena sp. strain ABRG5-3 undergoes autolysis, which involves the accumulation of polyphosphate compounds and disintegration of thylakoid membranes in cells, as a unique feature that occurs due to growth conditions. In this study, the overexpression and easy recovery of alkane (a saturated hydrocarbon, C 17 H 36 ) as a biofuel were examined in recombinants of the cyanobacteria ABRG5-3 and Synechocystis sp. strain PCC6803. The results obtained indicated that the accumulated mass of alkane accounted for ~50 or 60% of the dry weight of ABRG5-3 or PCC6803 recombinant cells, respectively. Furthermore, cultivating cells in liquid medium BG11 in which the nitrogen resource had been depleted promoted the production of alkane and cell lysis, resulting in the easy recovery of target products from the supernatant.

Description: Leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for Parkinson’s disease (PD). LRRK2 contains a kinase and a GTPase domain, both of which provide critical intracellular signal-transduction functions. We showed previously that Rab5b, a small GTPase protein that regulates the motility and fusion of early endosomes, interacts with LRRK2 and co-regulates synaptic vesicle endocytosis. Using recombinant proteins, we show here that LRRK2 phosphorylates Rab5b at its Thr6 residue in in vitro kinase assays with mass spectrophotometry analysis. Phosphorylation of Rab5b by LRRK2 on the threonine residue was confirmed by western analysis using cells stably expressing LRRK2 G2019S. The phosphomimetic T6D mutant exhibited stronger GTPase activity than that of the wild-type Rab5b. In addition, phosphorylation of Rab5b by LRRK2 also exhibited GTPase activity stronger than that of the unphosphorylated Rab5b protein. Two assays testing Rab5’s activity, neurite outgrowth analysis and epidermal growth factor receptor degradation assays, showed that Rab5b T6D exhibited phenotypes that were expected to be observed in the inactive Rab5b, including longer neurite length and less degradation of EGFR. These results suggest that LRRK2 kinase activity functions as a Rab5b GTPase activating protein and thus, negatively regulates Rab5b signalling.

Description: The cellular Src (c-Src) tyrosine kinase is upregulated and believed to play a pivotal role in various human cancers. However, the molecular mechanism underlying c-Src-mediated tumour progression remains elusive. Recent studies have revealed that several microRNAs (miRNAs) function as tumour suppressors by regulating the malignant expression of signalling molecules. Aberrant expression of miRNAs is frequently observed in human cancers and should be exploited to seek related molecular targets. In this review, we focus on miRNAs found to be involved in Src signalling in various cancers. We summarize recent findings on Src-related miRNAs, their target genes, mechanisms behind their interplay and their implications for cancer therapeutics.

Description: Tail-anchored (TA) proteins, a class of membrane proteins having an N-terminal cytoplasmic region anchored to the membrane by a single C-terminal transmembrane domain, are posttranslationally inserted into the endoplasmic reticulum (ER) membrane. In yeasts, the posttranslational membrane insertion is mediated by the Guided Entry of TA Proteins (GET) complex. Get3, a cytosolic ATPase, targets newly synthesized TA proteins to the ER membrane, where Get2 and Get3 constitute the Get3 receptor driving the membrane insertion. While mammalian cells employ TRC40 and WRB, mammalian homologs of Get3 and Get1, respectively, they lack the gene homologous to Get2. We recently identified calcium-modulating cyclophilin ligand (CAML) as a TRC40 receptor, indicating that CAML was equivalent to Get2 in the context of the membrane insertion. On the other hand, CAML has been well characterized as a signaling molecule that regulates various biological processes, raising the question of how the two distinct actions of CAML, the membrane insertion and the signal transduction, are assembled. In this review, we summarize recent progress of the molecular mechanism of the membrane insertion of TA proteins and discuss the possibility that CAML could sense the various signals at the ER membrane, thereby controlling TA protein biogenesis.

Description: In bacterial organisms, the oriC -independent primosome plays an essential role in replication restart after dissociation of the replication DNA-protein complex following DNA damage. PriC is a key protein component in the oriC -independent replication restart primosome. Our previous study suggested that PriC was divided into an N-terminal domain and a C-terminal domain, with the latter domain being the major contributor to single-stranded DNA (ssDNA) binding capacity. In this study, we prepared several PriC mutants in which basic and aromatic amino acid residues were mutated to alanine. Five of these residues, Arg107, Lys111, Phe118, Arg121 and Lys165 in the C-terminal domain, were shown to be involved in ssDNA binding. Moreover, we evaluated the binding of the PriC mutants to the ssDNA-binding protein (SSB) complex. Five residues, Phe118, Arg121, Arg129, Tyr152 and Arg155 in the C-terminal domain of PriC, were shown to be involved in SSB binding in the presence of ssDNA. On the basis of these results, we propose a structural model of the C-terminal domain of PriC and discuss how the interactions of PriC with SSB and ssDNA may contribute to the regulation of PriC-dependent replication restart.

Description: L -Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L -lysine to form α-keto--aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L -amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

Description: For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed low immunogenic streptavidin mutant No. 314 (LISA-314), was produced previously as a drug delivery tool. However, endogenous biotins (BTNs) with high affinity ( K d 〈 10 –10 M) for the binding pocket of LISA-314 prevents access of exogenous BTN-labelled anticancer drugs. In this study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous BTN species, therefore ensuring that the newly designed LISA-314 binds only artificial BTN analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3a S ,4 S ,6a R )-2-iminohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamido)hexanoic acid (iminobiotin long tail, IMNtail). Surface plasmon resonance results showed that V212 has a K d value of 5.9 x 10 –7 M towards IMNtail, but no binding affinity for endogenous BTN species. This V212/IMNtail system will be useful as a novel delivery tool for anticancer therapy.

Description: A number of gene mutations are detected in cells derived from human cancer tissues, but roles of these mutations in cancer cell development are largely unknown. We examined G364R mutation of MCM4 detected in human skin cancer cells. Formation of MCM4/6/7 complex is not affected by the mutation. Consistent with this notion, the binding to MCM6 is comparable between the mutant MCM4 and wild-type MCM4. Nuclear localization of this mutant MCM4 expressed in HeLa cells supports this conclusion. Purified MCM4/6/7 complex containing the G364R MCM4 exhibited similar levels of single-stranded DNA binding and ATPase activities to the complex containing wild-type MCM4. However, the mutant complex showed only 30–50% of DNA helicase activity of the wild-type complex. When G364R MCM4 was expressed in HeLa cells, it was fractionated into nuclease-sensitive chromatin fraction, similar to wild-type MCM4. These results suggest that this mutation does not affect assembly of MCM2-7 complex on replication origins but it interferes some step at function of MCM2-7 helicase. Thus, this mutation may contribute to cancer cell development by disturbing DNA replication.

Description: Importin α performs the indispensable role of ferrying proteins from the cytoplasm into the nucleus with a transport carrier, importin β1. Mammalian cells from mouse or human contain either six or seven importin α subtypes, respectively, each with a tightly regulated expression. Therefore, the combination of subtype expression in a cell defines distinct signaling pathways to achieve progressive changes in gene expression essential for cellular events, such as differentiation. Recent studies reveal that, in addition to nucleocytoplasmic transport, importin αs also serve non-transport functions. In this review, we first discuss the physiological significance of importin α as a nuclear transport regulator, and then focus on the functional diversities of importin αs based on their specific subcellular and cellular localizations, such as the nucleus and plasma membrane. These findings enrich our knowledge of how importin αs actively contribute to various cellular events.

Description: The structure of the complex of maize sulfite reductase (SiR) and ferredoxin (Fd) has been determined by X-ray crystallography. Co-crystals of the two proteins prepared under different conditions were subjected to the diffraction analysis and three possible structures of the complex were solved. Although topological relationship of SiR and Fd varied in each of the structures, two characteristics common to all structures were found in the pattern of protein-protein interactions and positional arrangements of redox centres; (i) a few negative residues of Fd contact with a narrow area of SiR with positive electrostatic surface potential and (ii) [2Fe-2S] cluster of Fd and [4Fe-4S] cluster of SiR are in a close proximity with the shortest distance around 12 Å. Mutational analysis of a total of seven basic residues of SiR distributed widely at the interface of the complex showed their importance for supporting an efficient Fd-dependent activity and a strong physical binding to Fd. These combined results suggest that the productive electron transfer complex of SiR and Fd could be formed through multiple processes of the electrostatic intermolecular interaction and this implication is discussed in terms of the multi-functionality of Fd in various redox metabolisms.

Description: Analysis of replicating mammalian mitochondrial DNA (mtDNA) suggested that initiation of the replication occurs not only at the specific position, Ori-H but also across a broad zone in mtDNA. We investigated relationship of mitochondrial transcription initiation which takes place upstream of Ori-H and mtDNA replication initiation through analysing the effect of knockdown of mitochondrial transcription factor B2, TFB2M and mitochondrial RNA polymerase, POLRMT, components of the transcription initiation complexes in cultured human cells. Under the conditions where suppression of the transcription initiation complexes was achieved by simultaneous depletion of TFB2M and POLRMT, decrease of replication intermediates of mtDNA RITOLS replication mode accompanied reduction in mtDNA copy number. On the other hand, replication intermediates of coupled leading and lagging strand DNA replication, another proposed replication mode, appeared to be less affected. The findings support the view that the former mode involves transcription from the light strand promoter (LSP), and suggest that initiation of the latter mode is independent from the transcription and has distinct regulation. Further, knockdown of TFB2M alone caused significant decrease of 7S DNA, which implies that transcription initiation complexes formed at the LSP engage 7S DNA synthesis more frequently than the initiation of productive replication and transcription.

