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Additive manufacturing (AM) or industrial 3D printing uses cutting-edge technologies and materials to produce a variety of complex products. However, the effects of the unintentionally emitted AM (nano)particles (AMPs) on human cells following inhalation, require further investigations. The physicochemical characterization of the AMPs, extracted from the filter of a Laser Powder Bed Fusion (L-PBF) 3D printer of iron-based materials, disclosed their complexity, in terms of size, shape, and chemistry. Cell Painting, a high-content screening (HCS) assay, was used to detect the subtle morphological changes elicited by the AMPs at the single cell resolution. The profiling of the cell morphological phenotypes, disclosed prominent concentration-dependent effects on the cytoskeleton, mitochondria, and the membranous structures of the cell. Furthermore, lipidomics confirmed that the AMPs induced the extensive membrane remodeling in the lung epithelial and macrophage co-culture cell model. To further elucidate the biological mechanisms of action, the targeted metabolomics unveiled several inflammation-related metabolites regulating the cell response to the AMP exposure. Overall, the AMP exposure led to the internalization, oxidative stress, cytoskeleton disruption, mitochondrial activation, membrane remodeling, and metabolic reprogramming of the lung epithelial cells and macrophages. We propose the approach of integrating Cell Painting with metabolomics and lipidomics, as an advanced nanosafety methodology, increasing the ability to capture the cellular and molecular phenotypes and the relevant biological mechanisms to the (nano)particle exposure., QC 20230215

BACKGROUND: Diabetes is a chronic disease with several long-term complications. Several glucose-lowering drugs are used to treat type 2 diabetes mellitus (T2DM), e.g., glimepiride and liraglutide, in which both having different modes of action. Circulating microRNAs (miRNAs) are suggested as potential biomarkers that are associated with the disease development and the effects of the treatment. In the current study we evaluated the effect of glimepiride, liraglutide on the expression of the circulating miRNAs. METHODS: The present study is a post hoc trial from a previously randomized control trial comparing liraglutide versus glimepiride both in combination with metformin in subjects with T2DM, and subclinical heart failure. miRNAs were determined in the subjects' serum samples with next generation sequencing. Expression patterns of the circulating miRNAs were analyzed using bioinformatic univariate and multivariate analyses (clinical trial registration: NCT01425580). RESULTS: Univariate analyses show that treatment with glimepiride altered expression of three miRNAs in patient serum, miR-206, miR-182-5p, and miR-766-3p. Both miR-182-5p and miR-766-3p were also picked up among the top contributing miRNAs with penalized regularised logistic regressions (Lasso). The highest-ranked miRNAs with respect to Lasso coefficients were miR-3960, miR-31-5p, miR-3613-3p, and miR-378a-3p. Liraglutide treatment did not significantly influence levels of circulating miRNAs. CONCLUSION: Present study indicates that glucose-lowering drugs differently affect the expression of circulating miRNAs in serum in individuals with T2DM. More studies are required to investigate possible mechanisms by which glimepiride is affecting the expression of circulating miRNAs.

The mucosal surfaces throughout the body are constantly exposed to microorganisms. The mucosal surfaces accommodate a large part of the body’s immune system. For effective vaccination, it is believed that direct immunization on mucosal surfaces will be more effective than the more conventional systemic immunization. Certain probiotic bacteria provide significant adjuvant effects that may be utilized for the desired immune response. Virus-like particles (VLPs) are complexes of viral proteins that without being infectious are efficient in mimicking the natural viral structures. While presenting the patterns of viral antigens, the VLPs have been shown to efficiently interact with dendritic cells and be effective in triggering the B and T-cell immunity. HIV-1 Gag is one of the most highly conserved structural antigens and contains several immunodominant T- and B-cell epitopes. Mosaic Gag protein matches 74% of 9-amino-acid potential epitopes in global Gag sequences thus maximizing the coverage of potential T-cell epitopes for a viral population.The current study aimed to develop probiotic strains of Lactobacillus plantarum NC8 and E. coli Nissle 1917 that produced recombinant mosaic HIV-1 Gag protein as VLPs.For the transient expression of the mosaic HIV-1 Gag protein (GagM), the gene-carrying expression vectors were transformed into the L. plantarum and in probiotic E.coli Nissle 1917. Protein expression of the GagM was shown to lead to the formation of the VLPs of the recombinant HIV-1 Gag proteins. This was confirmed through immunoblotting and transmission electron microscopy (TEM).Our study shows that probiotic lactobacteria can be developed to express HIV-1 Gag VLPs, which may be used for VLP production and potentially for vaccine delivery. Further evaluation of the concept is merited., Developvaccines@oru

