Genotoxic effects of ethylene oxide and propylene oxide: a comparative study

The two alkylating agents ethylene oxide (EO) and propylene oxide (PO) were compared for genotoxic effectiveness in various test systems. The study was undertaken partly to shed light on the difference between the compounds found after chronic exposure of monkeys (Lynch et al., 1984) where EO but not PO was able to induce SCE and chromosomal aberrations. In the present study EO was found to be 5–10 times more effective than PO with respect to gene conversion and reverse mutation in Saccharomyces cerevisiae D7 and sister-chromatid conversion in S. cerevisiae RS112. In contrast, the abilities of the two compounds to induce point mutation in S. typhimurium strains and SCE in human lymphocytes were approximately equal. One possible cause of EO being more effective than PO in certain respects, discussed on the basis of inference from earlier studies, is an expected difference in ability to cause strand breaks via alkylation of DNA-phosphate groups.     

NrdH-redoxin protein mediates high enzyme activity in manganese-reconstituted ribonucleotide reductase from Bacillus anthracis

Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.     

Localization of NADPH-protochlorophyllide oxidoreductase in dark-grown wheat (Triticum aestivum) by immuno-electron microscopy before and after transformation of the prolamellar bodies

The localization of NADPH-protochlorophyllide oxidoreductase (PChlide reductase, EC 1.6.99.–) in dark-grown and in irradiated dark-grown leaves of wheat ( Triticum aestivum L. cv. Walde) was investigated by subjecting thin sections of Lowicryl K4M-embedded leaf pieces to a monospecific antiserum raised against PChlide reductase followed by protein A-gold. A well-preserved antigenicity of the tissue was achieved by polymerizing the resin under UV-light at low temperature. In dark-grown leaves PChlide reductase was found in prolamellar bodies only. In leaves irradiated for 30 min with white light PChlide reductase was found not only in the transformed prolamellar bodies but also to a large extent in connection with the prothylakoids. The localization of PChlide reductase is discussed in relation to fluorescence emission spectra of the dark-grown and greening leaves. We conclude that the light-dependent transformation of protochlorophyllide to chlorophyllide initiates a translocation of PChlide reductase from the prolamellar bodies to the prothylakoids.     

In vitro studies of the influence of glutathione transferases and epoxide hydrolase on the detoxification of acrylamide and glycidamide in blood

Enzymes involved in the metabolism of xenobiotic substances are often polymorphic in humans. Such genetic polymorphisms may result in inter-individual differences in detoxification of certain chemicals, and as a consequence, possibly affect health-risk assessments. This present work concerns studies of the influence of polymorphic enzymes in the detoxification of acrylamide and its metabolite glycidamide. Enzymes that enhance conjugation with glutathione (GSH), the glutathione transferases (GSTs), may influence the detoxification of both acrylamide and glycidamide, whereas the enzyme epoxide hydrolase (EH) should only catalyse the hydrolysis of glycidamide. In this study, the doses of acrylamide or glycidamide measured as specific adducts to hemoglobin (Hb) were analysed in blood samples after in vitro incubation with these compounds. Blood samples from individuals with different genotypes for GSTT1 and GSTM1 were studied. No significant differences in adduct levels depending on genotype were noted. In a parallel experiment, incubation with ethylene oxide was used as positive control. In this experiment individuals carrying GSTT1 showed lower adduct level increments from ethylene oxide than individuals lacking GSTT1. Furthermore, addition of ethacrynic acid or laurylamine, compounds which inhibit GST and EH, respectively, did not affect the adduct levels. These results suggest that neither GSTs nor EH have any significant effect on the blood dose, measured as Hb-adducts over time, after exposure to acrylamide or glycidamide.     

Microsomal long-chain acyl-CoA thioesterase (carboxylesterase ES-4) is regulated by thyroxine

Long chain acyl-CoA thioesterase activity is mainly located in microsomes after subcellular fractionation of liver from untreated rats. The physiological function and regulation of expression of this activity is not known. In the present study we have investigated the effect of thyroxine on expression of carboxylesterase ES-4, the major acyl-CoA thioesterase of liver microsomes. Thyroidectomy of rats decreased the palmitoyl-CoA thioesterase activity to about 25% of normal activity. This decrease was accompanied by similar decreases at the protein and mRNA levels (31% and 57%, respectively, of controls). Treatment with thyroxine completely reversed the effect of thyroidectomy and resulted in elevated levels in both thyroidectomized and control rats. For reasons of comparison we also studied the possibility that ES-10 and ES-2, two other members of the same gene family, are affected by thyroxine. ES-10 was not changed at the protein or mRNA level by any of the treatments, while ES-2 expression in liver was decreased by thyroxine treatment. The data shows that changes in activity and expression of ES-4 correlate to thyroxine status in the rat suggesting a physiological regulatory role by this hormone. Since thyroxine regulates the expression of lipogenic enzymes, these results are consistent with a function for this microsomal acyl-CoA thioesterase in fatty acid synthesis and/or secretion, rather than in oxidative degradation of fatty acids.     