Description: Cycas revoluta leaf lectin (CRLL) of mannose-recognizing jacalin-related lectin (mJRL) has two tandem repeated carbohydrate recognition domains, and shows the characteristic sugar-binding specificity toward high mannose-glycans, compared with other mJRLs. We expressed the N-terminal domain and C-terminal domain (CRLL-N and CRLL-C) separately, to determine the fine sugar-binding specificity of each domain, using frontal affinity chromatography, glycan array and equilibrium dialysis. The specificity of CRLL toward high mannose was basically derived from CRLL-N, whereas CRLL-C had affinity for α1-6 extended mono-antennary complex-type glycans. Notably, the affinity of CRLL-N was most potent to one of three Man 8 glycans and Man 9 glycan, whereas the affinity of CRLL-C decreased with the increase in the number of extended α1-2 linked mannose residue. The recognition of the Man 8 glycans by CRLL-N has not been found for other mannose recognizing lectins. Glycan array reflected these specificities of the two domains. Furthermore, it was revealed by equilibrium dialysis method that the each domain had two sugar-binding sites, similar with Banlec, banana mannose-binding Jacalin-related lectin.

Description: The ICH S6(R1) recommendations on safety evaluation of biotherapeutics have led to uncertainty in determining what would constitute a cause for concern that would require genotoxicity testing. A Health and Environmental Sciences Institute’s Genetic Toxicology Technical Committee Workgroup was formed to review the current practice of genotoxicity assessment of peptide/protein-related biotherapeutics. There are a number of properties of peptide/protein-related biotherapeutics that distinguish such products from traditional ‘small molecule’ drugs and need to be taken into consideration when assessing whether genotoxicity testing may be warranted and if so, how to do it appropriately. Case examples were provided by participating companies and decision trees were elaborated to determine whether and when genotoxicity evaluation is needed for peptides containing natural amino acids, non-natural amino acids and other chemical entities and for unconjugated and conjugated proteins. From a scientific point of view, there is no reason for testing peptides containing exclusively natural amino acids irrespective of the manufacturing process. If non-natural amino acids, organic linkers and other non-linker chemical components have already been tested for genotoxicity, there is no need to re-evaluate them when used in different peptide/protein-related biotherapeutics. Unless the peptides have been modified to be able to enter the cells, it is generally more appropriate to evaluate the peptides containing the non-natural amino acids and other non-linker chemical moieties in vivo where the cleavage products can be formed. For linkers, it is important to determine if exposure to reactive forms are likely to occur and from which origin. When the linkers are anticipated to be potential mutagenic impurities they should be evaluated according to ICH M7. If linkers are expected to be catabolic products, it is recommended to test the entire conjugate in vivo , as this would ensure that the relevant ‘free’ linker forms stemming from in vivo catabolism are tested.

Description: Genome sequences that contain tandem repeats of guanine can form stable four-stranded structures known as G-quadruplex, or G4 DNA. While the molecular mechanisms are not fully defined, such guanine-rich loci are prone to mutagenesis and recombination. Various repair pathways function to reduce the potential for genome instability by correcting base damage and replication errors; however, it is not yet fully defined how well these processes function at G4 DNA. One frequent form of base damage occurs from cytidine deamination, resulting in deoxyuracil and UG mismatches. In duplex and single-stranded DNA, uracil bases are recognised and excised by uracil glycosylases. Here, we tested the efficiency of uracil glycosylase activity in vitro on uracil bases located directly adjacent to guanine repeats and G4 DNA. We show that uracil excision by bacterial UDG and human hUNG2 is reduced at uracils positioned directly 5' or 3' of a guanine tetrad. Control reactions using oligonucleotides disrupted for G4 formation or reaction conditions that do not favour G4 formation resulted in full uracil excision activity. Based on these in vitro results, we suggest that folding of guanine-rich DNA into G4 DNA results in a DNA conformation that is resistant to uracil glycosylase-initiated repair and this has the potential to increase the risk of instability at guanine repeats in the genome.

Description: Galectin-4 is a member of the galectin family which consists of 15 galactoside-binding proteins. Previously, galectin-4 has been shown to have a role in cancer progression and metastasis and it is found upregulated in many solid tumours, including colorectal cancer (CRC). Recently, the role in the metastatic process was suggested to be via promoting cancer cells to adhere to blood vascular endothelium. In the present study, the regulatory region of LGALS4 (galectin-4) in seven colon cell lines was investigated with respect to genetic variation that could be linked to expression levels and therefore a tumourigenic effect. Interestingly, qRT-PCR and sequencing results revealed that galectin-4 upregulation is associated with SNPs rs116896264 and rs73933062. By use of luciferase reporter- and pull-down assays, we confirmed the association between the gene upregulation and the two SNPs. Also, using pull-down assay followed by mass spectrometry, we found that the presence rs116896264 and rs73933062 is changing transcription factors binding sites. In order to assess the frequencies of the two SNPs among colon cancer patients and healthy individuals, we genotyped 75 colon cancer patients, 12 patients with adenomatous polyposis and 17 patients with ulcerative colitis and we performed data mining in the 1000 genomes databank. We found the two SNPs co-occuring in 21% of 75 CRC patients, 0 out of 12 patients of adenomatous polyposis, and 6 out of 17 patients (35%) with ulcerative colitis. Both in the patient samples and in the 1000 genomes project, the two SNPs were found to co-occur whenever present (D' = 1).

Description: The dose effect between nucleoplasmic bridges (NPB) and relatively low doses of ionising radiation remains unknown. Accordingly, this study investigated the NPB frequencies in human peripheral blood lymphocytes exposed to low-dose 60 Co -rays. Complex anomalies, including fused nuclei (FUS), horse-shoe nuclei (HS) and circular nuclei (CIR), which possibly originated from multiple NPBs, were also scored. Human peripheral blood samples were collected from three healthy males and irradiated with 0–1 and 0–0.4 Gy 60 Co -rays. A cytokinesis-block micronucleus cytome assay was then conducted to analyse NPB, PFHC (NPB plus three complex nuclear anomalies) and micronucleus (MN) in binucleated cells. All dose–response curves followed the linear model for both NPB frequency and PFHC cell frequency. The dose–response curves between NPB frequency and absorbed dose at 0–1 and 0–0.4 Gy were y = 0.0037 x + 0.0005 ( R 2 = 0.979, P 〈 0.05) and y = 0.0043 x + 0.0004 ( R 2 = 0.941, P 〈 0.05), respectively. The dose–response curves between PFHC cell frequency and absorbed dose at 0–1 and 0–0.4 Gy were y = 0.0044 x + 0.0007 ( R 2 = 0.982, P 〈 0.05) and y = 0.0059 x + 0.0005 ( R 2 = 0.969, P 〈 0.05), respectively. The statistical significance of differences between the irradiated groups (0–0.4 Gy) and background levels of NPB, PFHC and MN were also analysed. The lowest analysable doses of NPB, PFHC and MN were 0.12, 0.08 and 0.08 Gy, respectively. In conclusion, NPBs and PFHC positively correlated with the absorbed radiation at a relatively low dose.

Description: The aim of the study was to investigate how coadministration of resveratrol (RSV) at different time after the start of irradiation influences the frequency of micronuclei (MN) in reticulocytes of bone marrow and peripheral blood, and if the RSV supplementation after termination of irradiation may influence the recovery process of damaged cells. Coadministration of RSV with 1-day delay after 1 Gy irradiation significantly decreased the levels of MN in bone marrow and in peripheral blood, whereas with 1-week delay, only in bone marrow reticulocytes. Above combined treatment did not improve the process of recovery. RSV supplementation with 1-day delay relatively to 0.5 Gy irradiation, significantly decreased the frequencies of MN, especially after coadministration with 28mg/kg bw of RSV. Coadministration of RSV since eighth day did not influence the frequencies of MN compared to irradiated cells. The recovery process in the presence of RSV proceeded faster. Supplementation of RSV following initiation of irradiation is beneficial in case of irradiation with lower doses. RSV should be supplemented as soon as possible. Supplementation of RSV after termination of irradiation significantly speed up the recovery. Current results confirmed the ability of RSV to mitigate the effect of irradiation.

Description: Various naturally occurring stilbene-like compounds that are related to resveratrol (RSV) possess some of the beneficial effects of the parent molecule and provide even further benefits. Therefore, a series of methoxylated analogues of RSV were prepared with the aim of increasing antitumour and proapoptotic activity. In a previous article, we studied two methoxy-derivatives, pterostilbene (PTERO) and trimethoxystilbene (TRIMETHOXY), in which the first was formed by the substitution of two hydroxyl groups with two methoxy groups ( trans -3,5-dimethoxy-4'-hydroxystilbene) and the second was formed by the replacement of all three OH groups with methoxy groups ( trans -3,5,4'-trimethoxystilbene). Both methoxy-derivatives showed stronger antioxidant activity when compared with RSV. In the present article, we focused on the analysis of the ability of RSV and its two methoxylated derivatives to protect proliferating non-tumoural cells from the damage induced by ionising radiation (IR). First we showed that the methoxy derivatives, contrary to their parental compound, are unable to affect topoisomerase enzyme and consequently are not clastogenic per se . Second we showed that both PTERO and TRIMETHOXY more efficiently reduce the chromosome damage induced by IR. Furthermore, TRIMETHOXY, but not PTERO, causes a delay in cell proliferation, particularly in mitosis progression increasing the number of cells in metaphase at the expense of prophases and ana/telophases.

Description: α-, β- and -asarone are naturally occurring phenylpropenes that occur in different plant families, mainly in Aristolochiaceae , Acoraceae and Lauraceae. Plants containing asarones are used as flavouring ingredients in alcoholic beverages (bitters), traditional phytomedicines and the rhizome of e.g. Acorus calamus is used to prepare tea. Although α- and β-asarone show a potential in the treatment of several diseases, previous studies have shown carcinogenicity in rodents (duodenum, liver). However, the mechanism of action remained unclear. Studies on the mutagenicity of propenylic α- and β-asarone are inconsistent and data on carcinogenicity and genotoxicity of allylic -asarone are lacking completely. Thus, the present study determined the mutagenicity of the three asarone isomers using the Ames fluctuation assay with and without exogenous metabolic activation (S9 mix) in the standard Salmonella typhimurium strains TA98 and TA100. A concentration dependent increase in mutagenicity could be verified for α- and β-asarone in strain TA100 in the presence of rat liver homogenate. The side-chain epoxides of α- and β-asarone, major metabolites formed in liver microsomes, caused mutations in TA100, supporting the hypothesis that epoxidation of the side chain plays a key role in mutagenicity of the propenylic alkenylbenzenes. The allylic -asarone, not undergoing detectable side-chain epoxidation in liver microsomes, was supposed to be activated via side-chain hydroxylation and further sulphonation, a typical pathway for other allylic alkenylbenzenes like estragole or methyleugenol. However, neither y-asarone nor 1'-OH--asarone showed any mutagenic effect even in the human SULT-expressing Salmonella strains (TA100-hSULT1A1 and TA100-hSULT1C2), while 1'-OH-methyleugenol used as a positive control was mutagenic under these conditions. These results indicate that the propenylic asarones are genotoxic via metabolic formation of side-chain epoxides while the side-chain hydroxylation/sulphonation pathway is either not operative or does not lead to mutagenicity with the allylic -asarone.