The pyrethroid insecticide permethrin is widely used for agricultural and domestic purposes. Previous data indicated that it acts as a developmental neurotoxicant and can induce transgenerational effects in non-target organisms. However, associated underlying mechanisms remain unclear. The aim of this study was to investigate permethrin-related transgenerational effects in the zebrafish model, and to identify possible molecular mechanisms underlying inheritance. Zebrafish (F0) were exposed to permethrin during early-life (2 h post-fertilization up to 28 days). The F1 and F2 offspring generations were obtained by pairing exposed F0 males and females, and were bred unexposed. Locomotor and anxiety behavior were investigated, together with transcriptomic and epigenomic (DNA methylation) changes in brains. Permethrin exposed F0 fish were hypoactive at adulthood, while males from the F1 and F2 generations showed a specific decrease in anxiety-like behavior. In F0, transcriptomic data showed enrichment in pathways related to glutamatergic synapse activity, which may partly underlie the behavioral effects. In F1 and F2 males, dysregulation of similar pathways was observed, including a subset of differentially methylated regions that were inherited from the F0 to the F2 generation and indicated stable dysregulation of glutamatergic signaling. Altogether, the present results provide novel evidence on the transgenerational neurotoxic effects of permethrin, as well as mechanistic insight: a transient exposure induces persistent transcriptional and DNA methylation changes that may translate into transgenerational alteration of glutamatergic signaling and, thus, into behavioral alterations.

Transgenerational effects induced by environmental stressors are a threat to ecosystems and human health. However, there is still limited observation and understanding of the potential of chemicals to influence life outcomes over several generations. In the present study, we investigated the effects of two environmental contaminants, coumarin 47 and permethrin, on exposed zebrafish (F0) and their progeny (F1–F3). Coumarin 47 is commonly found in personal care products and dyes, whereas permethrin is used as a domestic and agricultural pyrethroid insecticide/insect repellent. Zebrafish (F0) were exposed during early development until 28 days post-fertilization and their progeny (F1–F3) were bred unexposed. On one hand, the effects induced by coumarin 47 suggest no multigenerational toxicity. On the other hand, we found that behavior of zebrafish larvae was significantly affected by exposure to permethrin in F1 to F3 generations with some differences depending on the concentration. This suggests persistent alteration of the neural or neuromuscular function. In addition, lipidomic analyses showed that permethrin treatment was partially correlated with lysophosphatidylcholine levels in zebrafish, an important lipid for neurodevelopment. Overall, these results stress out one of the most widely used pyrethroids can trigger long-term, multi- and possibly transgenerational changes in the nervous system of zebrafish. These neurobehavioral changes echo the effects observed under direct exposure to high concentrations of permethrin and therefore call for more research on mechanisms underlying effect inheritance.

Hepatotoxicity is a major reason for the withdrawal or discontinuation of drugs from clinical trials. Thus, better tools are needed to filter potential hepatotoxic drugs early in drug discovery. Our study demonstrates utilization of HCI phenotypes, chemical descriptors, and both combined (hybrid) descriptors to construct random forest classifiers (RFCs) for the prediction of hepatotoxicity. HCI data published by Broad Institute provided HCI phenotypes for about 30 000 samples in multiple replicates. Phenotypes belonging to 346 chemicals, which were tested in up to eight replicates, were chosen as a basis for our analysis. We then constructed individual RFC models for HCI phenotypes, chemical descriptors, and hybrid (chemical and HCI) descriptors. The model that was constructed using selective hybrid descriptors showed high predictive performance with 5-fold cross validation (CV) balanced accuracy (BA) at 0.71, whereas within the given applicability domain (AD), independent test set and external test set prediction BAs were equal to 0.61 and 0.60, respectively. The model constructed using chemical descriptors showed a similar predictive performance with a 5-fold CV BA equal to 0.66, a test set prediction BA within the AD equal to 0.56, and an external test set prediction BA within the AD equal to 0.50. In conclusion, the hybrid and chemical descriptor-based models presented here should be considered as a new tool for filtering hepatotoxic molecules during compound prioritization in drug discovery., Funding Agencies:EnForce project (Environmental Forensic Laboratory)Örebro University, Sweden