Ascorbic acid alters cell fate commitment of human neural progenitors in a WNT/β-catenin/ROS signaling dependent manner

Background

Improving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/β-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis.

Methods

Effects of 200 μM AA were compared with the pro-neurogenic reagent and WNT/β-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/β-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET).

Results

In contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/β-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation.

Conclusions

Our findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/β-catenin signaling response that would enhance the ensuing neuronal cell differentiation.

     

Human memory B lymphocyte subsets fulfill distinct roles in the anti-polysaccharide and anti-protein immune response

There is controversy on the role of IgM memory and switched memory B lymphocytes in the Ab response to T cell-independent and T cell-dependent Ags. We transplanted SCID/SCID mice with human B lymphocyte subsets and immunized them with heat-inactivated Streptococcus pneumoniae or with a pneumococcal vaccine. Inactivated S. pneumoniae and soluble pneumococcal capsular polysaccharides elicited an IgM anti-polysaccharide and anti-protein Ab response from IgM memory B lymphocytes and an IgG anti-polysaccharide and anti-protein response from switched memory B lymphocytes. In addition to the IgM Ab response, IgM memory B cells elicited an IgG anti-polysaccharide and anti-protein Ab response after immunization with inactivated S. pneumoniae or soluble pneumococcal capsular polysaccharides. In conclusion, our findings provide evidence for a versatile role of IgM memory B cells in T-independent and T-dependent immune responses.     

Inferring Growth Control Mechanisms in Growing Multi-cellular Spheroids of NSCLC Cells from Spatial-Temporal Image Data

We develop a quantitative single cell-based mathematical model for multi-cellular tumor spheroids (MCTS) of SK-MES-1 cells, a non-small cell lung cancer (NSCLC) cell line, growing under various nutrient conditions: we confront the simulations performed with this model with data on the growth kinetics and spatial labeling patterns for cell proliferation, extracellular matrix (ECM), cell distribution and cell death. We start with a simple model capturing part of the experimental observations. We then show, by performing a sensitivity analysis at each development stage of the model that its complexity needs to be stepwise increased to account for further experimental growth conditions. We thus ultimately arrive at a model that mimics the MCTS growth under multiple conditions to a great extent. Interestingly, the final model, is a minimal model capable of explaining all data simultaneously in the sense, that the number of mechanisms it contains is sufficient to explain the data and missing out any of its mechanisms did not permit fit between all data and the model within physiological parameter ranges. Nevertheless, compared to earlier models it is quite complex i.e., it includes a wide range of mechanisms discussed in biological literature. In this model, the cells lacking oxygen switch from aerobe to anaerobe glycolysis and produce lactate. Too high concentrations of lactate or too low concentrations of ATP promote cell death. Only if the extracellular matrix density overcomes a certain threshold, cells are able to enter the cell cycle. Dying cells produce a diffusive growth inhibitor. Missing out the spatial information would not permit to infer the mechanisms at work. Our findings suggest that this iterative data integration together with intermediate model sensitivity analysis at each model development stage, provide a promising strategy to infer predictive yet minimal (in the above sense) quantitative models of tumor growth, as prospectively of other tissue organization processes. Importantly, calibrating the model with two nutriment-rich growth conditions, the outcome for two nutriment-poor growth conditions could be predicted. As the final model is however quite complex, incorporating many mechanisms, space, time, and stochastic processes, parameter identification is a challenge. This calls for more efficient strategies of imaging and image analysis, as well as of parameter identification in stochastic agent-based simulations.     

Increased or decreased levels of Caenorhabditis elegans lon-3, a gene encoding a collagen,cause reciprocal changes in body length

Body length in C. elegans is regulated by a member of the TGFbeta family, DBL-1. Loss-of-function mutations in dbl-1, or in genes encoding components of the signaling pathway it activates, cause worms to be shorter than wild type and slightly thinner (Sma). Overexpression of dbl-1 confers the Lon phenotype characterized by an increase in body length. We show here that loss-of-function mutations in dbl-1 and lon-1, respectively, cause a decrease or increase in the ploidy of nuclei in the hypodermal syncytial cell, hyp7. To learn more about the regulation of body length in C. elegans we carried out a genetic screen for new mutations causing a Lon phenotype. We report here the cloning and characterization of lon-3. lon-3 is shown to encode a putative cuticle collagen that is expressed in hypodermal cells. We show that, whereas putative null mutations in lon-3 (or reduction of lon-3 activity by RNAi) causes a Lon phenotype, increasing lon-3 gene copy number causes a marked reduction in body length. Morphometric analyses indicate that the lon-3 loss-of-function phenotype resembles that caused by overexpression of dbl-1. Furthermore, phenotypes caused by defects in dbl-1 or lon-3 expression are in both cases suppressed by a null mutation in sqt-1, a second cuticle collagen gene. However, whereas loss of dbl-1 activity causes a reduction in hypodermal endoreduplication, the reduction in body length associated with overexpression of lon-3 occurs in the absence of defects in hypodermal ploidy.