Description: Prior to the downstream development of chemical substances, including pharmaceuticals and cosmetics, their influence on the genetic apparatus has to be tested. Several in vitro and in vivo assays have been developed to test for genotoxicity. In a first tier, a battery of two to three in vitro tests is recommended to cover mutagenicity, clastogenicity and aneugenicity as main endpoints. This regulatory in vitro test battery is known to have a high sensitivity, which is at the expense of the specificity. The high number of false positive in vitro results leads to excessive in vivo follow-up studies. In the case of cosmetics it may even induce the ban of the particular compound since in Europe the use of experimental animals is no longer allowed for cosmetics. In this article, an alternative approach to derisk a misleading positive Ames test is explored. Hereto we first tested the performance of five existing computational tools to predict the potential mutagenicity of a data set of 132 cosmetic compounds with a known genotoxicity profile. Furthermore, we present, as a proof-of-principle, a strategy in which a combination of computational tools and mechanistic information derived from in vitro transcriptomics analyses is used to derisk a misleading positive Ames test result. Our data shows that this strategy may represent a valuable tool in a weight-of-evidence approach to further evaluate a positive outcome in an Ames test.

Description: Pioglitazone (PTZ) is an oral antidiabetic agent whose anti-cancer properties have been described recently. Since PTZ increases the production of reactive oxygen species in mammalian cells, the aim of current study was to evaluate the cytotoxic, mutagenic and recombinogenic effects of PTZ using respectively the in vitro mitotic index assay and the in vitro mammalian cell micronucleus test in human peripheral lymphocytes, and the in vivo homozygotization assay in Aspergillus nidulans , which detects the loss of heterozygosity due to somatic recombination. Although the lowest PTZ concentrations (4–36 μM) did not show any significant rise in the micronucleus production, the higher PTZ concentration (108 μM) produced a statistically higher number of micronuclei than the negative control and significantly altered the cell-proliferation kinetics, demonstrating the mutagenic and antiproliferative effects of PTZ, respectively. The recombinogenic activity of PTZ, demonstrated here for the first time, was observed at the highest tested concentration (400 μM) through the homozygotization index rates significantly different from the negative control. Taken together, our results show that PTZ is genotoxic at a concentration higher than the therapeutic plasma concentration. This PTZ genotoxicity may be a potential benefit to its previously described antitumour activity.

Description: Environmental pollutants are complex mixtures in which metals are ubiquitous. Metal mixtures of arsenic, cadmium and lead are present in the occupational environment and generate health effects such as cardiovascular, renal and cancer diseases. Cell transformation induced by metal mixtures that depend on reactive oxygen species (ROS) generation, cell viability maintenance and avoidance of senescence was previously reported by our group. The aim of the present study was to explore the role of a Obg-like ATPase1 (OLA1) in the cell transformation of BALB/c 3T3 A31-1-1 clonal cells induced by a metal mixture (2 µM NaAsO 2 , 2 µM CdCl 2 and 5 µM Pb(C 2 H 3 O 2 ) 2 . 3H 2 O) through ROS generation. The interest in OLA1 is justified because this protein has been proposed to be a negative regulator of the cellular antioxidant response. Small interfering RNA (siRNA) was used to knockdown OLA1 before the initiation stage of the transformation assay. We evaluated (ROS) and OLA1 protein expression throughout the initiation and promotion stages of transformation. OLA1 knockdown modulated metal mixture-induced cell transformation more strongly when the metal mixture was an initiator stimulus than when it was a promoter. The ability of the metal mixture to initiate cell transformation was diminished by OLA1 knockdown, an effect that depended on intracellular ROS levels. The effect of OLA1 was synergistic with N -Acetyl- l -cysteine (NAC) co-treatment. Oxidative stress-associated transcription factors Egr1 and Smad were also down-regulated by the OLA1 knockdown, contributing to the rescue of metal mixture cell transformation.

Description: G-quadruplexes (G4) are highly stable tetra-stranded DNA secondary structures known to mediate gene regulation and to trigger genomic instability events during replication. G4 structural stability can be affected by DNA methylation and oxidation modifications; thus nutrients such as folate that have the ability to alter these processes could potentially modify the genomic occurrence of G4 elements. Hela cells were cultured in a range of folate concentrations or in the presence or absence of 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor. G4 structures were then quantified by immunofluorescence using an automated quantitative imaging system. G4 frequency in Hela cells and nuclei area mean were increased in 20nM folate medium compared with 2000nM folate, as well as in the presence of 5-aza-2'-deoxycytidine when compared to cells non-exposed to 5-aza-2'-deoxycytidine. These changes were exacerbated when pyridostatin, a G4 stabilising ligand, was added to the culture medium. G4 intensity in Hela cells cultured in deficient folate condition with pyridostatin was highly correlated with DNA damage as measured by H2AX immunofluorescence ( r = 0.71). This study showed for the first time that cellular G4 balance is modifiable by low folate concentrations and that these changes may occur as a consequence of DNA hypomethylation. Although the exact mechanism by which these changes occur is unclear, these findings establish the possibility that nutrients could be utilised as a tool for sustaining genome integrity by modifying G4 frequency at a cellular level.

Description: Metal oxide nanoparticles (NPs), including zinc oxide (ZnO) NPs have shown success for use as vehicles for drug delivery and targeting gene delivery in many diseases like cancer. Current anticancer chemotherapeutics fail to effectively differentiate between cancerous and normal cells. There is an urgent need to develop novel drug delivery system that can better target cancer cells while sparing normal cells and tissues. Particularly, ZnO NPs exhibit a high degree of cancer cell selectivity and induce cell death, oxidative stress, interference with the cell cycle progression and genotoxicity in cancerous cells. In this scenario, effective cellular uptake of NP seems to be crucial, which is shown to be affected by cell cycle progression. In the present study, the cytotoxic potential of ZnO NPs and the effect of different cell cycle phases on the uptake of ZnO NPs were examined in A431 cells. It is shown that the ZnO NPs led to cell death and reactive oxygen species generation and were able to induce cell cycle arrest in S and G 2 /M phase with the higher uptake in G 2 /M phase compared with other phases.

Description: Malondialdehyde (MDA), a biomarker of lipid peroxidation and oxidative stress, is a mutagenic and carcinogenic compound that can react with DNA to form several types of DNA adducts including the deoxyguanosine adduct (M 1 dG). The aim of this cross-sectional study was to evaluate the association between individual dietary and lifestyle habits and M 1 dG levels, measured in peripheral leukocytes in a large representative sample of the general population of Florence City (Italy). Selected anthropometric measurements, detailed information on dietary and lifestyle habits and blood samples were available for 313 adults of the Florence City Sample enrolled in the frame of European Prospective Investigation into Cancer and nutrition (EPIC) study. A multivariate regression analysis adjusted for selected individual characteristics possibly related to M 1 dG levels (sex, age, BMI, smoke, physical activity level, education level, total caloric intake and a Mediterranean dietary score) was performed to estimate the association between these parameters and M 1 dG levels. M 1 dG levels were significantly higher in women ( P = 0.014) and lower in moderately active or active subjects ( P = 0.037).We also found a significant inverse association with the Modified Mediterranean dietary score ( P for trend = 0.049), particularly evident for the highest categories of adherence. Our results indicate that M 1 dG levels can be modulated by selected individual characteristics such as gender, physical activity and a Mediterranean dietary pattern.

Description: A requirement for advancing antibody-based medicine is the development of proteins that can bind with high affinity to a specific epitope related to a critical protein activity site. As a part of generating such proteins, we have succeeded in creating a binding protein without changing epitope by complementarity-determining region 3 (CDR3) grafting (Inoue et al. , Affinity transfer to a human protein by CDR3 grafting of camelid VHH. Protein Sci. 20, 1971–1981). However, the affinity of the target-binding protein was low. In this manuscript, the affinity maturation of a target-binding protein was examined using CDR3-grafted camelid single domain antibody (VHH) as a model protein. Several amino acids in the CDR1 and CDR2 regions of VHH were mutated to tyrosines and/or serines and screened for affinity-matured proteins by using in silico analysis. The mutation of two amino acids in the CDR2 region to arginine and/or aspartic acid increased the affinity by decreasing the dissociation rate. The affinity of designed mutant increased by ~20-fold over that of the original protein. In the present study, candidate mutants were narrowed down using in silico screening and computational modelling, thus avoiding much in vitro analytical effort. Therefore, the method used in this study is expected to be one of the useful for promoting affinity maturation of antibodies.

Description: A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ~30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Description: The tripartite motif (TRIM) or RBCC proteins are characterized by the TRIM composed of a RING finger, B-box and coiled-coil domains. TRIM proteins often play roles in the post-translational protein modification, including ubiquitylation and other ubiquitin-like modifications. Evidence has accumulated in regard to the contribution of TRIM proteins to diverse cellular processes, including such as cell cycle progression, apoptosis, immunity and transcriptional regulation. In particular, some of the TRIM proteins have been characterized to exert oncogenic or tumour suppressor-like functions depending on the context. A recent report by Inoue and his colleagues has revealed that Terf/TRIM17 stimulates the degradation of a kinetochore protein ZWINT and regulates the proliferation of breast cancer cells. Terf has also been paid attention as a factor promoting neuronal apoptosis, by degrading a Bcl2-like anti-apoptotic protein Mcl-1. Like aircraft trim tabs, TRIM proteins trim the balance of homoeostasis by modulating various biological pathways through protein–protein interactions.