This paper presents morphological and molecular characterizations of the representative of the pantropic genus Brasilonema Fiore et al. first discovered in a European greenhouse. The genus described in 2007, using an integrated approach, is morphologically close to Scytonema Agardh; differing in the filaments forming ascending bundles, rarely occurring false branching, and the color of cells. To date, 12 species of Brasilonema have been described; all of them occur subaerophytically in humid tropical and subtropical regions. For several years in the greenhouse of the N.N. Grishko National Botanical Garden of the NAS of Ukraine, we observed abundant blackish mats with a fleecy surface, formed by falsely branching filaments of heterocytous cyanobacteria lilac, pale purple or gray in color. Cyanobacteria densely covered the vegetative organs of various tropical epiphytic plants from the Orchidaceae and Bromeliaceae families; sometimes inhabiting concrete and wooden substrates in the greenhouse. The morphological features of this newly discovered cyanobacteria were studied both in natural material greenhouse samples and culture. For cultivation, liquid and agarized N-free BG11 medium was used. The specimens from the mats and the cultures had some differences in thalli habitus, coloration and arrangement of filaments, frequency of false branching, trichome appearance, dimensional limits, etc. A comparative analysis of the original data with descriptions of the known Brasilonema species showed that the Kiev population coincides morphologically and ecologically with several species, of which it's closest relative is B. octagenarum Aguiar et al. Phylogenetic analysis of the nucleotide sequence of the gene encoding 16S rRNA confirmed affiliation of the original strains to Brasilonema. Analysis of the nucleotide sequence of the 16S-23S ITS region, as well as the secondary structure of its most informative helices, showed the closest proximity of the Kiev material to B. octage

Perfluorooctanesulfonate (PFOS) is a well-known contaminant in the environment and it has shown to disrupt multiple biological pathways, particularly those related with lipid metabolism. In this study, we have studied the impact of in ovo exposure to PFOS on lipid metabolism in livers in developing chicken embryos using lipidomics for detailed characterization of the liver lipidome. We used an avian model (Gallus gallus domesticus) for in ovo treatment at two levels of PFOS. The lipid profile of the liver of the embryo was investigated by ultra-high performance liquid chromatography combined with quadrupole-time-of-flight mass spectrometry and by gas chromatography mass spectrometry. Over 170 lipids were identified, covering phospholipids, ceramides, di- and triacylglycerols, cholesterol esters and fatty acid composition of the lipids. The PFOS exposure caused dose dependent changes in the lipid levels, which included upregulation of specific phospholipids associated with the phosphatidylethanolamine N-methyltransferase (PEMT) pathway, triacylglycerols with low carbon number and double bond count as well as of lipotoxic ceramides and diacylglycerols. Our data suggest that at lower levels of exposure, mitochondrial fatty acid β-oxidation is suppressed while the peroxisomal fatty acid β -oxidation is increased. At higher doses, however, both β -oxidation pathways are upregulated.

A number of chemicals have been shown to affect epigenetic patterning and functions. Since epigenetic mechanisms regulate transcriptional networks, epigenetic changes induced by chemical exposure can represent early molecular events for long-term adverse physiological effects. Epigenetics has thus appeared as a research field of major interest within (eco)toxicological sciences. The present study aimed at measuring effects on epigenetic-related mechanisms of selected environmental chemicals (bisphenols, perfluorinated chemicals, methoxychlor, permethrin, vinclozolin and coumarin 47) in zebrafish embryos and liver cells (ZFL). Transcription of genes related to DNA methylation and histone modifications was measured and global DNA methylation was assessed in ZFL cells using the LUMA assay. The differences in results gathered from both models suggest that chemicals affect different mechanisms related to epigenetics in embryos and cells. In zebrafish embryos, exposure to bisphenol A, coumarin 47, methoxychlor and permethrin lead to significant transcriptional changes in epigenetic factors suggesting that they can impact early epigenome reprogramming related to embryonic development. In ZFL cells, significant transcriptional changes were observed upon exposure to all chemicals but coumarin 47; however, only perfluorooctane sulfonate induced significant effects on global DNA methylation. Notably, in contrast to the other tested chemicals, perfluorooctane sulfonate affected only the expression of the histone demethylase kdm5ba. In addition, kdm5ba appeared as a sensitive gene in zebrafish embryos as well. Taken together, the present results suggest a role for kdm5ba in regulating epigenetic patterns in response to chemical exposure, even though mechanisms remain unclear. To confirm these findings, further evidence is required regarding changes in site-specific histone marks and DNA methylation together with their long-term effects on physiological outcomes., Funding Agency:EnForce platform - KK Foundation 201660019