Description: Cyclin-dependent kinase (CDK) that plays a central role in preventing re-replication of DNA phosphorylates several replication proteins to inactivate them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo . There are inversed correlations between the phosphorylation of these proteins and their chromatin binding. Here, we examined in vitro phosphorylation of human replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases. MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was examined by gel-shift analysis. The phosphorylation of RPA did not affect its DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of six amino acids of serine and threonine to alanines in the amino-terminal region of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These biochemical data suggest that phosphorylation of MCM4 at these sites by CDK plays a direct role in dislodging MCM2-7 from chromatin and/or preventing re-loading of the complex to chromatin.

Description: To determine the effects of alcohols on the low-frequency local motions that control slow changes in structural dynamics of native-like compact states of proteins, we have studied the effects of alcohols on structural fluctuation of M80-containing -loop by measuring the rate of thermally driven CO dissociation from a natively folded carbonmonoxycytochrome c under varying concentrations of alcohols (methanol, ethanol, 1-propanol, 2-propanol, 3°-butanol, 2,2,2-trifluoroethanol). As alcohol is increased, the rate coefficient of CO dissociation ( k diss ) first decreases in subdenaturing region and then increases on going from subdenaturing to denaturing milieu. This decrease in k diss is more for 2,2,2-trifluroethanol and 1-propanol and least for methanol, indicating that the first phase of motional constraint is due to the hydrophobicity of alcohols and intramolecular protein cross-linking effect of alcohols, which results in conformational entropy loss of protein. The thermal denaturation midpoint for ferrocytochrome c decreases with increase in alcohol, indicating that alcohol decrease the global stability of protein. The stabilization free energy ( G ) in alcohols’ solution was calculated from the slope of the Wyman–Tanford plot and water activity. The m -values obtained from the slope of G versus alcohols plot were found to be more negative for longer and linear chain alcohols, indicating destabilization of proteins by alcohols through disturbance of hydrophobic interactions and hydrogen bonding.

Description: Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-B (NF-B) signalling. NF-B is crucial for inflammatory and immune responses, and aberrant NF-B signalling is implicated in multiple disorders. We found that linear ubiquitin chain assembly complex (LUBAC), composed of HOIL-1L, HOIP and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-B pathway. Moreover, specific deubiquitinases, such as CYLD, A20 (TNFAIP3) and OTULIN/gumby, inhibit LUBAC-induced NF-B activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B-cell lymphomas and Parkinson’s disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-B activation pathway regulation by LUBAC.

Description: We screened circadian-regulated genes in rat cartilage by using a DNA microarray analysis. In rib growth-plate cartilage, numerous genes showed statistically significant circadian mRNA expression under both 12:12 h light–dark and constant darkness conditions. Type II collagen and aggrecan genes—along with several genes essential for post-translational modifications of collagen and aggrecan, including prolyl 4-hydroxylase 1, lysyl oxidase, lysyl oxidase-like 2 and 3'-phosphoadenosine 5'-phosphosulphate synthase 2—showed the same circadian phase. In addition, the mRNA level of SOX9, a master transcription factor for the synthesis of type II collagen and aggrecan, has a similar phase of circadian rhythms. The circadian expression of the matrix-related genes may be critical in the development and the growth of various cartilages, because similar circadian expression of the matrix-related genes was observed in hip joint cartilage. However, the circadian phase of the major matrix-related genes in the rib permanent cartilage was almost the converse of that in the rib growth-plate cartilage under light–dark conditions. We also found that half of the oscillating genes had conserved clock-regulatory elements, indicating contribution of the elements to the clock outputs. These findings suggest that the synthesis of the cartilage matrix macromolecules is controlled by cell-autonomous clocks depending upon the in vivo location of cartilage.

Description: Human chromosome 7 open reading frame 24 (C7orf24)/-glutamyl cyclotransferase has been suggested to be a potential diagnostic marker for several cancers, including carcinomas in the bladder urothelium, breast and endometrial epithelium. We here investigated the epigenetic regulation of the human C7orf24 promoter in normal diploid ARPE-19 and IMR-90 cells and in the MCF-7 and HeLa cancer cell lines to understand the transcriptional basis for the malignant-associated high expression of C7orf24. Chromatin immunoprecipitation analysis revealed that histone modifications associated with active chromatin were enriched in the proximal region but not in the distal region of the C7orf24 promoter in HeLa and MCF-7 cells. In contrast, elevated levels of histone modifications leading to transcriptional repression and accumulation of heterochromatin proteins in the C7orf24 promoter were observed in the ARPE-19 and IMR-90 cells, compared to the levels in HeLa and MCF-7 cancer cells. In parallel, the CpG island of the C7orf24 promoter was methylated to a greater extent in the normal cells than in the cancer cells. These results suggest that the transcriptional silencing of the C7orf24 gene in the non-malignant cells is elicited through heterochromatin formation in its promoter region; aberrant expression of C7orf24 associated with malignant alterations results from changes in chromatin dynamics.

Description: Abscisic acid (ABA) is a stress-inducible plant hormone comprising an inevitable component of the human diet. Recently, stress-induced accumulation of autocrine ABA was shown in humans, as well as ABA-mediated modulation of a number of disease-associated systems. Now, the application of a chemical proteomics approach to gain further insight into ABA mechanisms of action in mammalian cells is reported. An ABA mimetic photoaffinity probe was applied to intact mammalian insulinoma and embryonic cells, leading to the identification of heat shock protein 70 (HSP70) family members, (including GRP78 and HSP70-2) as putative human ABA-binding proteins. In vitro characterization of the ABA–HSP70 interactions yielded K d s in the 20–60 µM range, which decreased several fold in the presence of co-chaperone. However, ABA was found to have only variable- and co-chaperone-independent effects on the ATPase activity of these proteins. The potential implications of these ABA–HSP70 interactions are discussed with respect to the intracellular protein folding and extracellular receptor-like activities of these stress-inducible proteins. While mechanistic and functional relevance remain enigmatic, we conclude that ABA can bind to human HSP70 family members with physiologically relevant affinities and in a co-chaperone-dependent manner.

Description: Determination of the mode of action of carcinogenic agents is an important factor in risk assessment and regulatory practice. To assess the ability of the erythrocyte-based Pig-a mutation assay to discriminate between genotoxic and non-genotoxic modes of action, the mutagenic response of Sprague Dawley rats exposed to methyl carbamate (MC) or ethyl carbamate (EC) was investigated. EC, a potent carcinogen, is believed to induce DNA damage through the formation of a DNA-reactive epoxide group, whereas the closely structurally related compound, MC, cannot form this epoxide and its weaker carcinogenic activity is thought to be secondary to inflammation and promotion of cell proliferation. The frequency of Pig-a mutant phenotype cells was monitored before, during, and after 28 consecutive days of oral gavage exposure to either MC (doses ranging from 125 to 500mg/kg/day) or EC (250mg/kg/day). Significant increases in the frequency of mutant reticulocytes were observed from Days 15 through 43, with a peak mean frequency of 19.9 x 10 –6 on Day 29 (i.e. 24.9-fold increase relative to mean vehicle control across all four sampling times). As expected, mutant erythrocyte responses lagged behind mutant reticulocyte responses, with a maximal mean frequency of 8.2 x 10 –6 on Day 43 (i.e. 16.4-fold increase). No mutagenic effects were observed with MC. A second indicator of in vivo genotoxicity, peripheral blood micronucleated reticulocytes, was also studied. This endpoint was responsive to EC (3.3-fold mean increase), but not to MC. These results support the hypothesis that genotoxicity contributes to the carcinogenicity of EC but not of MC, and illustrates the value of the Pig-a assay for discriminating between genotoxic and non-genotoxic modes of action.

Description: As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RET CD59– and RBC CD59– , respectively) in peripheral blood of male Sprague Dawley ® rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RET CD59– and RBC CD59– (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies.

Description: The Pig-a assay has shown promise as a regulatory assay for evaluating in vivo gene mutation. A recent International Workshop on Genotoxicity Testing workgroup discussed the state of the assay and identified several knowledge gaps in assay development. This Mutagenesis Special Topic includes a collection of reports that addresses some of these knowledge gaps, including identifying the mutations responsible for the Pig-a mutant phenotype, the effect of sex on the response, probing the robustness of the assay and expanding the number of agents tested in the assay, especially agents expected to yield negative responses.

Description: The Pig-a assay has rapidly gained international interest as a useful tool for assessing the mutagenic potential of compounds in vivo . Although a large number of compounds, including both mutagens and non-mutagens, have been tested in the rat Pig-a assay in haematopoietic cells, there is limited understanding of how perturbations in haematopoiesis affect assay performance. Of particular concern is the possibility that regenerative haematopoiesis alone, without exposure to a genotoxic agent, could result in elevated Pig-a mutant cell frequencies. To address this concern, Wistar-Han rats were dosed by oral gavage with a non-genotoxic haemolytic agent, 2-butoxyethanol (2-BE). Dose levels ranging from 0 to 450mg/kg were tested using both single administration and 28-day treatment regimens. Haematology parameters were assessed at minimum within the first 24h of treatment and 8 days after the final administration. Pig-a mutant frequencies were assessed on Days 15 and ~30 for both treatment protocols and also on Days 43 and 57 for the 28-day protocol. Even at doses of 2-BE that induced marked intravascular lysis and strong compensatory erythropoiesis, the average Pig-a mutant phenotype red blood cell and reticulocyte frequencies were within the historical vehicle control distribution. 2-BE therefore showed no evidence of in vivo mutagenicity in these studies. The data suggest that perturbations in haematopoiesis alone do not lead to an observation of increased mutant frequency in the Pig-a assay.

Description: The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N -ethyl- N -nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect—gene mutation in the Pig-a gene.

Description: Validation of the Pig-a gene mutation assay has been based mainly on studies in male rodents. To determine if the mutagen-induced responses of the X-linked Pig-a gene differ in females compared to males, 7- or 14-week old male and female Sprague Dawley rats were exposed to N -ethyl- N -nitrosourea (ENU). In the study with the 7-week old rats, exposure was to 0, 1, 5 or 25mg ENU/kg/day for three consecutive days (study Days 1–3). Pig-a mutant phenotype reticulocyte (RET CD59– ) and mutant phenotype erythrocyte (RBC CD59– ) frequencies were determined on study Days –4, 15, 29 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis ( In Vivo MutaFlow ® ). Additionally, blood samples collected on Day 4 were analysed for micronucleated reticulocyte (MN-RET) frequency ( In Vivo MicroFlow ® ). The percentage of reticulocytes (%RET) was markedly higher in the 7-week old males compared to females through Day 15 (2.39-fold higher on Day –4). At 25mg/kg/day, ENU reduced Day 4 RET frequencies in both sexes, and the two highest dose levels resulted in elevated MN-RET frequencies, with no sex or treatment x sex interaction. The two highest dose levels significantly elevated the frequencies of mean RET CD59– and RBC CD59– in both sexes from Day 15 onward. RET CD59– and RBC CD59– frequencies were somewhat lower for females compared to males at the highest dose level studied, and differences in RET CD59– resulted in a statistically significant interaction effect of treatment x sex. In the study with 14-week old rats, treatment was for 3 days with 0 or 25mg ENU/kg/day. RET frequencies differed to a lesser degree between the sexes, and in this case there was no evidence of a treatment x sex interaction. These results suggest that the slightly higher response in younger males than in the younger females may be related to differences in erythropoiesis function at that age. In conclusion, while some quantitative differences were noted, there were no qualitative differences in how males and females responded to a prototypical mutagen, and support the contention that both sexes are equally acceptable for Pig-a gene mutation studies.

Description: Isothiocyanates are plant-derived compounds that may be beneficial in the prevention of certain chronic diseases. Yet, by stimulating the production of reactive oxygen species (ROS), isothiocyanates can be genotoxic. Whether antioxidants influence isothiocyanate-induced genotoxicity is unclear, but this situation was clarified appreciably herein. In HCT116 cells, phenethyl isothiocyanate (PEITC) increased ROS production, which was inhibited by N -acetylcysteine (NAC) and deferoxamine (DFO) but not by ascorbic acid (ASC) and trolox (TRX) that were found to be more potent radical scavengers. Surprisingly, ASC and TRX each intensified the DNA damage that was caused by PEITC, but neither ASC nor TRX by themselves caused any DNA damage. In contrast, NAC and DFO each not only attenuated PEITC-induced DNA damage but also attenuated the antioxidant-intensified, PEITC-induced DNA damage. To determine if the DNA damage could be related to possible changes in the major antioxidant defence system, glutathione (GSH) was investigated. PEITC lowered GSH levels, which was prevented by NAC, whereas ASC, TRX and DFO neither inhibited nor enhanced the GSH-lowering effect of PEITC. The GSH synthesis inhibitor, buthionine sulphoxime, intensified PEITC-induced DNA damage, although by itself buthionine sulphoxime did not directly cause DNA damage. The principal findings suggest that ASC and TRX make PEITC more genotoxic, which might be exploited in killing cancer cells as one approach in killing cancer cells is to extensively damage their DNA so as to initiate apoptosis.

Description: The mycotoxin aflatoxin B 1 (AFB 1 ) may initiate cancer by causing oxidatively damaged DNA, specifically by causing 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) lesions. Base excision repair removes these lesions, with 8-oxoguanine glycosylase (OGG1) being the rate-limiting enzyme. The aim of this study was to determine the effect of ogg1 deficiency on AFB 1 -induced oxidatively damaged DNA and tumourigenesis. Female wild-type, heterozygous and homozygous ogg1 null mice were given a single dose of 50mg/kg AFB 1 or 40 µl dimethyl sulfoxide (DMSO) ip. Neither ogg1 genotype nor AFB 1 treatment affected levels of oxidised guanine in lung or liver 2h post-treatment. AFB 1 -treated ogg1 null mice showed exacerbated weight loss and mortality relative to DMSO-treated ogg1 null mice, but AFB 1 treatment did not significantly increase lung or liver tumour incidence compared with controls, regardless of ogg1 genotype. Suspect lung masses from three of the AFB 1 -treated mice were adenomas, and masses from two of the mice were osteosarcomas. No osteosarcomas were observed in DMSO-treated mice. All liver masses from AFB 1 -treated mice were adenomas, and one also contained a hepatocellular carcinoma. In DNA from the lung tumours, the K- ras mutation pattern was inconsistent with initiation by AFB 1 . In conclusion, ogg1 status did not have a significant effect on AFB 1 -induced oxidatively damaged DNA or tumourigenesis, but deletion of one or both alleles of ogg1 did increase susceptibility to other aspects of AFB 1 toxicity.

Description: Integration of in vivo genotoxicity testing into standard toxicology studies presents multiple advantages as it reduces animal use and costs, accelerates data generation and provides concurrent data that are useful for interpreting results. The in vivo Pig-a assay is a mammalian gene mutation assay that utilises peripheral blood and thus has a high integration potential. Although inter-laboratory reproducibility has been well demonstrated, further characterisation is required for this assay. In this study, we evaluated intra-laboratory reproducibility of the in vivo Pig-a gene mutation assay (MutaFlow® kit) in rats through the conduct of an assay characterisation prior to subsequent use in Good Laboratory Practices (GLP) toxicology studies. To evaluate intra-laboratory reproducibility, intra-assay and inter-assay variability, ruggedness, robustness and blood storage stability were assessed. These assessments were performed using blood obtained from male Sprague–Dawley rats exposed to 0, 20 or 40mg/kg/day N -ethyl- N -nitrosourea via oral gavage for three consecutive days. Blood was collected from these rats on multiple occasions from Day 29 to Day 71 and samples were analysed for Pig-a mutation using the rat MutaFlow kit. Frequencies of reticulocytes (RET), mutant phenotype erythrocytes (RBC CD59– ) and mutant phenotype RET (RET CD59– ) were determined. Overall, the proportion of RET and frequencies of RBC CD59– and of RET CD59– were consistent throughout the different assessments. The assay demonstrated acceptable intra-run and inter-run variability with coefficients of variation of ≤4.8 and 20.6%, respectively. The method was shown to be independent of the analyst performing the assay and unaffected by small changes in assay conditions. Comparable results were obtained from freshly collected samples and samples refrigerated for up to 4 days. Although technically challenging, the rat Pig-a gene mutation assay using a high-throughput automated method was shown to be reliable. The different assay parameters evaluated during the conduct of this study yielded acceptable results. Thus, the method was considered suitable for use in GLP toxicology studies.

Description: Research over the years has generated enough evidence to implicate areca nut, as a carcinogen in humans. Besides oral, significant rise in the incidence of cancers of the oesophagus, liver and stomach was seen among areca nut chewers. Early diagnosis seems key to understand the initial processes of carcinogenesis which is highly curable. In North-East India, betel quid contains raw areca nut (RAN), lime and small portion of betel leaf without any other constituents. This study was not intended to isolate any active ingredients from the RAN and to look its action. The present objective is to validate the screening of precocious anaphase and analysis of expression of Securin and p53 in non-target cells like human peripheral blood lymphocytes (PBLs) and mouse bone marrow cells (BMCs) as early indicative parameters of RAN + lime-induced cancers. A total of 35 mice were examined at different time points for following ad libitum administration of RAN extract in drinking water with lime. Peripheral blood was collected from 32 human donors of which, 24 were RAN + lime heavy chewers. Expression of genes was assessed by immunoblotting and/or by immunohistochemistry. Histological preparation of stomach tissue of mice revealed that RAN + lime induced stomach cancer. A gradual increase in the frequency of precocious anaphases and aneuploid cells was observed in both RAN + lime-treated mouse BMC and human PBL of RAN heavy chewers. Levels of p53 and Securin were increased in these cells during early days of RAN + lime exposure. The level of Securin was significantly higher in human tumour samples than their adjacent normal counterpart. The expression of Securin was increased significantly in RAN + lime-administered mice as well as in stomach tumour. Present study revealed that precocious anaphase and expression of p53 and Securin in non-target cells are significantly associated with an increased risk of RAN-induced cancer and thus these parameters can be of early diagnostic value.

Description: Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials due to their antibacterial properties. Owing to the recent boost in the usage of AgNPs-containing products, human exposure to AgNPs is increasing, highlighting the need for careful evaluation of AgNPs toxicity in humans. We used two cellular models, hepatic HepG2 and epithelial A549 cell lines, to study the mechanism of AgNPs-induced toxicity at the cellular level. These two cell lines differ significantly in their response to AgNPs treatment. In the case of A549 cells, a minor decrease in viability and increase in the extent of DNA breakage were observed. A markedly different response to AgNPs was observed in HepG2 cells. In short term, a massive induction of DNA breakage was observed, suggesting that the basal activity of antioxidant defence in these cells was not sufficient to effectively protect them from the nanoparticle-induced oxidative stress. After prolonged exposure, the extent of DNA breakage decreased to the level observed in the control cells proving that a successful adaptation to the new conditions had taken place. The cells that were unable to adapt must have died, as revealed by the Neutral Red assay that indicated less than half viable cells after 24-h treatment with 100 µg/ml of 20nm AgNPs. The gene expression analysis revealed that the observed adaptation was underlain by a pro-proliferative, anti-apoptotic signal leading to up-regulation of the genes promoting proliferation and inflammatory response ( EGR1 , FOS , JUN , HK2 , IL4 , MMP10 , VEGFA , WISP1 , CEBPB , IL8 , SELPLG ), genes coding the anti-apoptotic proteins ( BCL2A1 , CCL2 ) and factors involved in the response to stress ( HSPB1 , GADD45A ). Such a selection of highly resistant population of cells should be taken into account in the case of medical applications of nanoparticles since the sustained proliferative signalling and resistance to cell death are hallmarks of cancer, acquired by the cells in the process of carcinogenesis.