OBJECTIVE: The aim of this study was to investigate whether protein expression of the extracellular matrix-degrading protease ADAMTS5 can be demonstrated in the urinary bladder of healthy rats, and, if so, to determine the localization of this enzyme. MATERIALS AND METHODS: The experiments were conducted with eight inbred male Sprague-Dawley rats. Immunohistochemistry was used to investigate the expression of ADAMTS5 in the urinary bladder. Negative controls were established by either excluding the primary antibody or applying the antibody after it had been preabsorbed with its immunogenic peptide. Confocal microscopy was used to visualize the distribution of ADAMTS5 in the urinary bladder tissue. RESULTS: Immunoreactivity for ADAMTS5 was demonstrated in the urothelium and in the detrusor. This expression was localized not only in the cytoplasm, but also in the nuclei. Confocal microscopy corroborated these findings. CONCLUSION: Expression of ADAMTS5 was demonstrated in the cytoplasm as well as in the nuclei of the urothelium and detrusor cells, suggesting that it may play a role at the transcriptional level., Funding Agencies:Anna-Lisa and Bror Björnsson's Research Foundation Martha and Gustaf Ågren's Research Foundation Örebro University

Perfluorooctane sulfonic acid (PFOS) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) are persistent organic pollutants of high concern because of their environmental persistence, bioaccumulation and toxic properties. Besides, the amphiphilic properties of fluorinated compounds such as PFOS and perfluorohexanoic acid (PFHxA) suggest a role in increasing cell membrane permeability and solubilizing chemicals. The present study aimed at investigating whether PFOS and PFHxA are capable of modifying the activation of PCB126 toxicity-related pathways. For this purpose, zebrafish embryos were exposed in semi-static conditions to 7.5 μg/L of PCB126 alone, in the presence of 25 mg/L of PFOS, 15.7 mg/L of PFHxA or in the presence of both PFOS and PFHxA. Quantitative PCR was performed on embryos aged from 24 h post fertilization (hpf) to 96 hpf to investigate expression changes of genes involved in metabolism of xenobiotics (ahr2, cyp1a), oxidative stress (gpx1a, tp53), lipids metabolism (acaa2, osbpl1a), and epigenetic mechanisms (dnmt1, dnmt3ba). Cyp1a and ahr2 expression were significantly induced by the presence of PCB126. However, after 72 and 78 h of exposure, induction of cyp1a expression was significantly lower when embryos were co-exposed to PCB126 + PFOS + PFHxA when compared to PCB126-exposed embryos. Significant upregulation of gpx1a occurred after exposure to PCB126 + PFHxA and to PCB126 + PFOS + PFHxA at 30 and 48 hpf. Besides, embryos appeared more sensitive to PCB126 + PFOS + PFHxA at 78 hpf: acaa2 and osbpl1a were significantly downregulated; dnmt1 was significantly upregulated. While presented as environmentally safe, PFHxA demonstrated that it could affect gene expression patterns in zebrafish embryos when combined to PFOS and PCB126, suggesting that such mixture may increase PCB126 toxicity. This is of particular relevance since PFHxA is persistent and still being ejected into the environment. Moreover, it provides additional information as to the importance to, Funding agencies:ERASMUS program

The ability of commensal bacteria to influence gene expression in host cells under the influence of pathogenic bacteria has previously been demonstrated, however the extent of this interaction is important for understanding how bacteria can be used as probiotics. Real-time quantitative polymerase chain reaction is the most sensitive tool for evaluating relative changes to gene expression levels. However as a result of its sensitivity an appropriate method of normalisation should be used to account for any variation incurred in preparatory experimental procedures. These variations may result from differences in the amount of starting material, quality of extracted RNA, or in the efficiency of the reverse transcriptase or polymerase enzymes. Selection of an endogenous control gene is the preferred method of normalisation, and ideally a proper validation of the gene's appropriateness for the study in question should be performed. In this study we used quantitative polymerase chain reaction data and applied four different algorithms (geNorm, BestKeeper, NormFinder, and comparative ΔCq) to evaluate eleven different genes as to their suitability as endogenous controls for use in studies involving colonic (HT-29) and vaginal (VK2/E6E7) human mucosal epithelial cells treated with probiotic and pathogenic bacteria. We found phosphoglycerate kinase 1 to be most appropriate for HT-29 cells, and ribosomal protein large P0 to be the best choice for VK2/E6E7 cells. We also showed that use of less stable reference genes can lead to less accurate quantification of expression levels of gene of interest (GOI) and also can result in decreased statistical significance for GOI expression levels when compared to control. Additionally, we found the cell type being analysed had greater influence on reference gene selection than the treatment performed. This study provides recommendations for stable endogenous control genes for use in further studies involving colonic and vaginal cell lines