Description: Ionising radiation induces single-strand breaks, double-strand breaks (DSB) and base damages in human cell. DSBs are the most deleterious and if not repaired may lead to genomic instability and cell death. DSB can be repaired through non-homologous end joining (NHEJ) pathway in resting lymphocytes. In this study, NHEJ genes and proteins were studied in irradiated human peripheral blood mononuclear cells (PBMC) at resting stage. Dose-response, time point kinetics and adaptive-response studies were conducted in irradiated PBMC at various end points such as DNA damage quantitation, transcription and protein expression profile. Venous blood samples were collected from 20 random, normal and healthy donors with written informed consent. PBMC was separated and irradiated with various doses between 0.1 and 2.0 Gy ( 60 CO- source) for dose-response study. Repair kinetics of DNA damage and time point changes in expression of genes and proteins were studied in post-irradiated PBMC at 2.0 Gy at various time points up to 240min. Adaptive-response study was conducted with a priming dose of 0.1 Gy followed by a challenging dose of 2.0 Gy after 4-h incubation. Our results revealed that Ku70, Ku80, XLF and Ligase IV were significantly upregulated ( P 〈 0.05) at 4-h post-irradiation at transcript and protein level. Adaptive-response study showed significantly increased expression of the proteins involved in NHEJ, suggesting their role in adaptive response in human PBMC at G 0 /G 1, which has important implications to human health.

Description: Excision repair cross complementing group 1 (ERCC1) and X-ray repair cross-complementing groups 1 (XRCC1) are DNA repair enzymes. Polymorphisms in DNA repair genes may be important factors affecting cancer susceptibility, prognosis and therapy outcome. The purpose of this study was to investigate the correlation of ERCC1 and XRCC1 polymorphisms with colorectal cancer (CRC) risk, and explore the effect of polymorphisms on event-free, overall survival and oxaliplatin-based therapy in CRC patients. Genotyping was examined with the iMLDR technique. An unconditional logistic regression model was used to estimate the association of certain polymorphisms with CRC risk. The Kaplan–Meier method, log-rank test and Cox regression model were employed to evaluate the effects of polymorphisms on survival analysis. Results showed that Trp/Trp genotype of XRCC1 Arg194Trp and AA genotype of ERCC1 rs2336219 have a significantly increased risk of CRC; Trp allele of XRCC1 Arg194Trp and CC genotype of ERCC1 rs735482 were associated with lower response to oxaliplatin-based chemotherapy, a shorter survival and a higher risk of relapse or metastasis. 194Trp/280Arg/399Arg haplotype was associated with a significant resistance, and the ERCC1 protein expression was statistically higher in tumours with rs735482 CC genotype than with AA genotype. Our studies indicate that XRCC1 and ERCC1 polymorphisms probably affect susceptibility, chemotherapy response and survival of CRC patients.

Description: Epigenetic control of gene expression in children remains poorly understood, but new technologies can help elucidate the relationship between expression and DNA methylation. Here, we utilized the nCounter Analysis System to characterise the expression of 60 genes in 69 9-year-old children from a cohort with a high prevalence of obesity. nCounter expression levels ranged broadly (from 3 to over 10000 messenger RNA counts) and were divided into four categories: high (〉2000 counts), moderate (200–1000 counts), low (100–200 counts) and marginal (〈100 counts). For a subset of five genes ( ADIPOR1 , PPARG1 , GSTM1 , PON1 and ACACA ) from different expression level categories, we validated nCounter data using reverse transcription-polymerase chain reaction (RT-PCR), and expanded RT-PCR analysis of ADIPOR1 to include 180 children. Expression data from the two methodologies were correlated for all five genes included in the validation experiment, with estimates ranging from r s = 0.26 ( P = 0.02) to r s = 0.88 ( P 〈 5 x 10 –6 ). ADIPOR1 and PPARG1 nCounter expression levels were negatively correlated ( r = –0.60, P 〈 5 x 10 –5 ), and this relationship was stronger in overweight children ( r = –0.73, P 〈 5 x 10 –5 ) than in normal weight children ( r = –0.42, P = 0.016). Using methylation data from the Infinium HumanMethylation450 BeadChip ( n = 180), we found eight CpG sites in ADIPOR1 and PPARG where methylation level was associated with expression by RT-PCR ( P 〈 0.05). Hypomethylation of PPARG gene body site cg10499651 was associated with increased expression as measured by both RT-PCR and nCounter ( P 〈 0.05). We found no statistically significant relationships between either expression or methylation of ADIPOR1 and PPARG and body mass index or waist circumference. In addition to demonstrating the validity of expression data derived from nCounter, our results illustrate the use of new technologies in assessing epigenetic effects on expression in children.

Description: The in vitro micronucleus test is a well-known test for the screening of genotoxic compounds. However until now, most studies have been performed on either human peripheral lymphocytes or established cancer cell lines. This study provides human mesenchymal stem cells as an alternative to the conventional micronucleus test. We grew umbilical cord mesenchymal stem cells (UC-MSCs) on coverslips eliminating the cumbersome technique involving hypotonic treatment, fixation and preparing smears required for suspension culture (lymphocytes). The background frequency of nuclear blebs and micronuclei in UC-MSCs was found to be 7±5, in lymphocytes 16±3.5 and 9±3 and that for A549 cell line was 65±5 and 15±5 per 1000 cells, respectively, suggesting differences in the repair mechanism of normal and cancer cell lines. We inspected the cytotoxic and genotoxic effects of two known mutagens, mitomycin-C and hydrogen peroxide (H 2 O 2 ), on UC-MSCs, lymphocytes and A549 cells. Treatment with mitomycin-C and H 2 O 2 demonstrated drastic differences in the degree of cytotoxicity and genotoxicity suggesting a constitutional difference between normal and cancer cells. In addition we tested two solvents, dimethyl sulfoxide (DMSO) and ethanol, and two drugs, metformin and rapamycin. DMSO above 1% was found to be cytotoxic and genotoxic, whereas ethanol at same concentration was neither cytotoxic nor genotoxic indicating the minimal non-toxic level of the solvents. This study thus offers UC-MSCs as a better substitute to peripheral lymphocytes and cancer cell lines for high throughput screening of compounds and reducing the animal studies.

Description: Cells exhibiting radiation-induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during n ormal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesis that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.

Description: Sequencing by ligation (SBL) is a straightforward enzymatic method for interrogating DNA sequence, in which the ligation efficiency and specificity of each probe play an essential role. Here, the number of labelled dyes in the probe, probe length and probe constituent were investigated to optimize the ligation efficiency and specificity. First, the performance of double- and single-labelled fluorescent probes in SBL was evaluated. The experimental results showed that double-labelled fluorescent probes could yield a remarkable increase in the fluorescence intensities and avoid higher background compared with single-labelled fluorescent probes. Second, probes between 7- and 9-mers in length were designed to uniform T m difference. We hoped the uniformed probes with smaller T m difference could improve the ligation efficiency. However, 8-mer probes with larger T m difference showed stronger fluorescence intensities. Third, we evaluated whether probes containing deoxyinosines either in the 5' or the 3' end had influence on the ligation efficiency. Consequently, probes containing deoxyinosines at the 5' termini might decrease the ligation efficiency, and the accumulation of 3' terminal deoxyinosines in the sequencing primers was likely to reduce the fluorescence intensity and the ligation efficiency, which was inconsistent with the traditional viewpoint. The optimized probes will improve the ligation efficiency and accuracy in SBL.

Description: In this study, bleomycin-Fe 3+ steadily oxidized tetramethylbenzidine (TMB) in the presence of peroxides. However, the ability of bleomycin-Fe 3+ to function as a peroxidase was extremely low compared with that of other peroxidases. A characteristic property of bleomycin-Fe 3+ different from that observed for other peroxidases is its ability to oxidize TMB at the similar rate at both a pH 5 and 8 in the presence of lipid hydroperoxide (LOOH). In the present experiments, hydroxyl radicals (HO•) were generated only when bleomycin-Fe 3+ was incubated with H 2 O 2 at a pH of 5. No generation of HO• was observed during the incubation of bleomycin-Fe 3+ with LOOH. Meanwhile, bleomycin-Fe 3+ induced the formation of LOOH from linoleic acid and alcohol dehydrogenase was inactivated by bleomycin-Fe 3+ with peroxides. Thiobarbituric acid reactive substances were formed from DNA by bleomycin-Fe 3+ with H 2 O 2 , and strand breaks were caused by bleomycin-Fe 3+ with LOOH. The oxidative substrates for bleomycin-Fe 3+ blocked the damage to biological components induced by bleomycin-Fe 3+ . These results suggest that compound I-like species contribute to the process of damage to biological components induced by bleomycin-Fe 3+ .

Description: The Ral guanosine triphosphatases (GTPases), RalA and RalB, are members of the Ras superfamily of small GTPases. Research on Ral GTPases and their functions over the past 25 years has revealed the essential involvement of these GTPases in unique and diverse cellular processes including exocyst-mediated exocytosis and related cellular activities. Moreover, it is increasingly appreciated that the aberrant activation of Ral GTPases is one of the major causes of human tumourigenesis induced by oncogenic Ras. Recent evidence suggests that Ral signalling pathways may be potential therapeutic targets for the treatment of human cancers. This review summarizes recent advance in the investigation of Ral GTPases.

Description: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro . We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF.

Description: In a previous study, we validated an in vitro genotoxicity assay based on H2AX quantification using the In-Cell Western (ICW) method in HepG2 cells. The assay demonstrated high sensitivity and specificity but failed to detect genotoxicity for few compounds that require specific metabolic bioactivation not sufficiently covered by HepG2 cells. The aim of the present study was to assess H2AX ICW sensitivity using a broader range of genotoxic molecules with HepG2 cells and three additional human cell lines with distinct biotransformation properties: two cell lines expressing some phase I and II bioactivation capabilities (LS-174T and Hep3B), and one with poor general bioactivation properties (ACHN). We evaluated the four cell lines by testing 24 compounds recommended by European Centre for the Validation of Alternative Methods and a set of 24 additional chemicals with different mode of genotoxic action (MOA) (aneugenicity, DNA adducts formation, induction of oxidative stress), including some known to require specific cytochrome P450 metabolic bioactivation. Results for the 48 compounds tested showed that the H2AX ICW assay was more sensitive with LS-174T and HepG2 cells than with Hep3B or ACHN cell lines. Among the 38 compounds tested with positive or equivocal carcinogenicity data, 36 (95%) showed a positive genotoxic response with the H2AX ICW assay compared to only 27 (71%) using the Ames assay. We confirm that the H2AX ICW assay on HepG2 cells, without an exogenous metabolic activation system, may be a suitable test to predict the in vivo genotoxicity of chemicals with different genotoxic MOA. Moreover, the use of the ACHN cell line in combination with LS-174T and HepG2 cells may permit in many cases to discriminate direct from bioactivated genotoxins. Overall, our results confirm the high sensitivity of the H2AX ICW assay which, in turn, should reduce the number of animals used for genotoxicity assessment.

Description: Quantum dots (QD) have unique electronic and optical properties promoting biotechnological advances. However, our understanding of the toxicological structure–activity relationships remains limited. This study aimed to determine the biological impact of varying nanomaterial surface chemistry by assessing the interaction of QD with either a negative (carboxyl), neutral (hexadecylamine; HDA) or positive (amine) polymer coating with human lymphoblastoid TK6 cells. Following QD physico-chemical characterisation, cellular uptake was quantified by optical and electron microscopy. Cytotoxicity was evaluated and genotoxicity was characterised using the micronucleus assay (gross chromosomal damage) and the HPRT forward mutation assay (point mutagenicity). Cellular damage mechanisms were also explored, focusing on oxidative stress and mitochondrial damage. Cell uptake, cytotoxicity and genotoxicity were found to be dependent on QD surface chemistry. Carboxyl-QD demonstrated the smallest agglomerate size and greatest cellular uptake, which correlated with a dose dependent increase in cytotoxicity and genotoxicity. Amine-QD induced minimal cellular damage, while HDA-QD promoted substantial induction of cell death and genotoxicity. However, HDA-QD were not internalised by the cells and the damage they caused was most likely due to free cadmium release caused by QD dissolution. Oxidative stress and induced mitochondrial reactive oxygen species were only partially associated with cytotoxicity and genotoxicity induced by the QD, hence were not the only mechanisms of importance. Colloidal stability, nanoparticle (NP) surface chemistry, cellular uptake levels and the intrinsic characteristics of the NPs are therefore critical parameters impacting genotoxicity induced by QD.

Description: We previously reported that a urate-null strain of Drosophila is hypersensitive to cigarette smoke (CS), and we suggested that CS induces oxidative stress in Drosophila because uric acid is a potent antioxidant. Although the carcinogenic risk of CS exposure is widely recognized; documentation of in vivo genotoxic activity of environmental CS, especially gaseous-phase CS, remains inconclusive. To date, somatic-cell mutations in Drosophila resulting from exposure to CS have not been detected via the somatic mutation and recombination test (wing spot test) with wild-type flies, a widely used Drosophila assay for the detection of somatic-cell mutation; moreover, genotoxicity has not been documented via a DNA repair test that involves DNA repair-deficient Drosophila. In this study, we used a new Drosophila strain ( y v ma-l; mwh ) to examine the mutagenicity induced by gaseous-phase CS; these flies are urate-null due to a mutation in ma-l , and they are heterozygous for multiple wing hair ( mwh ), a mutation that functions as a marker for somatic-cell mutation. In an assay with this newly developed strain, a superoxide anion-producing weed-killer, paraquat, exhibited significant mutagenicity; in contrast, paraquat was hardly mutagenic with a wild-type strain. Drosophila larvae were exposed to CS for 2, 4 or 6h, and then kept at 25 ° C on instant medium until adulthood. After eclosion, mutant spots, which consisted of mutant hairs on wings, were scored. The number of mutant spots increased significantly in an exposure time-dependent manner in the urate-null females ( ma-l (–/–)), but not in the urate-positive females ( ma-l (+/–)). In this study, we showed that short-term exposure to CS was mutagenic in this in vivo system. In addition, we obtained suggestive data regarding reactive oxygen species production in larva after CS exposure using the fluorescence probe H 2 DCFDA. These results suggest that oxidative damage, which might be countered by uric acid, was partly responsible for induction of somatic cell mutations in Drosophila larvae exposed to CS.

Description: DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines ( KU70 –/– / RAD54 –/– and REV3 –/– ) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity—2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether—were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA.

Description: Cleidocranial dysplasia (CCD; MIM 119600) is an autosomal dominant skeletal dysplasia characterised by hypopalstic and/or aplastic clavicles, midface hypoplasia, absent or delayed closure of cranial sutures, moderately short stature, delayed eruption of permanent dentition and supernumerary teeth. The molecular pathogenesis can be explained in about two-thirds of CCD patients by haploinsufficiency of the RUNX2 gene. In our current study, we identified a novel and rare variant of the RUNX2 gene (c.181_189dupGCGGCGGCT) in a Japanese patient with phenotypic features of CCD. The insertion led an alanine tripeptide expansion (+3Ala) in the polyalanine tract. To date, a RUNX2 variant with alanine decapeptide expansion (+10Ala) is the only example of a causative variant of RUNX2 with polyalanine tract expansion to be reported, whilst RUNX2 (+1Ala) has been isolated from the healthy population. Thus, precise analyses of the RUNX2 (+3Ala) variant were needed to clarify whether the tripeptide expanded RUNX2 is a second disease-causing mutant with alanine tract expansion. We therefore investigated the biochemical properties of the mutant RUNX2 (+3Ala), which contains 20 alanine residues in the polyalanine tract. When transfected in COS7 cells, RUNX2 (+3Ala) formed intracellular ubiquitinated aggregates after 24h, and exerted a dominant negative effect in vitro . At 24h after gene transfection, whereas slight reduction was observed in RUNX2 (+10Ala), all of these mutants significantly activated osteoblast-specific element-2, a cis-acting sequence in the promoter of the RUNX2 target gene osteocalcin. The aggregation growth of RUNX2 (+3Ala) was clearly lower and slower than that of RUNX2 (+10Ala). Furthermore, we investigated several other RUNX2 variants with various alanine tract lengths, and found that the threshold for aggregation may be RUNX2 (+3Ala). We conclude that RUNX2 (+3Ala) is the cause of CCD in our current case, and that the accumulation of intracellular aggregates in vitro is related to the length of the alanine tract.

Description: In Saccharomyces cerevisiae , disruption of genes by deletion allowed elucidation of the molecular mechanisms of a series of human diseases, such as in Wilson disease (WD). WD is a disorder of copper metabolism, due to inherited mutations in human copper-transporting ATPase ( ATP7B ). An orthologous gene is present in S. cerevisiae , CCC2 gene. Copper is required as a cofactor for a number of enzymes. In excess, however, it is toxic, potentially carcinogenic, leading to many pathological conditions via oxidatively generated DNA damage. Deficiency in ATP7B (human) or Ccc2 (yeast) causes accumulation of intracellular copper, favouring the generation of reactive oxygen species. Thus, it becomes important to study the relative importance of proteins involved in the repair of these lesions, such as Ogg1 . Herein, we addressed the influence Ogg1 repair in a ccc2 deficient strain of S. cerevisiae . We constructed ccc 2-disrupted strains from S. cerevisiae ( ogg 1 ccc2 and ccc 2), which were analysed in terms of viability and spontaneous mutator phenotype. We also investigated the impact of 4-nitroquinoline-1-oxide (4-NQO) on nuclear DNA damage and on the stability of mitochondrial DNA. The results indicated a synergistic effect on spontaneous mutagenesis upon OGG1 and CCC2 double inactivation, placing 8-oxoguanine as a strong lesion-candidate at the origin of spontaneous mutations. The ccc2 mutant was more sensitive to cell killing and to mutagenesis upon 4-NQO challenge than the other studied strains. However, Ogg1 repair of exogenous-induced DNA damage revealed to be toxic and mutagenic to ccc2 deficient cells, which can be due to a detrimental action of Ogg1 on DNA lesions induced in ccc2 cells. Altogether, our results point to a critical and ambivalent role of BER mediated by Ogg1 in the maintenance of genomic stability in eukaryotes deficient in CCC2 gene.

Description: Osteoclasts are giant multinucleated cells that differentiate from hematopoietic cells in the bone marrow and carry out important physiological functions in the regulation of skeletal homeostasis as well as hematopoiesis. Osteoclast biology shares many features and components with cells of the immune system, including cytokine-receptor interactions (RANKL-RANK), intracellular signalling molecules (TRAF6) and transcription factors (NFATc1). Although the roles of these molecules in osteoclast differentiation are well known, fundamental questions remain unsolved, including the exact location of the RANKL-RANK interaction and the in vivo temporal and spatial information on the transformation of hematopoietic cells into bone-resorbing osteoclasts. This review focuses on the importance of cell-cell contact and metabolic adaptation for differentiation, relatively overlooked aspects of osteoclast biology and biochemistry.

Description: Although monoclonal antibodies have been used not only as analytical tools but also as biologic therapeutics, they cannot target intracellular proteins due to their large molecular size and low membrane permeability, which limit their applications. During previous attempts to delivery antibodies intracellularly, the low efficiency of escape from endosomes to the cytosol reduced the bioavailability of antibodies or antibody-conjugated effectors. Recently, we found that the fusogenic peptides (FPs) B18 and B55 from bindin, a sea urchin gamete recognition protein, facilitated the endosomal escape of FP-fused enhanced green fluorescent protein (eGFP) and/or of co-administered cargos such as dextrans [Niikura et al. A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape. J. Control. Release 2015;212:85-93]. In this study, we constructed FP-fused anti-epidermal growth factor receptor (EGFR) single-chain Fv (αEGFR[scFv]) proteins and evaluated their endosomal escape efficiency by utilizing a nuclear localization signal). When the FP-fused αEGFR[scFv] proteins were incubated with A431 cells, the estimated endosomal escape efficiency of αEGFR[scFv]-B18 was significantly higher than that of αEGFR[scFv] alone, suggesting that the B18 peptide facilitates endosomal escape of the conjugated scFv in cis . Moreover, αEGFR[scFv]-B55 promoted the intracellular uptake of co-administered eGFP and dextrans in trans . These results imply that B18- and B55-fused antibodies may be useful for the cell-specific intracellular delivery of biomacromolecules.

Description: Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S -adenosyl- l -methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygen-tolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus [4Fe-4S] 2+ cluster bound at the N-terminus region. Mössbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of [4Fe-4S] 2+ and [2Fe-2S] 2+ clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions.