Lactobacillus rhamnosus GR-1 is a probiotic bacterium used to maintain urogenital health. The putative mechanism for its probiotic effect is by modulating the host immunity. Urinary tract infections (UTI) are often caused by uropathogenic Escherichia coli that frequently evade or suppress immune responses in the bladder and can target pathways, including nuclear factor-kappaB (NF-κB). We evaluated the role of L. rhamnosus GR-1 on NF-κB activation in E. coli-stimulated bladder cells. Viable L. rhamnosus GR-1 was found to potentiate NF-κB activity in E. coli-stimulated T24 bladder cells, whereas heat-killed lactobacilli demonstrated a marginal increase in NF-κB activity. Surface components released by trypsin- or LiCl treatment, or the resultant heat-killed shaved lactobacilli, had no effect on NF-κB activity. Isolation of released products from L. rhamnosus GR-1 demonstrated that the induction of NF-κB activity was owing to released product(s) with a relatively large native size. Several putative immunomodulatory proteins were identified, namely GroEL, elongation factor Tu and NLP/P60. GroEL and elongation factor Tu have previously been shown to elicit immune responses from human cells. Isolating and using immune-augmenting substances produced by lactobacilli is a novel strategy for the prevention or treatment of UTI caused by immune-evading E. coli., Funding Agencies:Knowledge FoundationMagnus Bergvalls Foundation Sparbanksstiftelsen Nya Carl Tryggers Foundation, Sweden

Background: Epithelial cells of the urinary tract recognize pathogenic bacteria through pattern recognition receptors on their surface, such as toll-like receptors (TLRs), and mount an immune response through the activation of the NF-kappaB pathway. Some uropathogenic bacteria can subvert these cellular responses, creating problems with how the host eliminates pathogens. Lactobacillus is a genus of lactic acid bacteria that are part of the microbiota and consist of many probiotic strains, some specifically for urogenital infections. Immunomodulation has emerged as an important mode of action of probiotic and commensal lactobacilli and given the importance of epithelial cells, we evaluated the effect of the urogenital probiotic Lactobacillus rhamnosus GR-1 on epithelial immune activation. Results: Immune activation through the NF-kappaB pathway was initiated by stimulation of T24 urothelial cells with heat-killed Escherichia coli and this was further potentiated when cells were co-cultured with live L. rhamnosus GR-1. Heat-killed lactobacilli were poor activators of NF-kappaB. Concomitant stimulation of bladder cells with E. coli and L. rhamnosus GR 1 increased the levels of the pro-inflammatory cytokine TNF, whereas IL-6 and CXCL8 levels were reduced. Another probiotic, L. rhamnosus GG, was also able to potentiate NF-kappaB in these cells although at a significantly reduced level compared to the GR 1 strain. The transcript numbers and protein levels of the lipopolysaccharide receptor TLR4 were significantly increased after co-stimulation with E. coli and lactobacilli compared to controls. Furthermore, inhibition of TLR4 activation by polymixin B completely blocked the lactobacilli potentiation of NF-kappaB. Conclusions: The immunological outcome of E. coli challenge of bladder cells was influenced by probiotic L. rhamnosus GR 1, by enhancing the activation of NF-kappaB and TNF release. Thus the urogenital probiotic L. rhamnosus GR-1 modulated the activation of the N

in vivo 24 (2010).

Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during the breeding season and is subsequently secreted into the urinary bladder from where it is employed for nest building. Since the production of the protein has been shown to be under androgenic control, spiggin has been suggested to be a useful biomarker for androgenic substances in the environment. In this study, two polyclonal spiggin antibodies based on synthetic peptides and one polyclonal antibody directed against native spiggin have been characterized. The antibodies ability to identify spiggin was investigated by quantitative immunoassay. For both peptide antibodies the quantification range was determined to be between 1 and 80 ng spiggin and determination of renal spiggin levels from immature and mature males displayed a 15-fold increase in total spiggin content of the kidney resulting in a 6-fold increase in male kidney weight due to hypertrophy. The kidney somatic index (KSI) was found to correlate well with the total renal spiggin content and therefore it appears that KSI in sticklebacks could be used as an initial method to identify substances displaying androgenic effects. Furthermore, western blot analysis revealed that the polyclonal antibodies recognize different spiggin isoforms and that spiggin can be detected in the urinary bladder and kidney of both males and female sticklebacks. In order to develop a quantitative detection method for native spiggin it is necessary to produce a standard that can be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin the protein is difficult to use as a standard in bioassays. So far spiggin has been shown to exist in at least 14 isoforms, all of which contain polymerization domains. To overcome the solubility problem we have produced recombinant spiggin gamma, with only one polymerization domain, that can be expressed in E. coli. Western blot analysis demonstrated that the polyclonal antibodies were a

In Pisum sativum, the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). Expression of two of these genes (SAD-A and -C) in Escherichia coli or Pichia pastoris resulted in full-length soluble proteins. Purified SAD-A was used as antigen for antibody production in rabbits. With these antibodies the recombinant SAD-C protein (which was most highly expressed of the two isoforms) was shown to be a tetramer consisting of a dimer of dimers. The SAD genes are transiently expressed in plants by short exposures to ultraviolet-B radiation (UV-B), as judged by northern blotting. In turn, mRNA accumulation leads to formation of SAD protein in leaf and stem tissue upon prolonged UV-B irradiation.

Androgens play an important role in male sexual differentiation and development. They exert their function by binding to and activating the androgen receptor (Ar), a member of the steroid hormone receptor superfamily. Here, we report on the isolation and characterization of zebrafish Ar. The complete transcript of zebrafish ar is 5.3 kb long encoding a putative polypeptide of 868 amino acids. Our experimental and bioinformatic analysis has found a single ar locus in zebrafish. Phylogenetic analysis using the ligand-binding domain showed that the zebrafish Ar clustered with its cyprinid orthologs to form a separate group, which was closer to the beta clade than to the alpha clade. Tissue-specific expression analysis revealed that the ar mRNA was expressed ubiquitously in all adult tissues tested, with sexually dimorphic expression in the gonad and muscle. While the ar transcript was maternally deposited into the embryo, signs of zygotic expression could be detected as early as 24 h after fertilization, and the expression level increased substantially afterwards. When analyzed during gonad development, the expression level of ar mRNA at 4 wk after fertilization was similar in both developing gonads but later became higher in the transforming testis, suggesting a potential role during male gonad differentiation. We also combined theoretical modeling with in vitro experiments to show that the zebrafish Ar is preferentially activated by 11-ketotestosterone.

In pea (Pisum sativum), the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). The SAD genes are transiently expressed in plants after short exposures to ultraviolet-B radiation, which in turn leads to formation of SAD protein in leaf and stem tissue upon prolonged irradiation. SAD gene expression is also seen as a result of wounding stress. The recombinant SAD-C protein (which was the most highly over-expressed isoform in Escherichia coli of the isoforms) was shown to be a tetramer that probably consists of a dimer of dimers and which possesses quinone-reducing capability. The enzyme shows approximately the same NADH- and NADPH-dependent activity with 2,5- and 2,6-dimethylbenzoquinone and menadione as substrates. Western blotting and immunohistochemistry (IHC) shows that the SAD protein is present to a smaller or larger extent in all the different pea tissues examined. Environmental stress such as UV-B radiation clearly increases the content of SAD in leaf and stem tissue but not in roots. This indicates that increased expression of the SAD genes, as a result of UV-B exposure, is limited to the exposed tissue. This is substantiated by the heterologous GUS expression from the pea SAD-C promoter in Arabidopsis during wounding. Only the wound site and the vicinal tissue show transcription from this promoter. In non-stressed tissue (as well as in UV-B-stressed leaves and stem), SAD predominantly occurred in epidermal or sub-epidermal cells as judged by IHC. The protoderm of the pea seed cotyledonary axis contains the most heavily stained cells. This indicates a possible role for the SAD protein in development as well as in protection against environmental stress. Also, discrete staining was obtained in particular cell types of the ovary. To be able to understand the biochemical and physiological role of the SAD enzyme, an in silico modeling study of the SAD protein structure was performed. The simula