Description: Pho Pop5 and Pho Rpp30 in the hyperthermophilic archaeon Pyrococcus horikoshii , homologues of human ribonuclease P (RNase P) proteins hPop5 and Rpp30, respectively, fold into a heterotetramer [ Pho Rpp30–( Pho Pop5) 2 – Pho Rpp30], which plays a crucial role in the activation of RNase P RNA ( Pho pRNA). Here, we examined the functional implication of Pho Pop5 and Pho Rpp30 in the tetramer. Surface plasmon resonance (SPR) analysis revealed that the tetramer strongly interacts with an oligonucleotide including the nucleotide sequence of a stem-loop SL3 in Pho pRNA. In contrast, Pho Pop5 had markedly reduced affinity to SL3, whereas Pho Rpp30 had little affinity to SL3. SPR studies of Pho Pop5 mutants further revealed that the C-terminal helix (α4) in Pho Pop5 functions as a molecular recognition element for SL3. Moreover, gel filtration indicated that Pho Rpp30 exists as a monomer, whereas Pho Pop5 is an oligomer in solution, suggesting that Pho Rpp30 assists Pho Pop5 in attaining a functionally active conformation by shielding hydrophobic surfaces of Pho Pop5. These results, together with available data, allow us to generate a structural and mechanistic model for the Pho pRNA activation by Pho Pop5 and Pho Rpp30, in which the two C-terminal helices (α4) of Pho Pop5 in the tetramer whose formation is assisted by Pho Rpp30 act as binding elements and bridge SL3 and SL16 in Pho pRNA.

Description: Wnt ligands play a central role in the development and homeostasis of various organs through β-catenin-dependent and -independent signalling. The crucial roles of Wnt/β-catenin signals in bone mass have been established by a large number of studies since the discovery of a causal link between mutations in the low-density lipoprotein receptor-related protein 5 ( Lrp5 ) gene and alternations in human bone mass. The activation of Wnt/β-catenin signalling induces the expression of osterix, a transcription factor, which promotes osteoblast differentiation. Furthermore, this signalling induces the expression of osteoprotegerin, an osteoclast inhibitory factor in osteoblast-lineage cells to prevent bone resorption. Recent studies have also shown that Wnt5a, a typical non-canonical Wnt ligand, enhanced osteoclast formation. In contrast, Wnt16 inhibited osteoclast formation through β-catenin-independent signalling. In this review, we discussed the current understanding of the Wnt signalling molecules involved in bone formation and resorption.

Description: Parathyroid hormone-related protein (PTHrP) has two different targeting signals: an N-terminal signal peptide for the endoplasmic reticulum (ER) targeting and an internal nuclear localization signal. The protein not only functions as a secretory protein, but is also found in the nucleus and/or nucleolus under certain conditions. PTHrP signal peptide is less hydrophobic than most signal peptides mainly due to its evolutionarily well-conserved region (QQWS). The substitution of four tandem leucine residues for this conserved region resulted in a significant inhibition of the signal peptide cleavage. At the same time, proportion of nuclear and/or nucleolar localization decreased, probably due to tethering of the protein to the ER membrane by the uncleaved mutant signal peptide. Almost complete cleavage of the signal peptide accompanied by a lack of nuclear/nucleolar localization was achieved by combining the hydrophobic h-region and an optimized sequence of the cleavage site. In addition, mutational modifications of the distribution of charged residues in and around the signal peptide affect its cleavage and/or nuclear/nucleolar localization of the protein. These results indicate that the well-conserved region in the signal peptide plays an essential role in the dual localization of PTHrP through ER targeting and/or the membrane translocation.

Description: Distribution of the isoelectric point (pI) was calculated for the hypervariable regions of Fab fragments of the antibody molecules, which structure is annotated in the structural antibody database SabDab. The distribution is consistent with the universal for all organisms dividing the proteome into two sets of acidic and basic proteins. It shows the additional fine structure in a form of the narrow-sized peaks of pI values. This is an explanation why a small change of the environmental pH can have a strong effect on the antibody-antigen affinity. To show this, a typical enzyme-linked immunospecific assay experiment for testing the reaction of goat anti-human IgA antibodies with human IgA immunoglobulins of saliva as antigens was modified in such a way that Fe 3 O 4 magnetic nanoparticles were added to PBS buffer. The magnetic nanoparticles were remotely heated by the radio frequency magnetic field providing the local change of temperature and pH. It was observed that short times of the heating were significantly increasing the antibody-antigen binding strength while it was not the case for a longer time. The finding discussed in the study can be useful for biopharmaceuticals using antibodies, the immunoassay techniques as well as for control over the use of hyperthermia.

Description: The 5'-AMP-activated protein kinase (AMPK) functions as a cellular energy sensor. 5-Aminoimidazole-4-carboxyamide-1-β-D-ribofranoside (AICAR) is a chemical activator of AMPK. In the liver, AICAR suppresses expression of the phosphoenolpyruvate carboxykinase ( PEPCK ) gene. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcriptional repressor and its target is the PEPCK gene. In this study, we examined an issue of whether the SHARP-2 gene expression is regulated by AICAR via the AMPK. AICAR increased the level of SHARP-2 mRNA in H4IIE cells. Whereas an AMPK inhibitor, compound-C, had no effects on the AICAR-induction, inhibitors for both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) completely diminished the effects of AICAR. Western blot analyses showed that AICAR rapidly activated atypical PKC lambda (aPKC). In addition, when a dominant negative form of aPKC was expressed, the induction of SHARP-2 mRNA level by AICAR was inhibited. Calcium ion is not required for the activation of aPKC. A calcium ion-chelating reagent had no effects on the AICAR-induction. Furthermore, the AICAR-induction was inhibited by treatment with an RNA polymerase inhibitor or a protein synthesis inhibitor. Thus, we conclude that the AICAR-induction of the SHARP-2 gene is mediated at transcription level by a PI 3-K/aPKC pathway.

Description: Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson’s disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1–Parkin–ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling.

Description: A method to express, purify and modify the Peptidyl-Lys metallopeptidase (LysN) of Armillaria mellea in Pichia pastoris was developed to enable functional studies of the protease. Based on prior work, we propose a mechanism of action of LysN. Catalytic residues were investigated by site-directed mutagenesis. As anticipated, these mutations resulted in significantly reduced catalytic rates. Additionally, based on molecular modelling eleven mutants were designed to have altered substrate specificity. The S 1 ' binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. To allow for arginine specificity in S 1 ', it was proposed to extend the S 1 ' binding pocket by mutagenesis, however the resulting mutant did not show any activity with arginine in P 1 '. Two mutants, A101D and T105D, showed increased specificity towards arginine in subsites S 2 '–S 4 ' compared to the wild type protease. We speculate that the increased specificity to result from the additional negative charge which attract and interact with positively charged residues better than the wild type.

Description: DNA polymerase (Pol), one of the typical member of the Y-family DNA polymerases, has been demonstrated to bypass the 10 S (+)- trans-anti -benzo[ a ]pyrene diol epoxide- N 2 -deoxyguanine adducts (BPDE-dG) efficiently and accurately. A large structural gap between the core and little finger as well as an N-clasp domain are essential to its unique translesion capability. However, whether the extreme N-terminus of Pol is required for its activity is unclear. In this work, we constructed two mouse Pol deletions, which have either a catalytic core (mPol 1-516 ) or a core without the first 21-residues (mPol 22-516 ), and tested their activities in the replication of normal and BPDE-DNA. These two Pol deletions are nearly as efficient as the full length protein (Pol 1-852 ) in normal DNA synthesis. However, steady-state kinetics reveals a significant reduction in efficiency of dCTP incorporation opposite the lesion by Pol 22-516 , along with increased frequencies for misinsertion compared with Pol 1-852 . The next nucleotide insertion opposite the template C immediately following the BPDE-dG was also examined, and the bypass differences induced by deletions were highlighted in both insertion and extension step. We conclude that the extreme N-terminal part of Pol is required for the processivity and fidelity of Pol during translesion synthesis of BPDE-dG lesions.

Description: Tumour suppressor p53, which is encoded by the TP53 gene, is widely known to play an important role in response to DNA damage and various stresses. It has recently been reported that p53 regulates glucose metabolism and that an increase in p53 protein level is induced after serum deprivation or treatments with a natural compound, trans -Resveratrol (Rsv). In this study, we constructed a Luciferase expression vector, pGL4-TP53-551, containing 551 bp of the 5’-upstream region of the human TP53 gene, which was then transfected into HeLa S3 cells. A Luciferase assay showed that Rsv treatment increased the promoter activity of the TP53 gene in comparison to that of PIF1 . Detailed deletion and mutation analyses revealed that Nkx-2.5 and E2F-binding elements are required in addition to duplicated GGAA (TTCC), for the regulation of TP53 promoter activity. In this study, it is suggested that the transient induction of TP53 gene expression by Rsv treatment might be partly involved in its anti-aging effect through maintenance of chromosomal DNAs.

Description: To explore the phosphoproteome profiles during Xenopus egg activation by Ca 2+ -stimulation, an automated phosphopeptide purification system involving a titania column was improved by introducing 4-step elution with phosphate buffers. The number of detected phosphopeptides in the tryptic digest of a Xenopus egg cytosol fraction on mass spectrometry (MS) was increased 1.5-fold and the percentage of multiply phosphorylated peptides increased from 17 to 24% with introduction of the 4-step elution method. Phosphopeptides were purified by the improved method from tryptic digests of cytosol fractions of Xenopus eggs without and with a Ca 2+ -stimulus, and then, analysed by MS. One thousand three hundred and seventy-five and 994 phosphopeptides were reproducibly detected on duplicate MS, respectively. They included 818 and 437 phosphopeptides specific to each digest, respectively. A method involving isobaric tags for relative and absolute quantitation (iTRAQ) was also applied to compare the phosphorylation levels in Xenopus eggs without and with a Ca 2+ -stimulus, the ratios for 112 phosphopeptides in tryptic digests of these egg cytosol fractions being obtained. It was suggested from all the results that the phosphorylation sites and levels change during Xenopus egg activation for many known and unknown sites on structural proteins, signalling related proteins, cell cycle-related proteins and others.

Description: Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts ( Cynops pyrrhogaster ). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37–38 kDa and 31–34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians.