Pollen-derived nonallergenic substances enhance Th2-induced IgE production in B cells.

BACKGROUND: B cells play a central role in IgE-mediated allergies. In damaged airway epithelium, they are exposed directly to aeroallergens. We aimed to assess whether direct exposure of B cells to pollen constituents affects allergic sensitization. METHODS: B cells from murine splenocytes and from blood samples of healthy donors were incubated for 8 days under Th2-like conditions with aqueous ragweed pollen extracts (Amb-APE) or its constituents. Secreted total IgM, IgG, and IgE was quantified by ELISA. Additionally, birch, grass, or pine-pollen extracts were tested. The number of viable cells was evaluated by ATP measurements. B-cell proliferation was measured by CFSE staining. IgE class switch was analyzed by quantitation of class switch transcripts. In an OVA/Alum i.p.-sensitization mouse model, Amb-APE was intranasally instilled for 11 consecutive days. RESULTS: Upon Th2 priming of murine B cells, ragweed pollen extract caused a dose-dependent increase in IgE production, while IgG and IgM were not affected. The low-molecular-weight fraction and phytoprostane E1 (PPE1) increased IgE production, while Amb a 1 did not. PPE1 enhanced IgE also in human memory B cells. Under Th1 conditions, Amb-APE did not influence immunoglobulin secretion. The IgE elevation was not ragweed specific. It correlated with proliferation of viable B cells, but not with IgE class switch. In vivo, Amb-APE increased total IgE and showed adjuvant activity in allergic airway inflammation. CONCLUSIONS: Aqueous pollen extracts, the protein-free fraction of Amb-APE, and the pollen-contained substance PPE1 specifically enhance IgE production in Th2-primed B cells. Thus, pollen-derived nonallergenic substances might be responsible for B-cell-dependent aggravation of IgE-mediated allergies.

Induction of DNA double-strand breaks by monochlorophenol isomers and ChKM in human gingival fibroblasts.

Phenol has been traditionally used in dental treatment as a sedative for the pulp or as disinfectant for carious cavity and root canal. However, phenol is regarded as a mutagenic and carcinogenic agent and its use in dental practice is now therefore restricted. Monochlorophenols are derivatives of phenol, which are still used clinically as root canal disinfectants, they are even more active antiseptics/disinfectants than phenol, and the so-called Walkhoff (ChKM) solution makes use of monochlorophenol for root canal disinfection. Ingredients in the ChKM solution are the monochlorophenol compound 4-chlorophenol (4-CP), camphor, and menthol. In literature, the use of the ChKM solution is controversial because of a possible DNA toxicity of the ingredient 4-CP. However, it is unknown whether ChKM can really induce DNA damage in human oral cells. In this study, the induction of DNA double-strand breaks (DSBs) by ChKM and monochlorophenol compounds (2-chlorophenol, 2-CP; 3-chlorophenol, 3-CP; and 4-chlorophenol, 4-CP) was tested in human gingival fibroblasts (HGFs). DNA DSBs (foci) induced in HGFs unexposed and exposed to monochlorophenols or ChKM solution were investigated using the γ-H2AX DNA focus assay, which is a direct marker for DSBs. DSBs result in the ATM-dependent phosphorylation of the histone H2AX. When cells were exposed to medium or medium + DMSO (1 %) (negative controls), an average of 3 foci per cell were found. In positive control cells (H2O2 + medium, or H2O2 + medium + DMSO (1 %), an average of 35 foci each were found. About 20 DSB foci per cell were found, when HGFs were exposed to 2-CP (4 mM), 3-CP (2.3 mM), 4-CP (2.1 mM), or ChKM (corresponding to 1.5 mM 4-CP). Our results show increasing DNA toxicities in the order of 2-CP < 3-CP < 4-CP < ChKM solution. An additive DNA toxicity was found for 4-CP in combination with camphor in the ChKM solution, compared to the 4-CP alone. No significant differences regarding multi-foci cells (cells that contain more than 40 foci) were found when HGFs were exposed to the EC50 concentrations (given in parenthesis) of ChKM (1.5 mM), 4-CP (2.1 mM), or 2-CP (4 mM). Significantly fewer multi-foci cells were found when HGFs were exposed to the EC50 concentration (given in parenthesis) of 3-CP (2.3 mM), compared to the EC50 concentrations of ChKM, 4-CP, or 2-CP. Monochlorophenol compounds and/or ChKM solution can induce DSBs in primary human oral (cavity) cells, which underscores their genotoxic capacity.

Nitric oxide as a signal in plants.

Molecular, genetic and biochemical studies have identified key players in the signaling pathways regulating growth and development, as well as defense responses in plants. Recently, nitric oxide (NO) - the versatile and powerful effector of animal redox-regulated signaling and immune responses - was shown to mediate plant defense responses against pathogens. Interestingly, several key components involved in NO-mediated signaling in animals also appear to be operative in plants.

Nitric oxide: comparative synthesis and signaling in animal and plant cells.

Since its identification as an endothelium-derived relaxing factor in the 1980s, nitric oxide has become the source of intensive and exciting research in animals. Nitric oxide is now considered to be a widespread signaling molecule involved in the regulation of an impressive spectrum of mammalian cellular functions. Its diverse effects have been attributed to an ability to chemically react with dioxygen and its redox forms and with specific iron- and thiol-containing proteins. Moreover, the effects of nitric oxide are dependent on the dynamic regulation of its biosynthetic enzyme nitric oxide synthase. Recently, the role of nitric oxide in plants has received much attention. Plants not only respond to atmospheric nitric oxide, but also possess the capacity to produce nitric oxide enzymatically. Initial investigations into nitric oxide functions suggested that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. These findings complement an emerging body of evidence indicating that many signal transduction pathways are shared between plants and animals.

Ketol-Acid Reductoisomerase from Barley (Hordeum vulgare) (Purification, Properties, and Specific Inhibition).

Ketol-acid reductoisomerase (KARI, EC 1.1.1.86) was purified to homogeneity from etiolated barley shoots (Hordeum vulgare) using anion exchange, Red-Sepharose, hydrophobic interaction, and chromatofocusing steps. Purification yielded 0.25 to 0.27 mg of pure KARI per 100 g fresh weight of starting material. The specific activity of the purified enzyme was 6 [mu]mol of NADPH oxidized min-1 mg-1 with acetohydroxybutyrate as substrate. The native enzyme had an apparent molecular weight of 115,000 as estimated by gel filtration and appeared to be a homodimer with a subunit molecular weight of 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Km values of the purified KARI for acetolactate, acetohydroxybutyrate, and NADPH (determined with acetohydroxybutyrate) were 11, 38, and 4.3 [mu]M, respectively. The Vmax obtained with acetohydroxybutyrate was 1.8 [mu]mol min-1 mg-1; the corresponding value for acetolactate was 0.16 [mu]mol min-1 mg-1. The enzyme showed optimum activity at pH 7.5. When either acetolactate or acetohydroxybutyrate was used as substrate, the experimental herbicidal compound 2-dimethyl-phosphinoyl-2-hydroxyacetic acid inhibited the purified KARI in a time-dependent and reversible manner. The initial inhibition was strictly competitive. The inhibition constant values were 0.46 (using acetolactate as substrate) and 0.19 [mu]M (acetohydroxybutyrate), respectively.

Nitric oxide inhibition of tobacco catalase and ascorbate peroxidase.

We used a variety of nitric oxide (NO) donors to demonstrate that NO inhibits the activities of tobacco catalase and ascorbate peroxidase (APX). This inhibition appears to be reversible because removal of the NO donor led to a significant recovery of enzymatic activity. In contrast, APX and catalase were irreversibly inhibited by peroxynitrite. The ability of NO and peroxynitrite to inhibit the two major H2O2-scavenging enzymes in plant cells suggests that NO may participate in redox signaling during the activation of defense responses following pathogen attack.

The oxygenase reaction of acetolactate synthase detected by chemiluminescence.

In addition to the synthesis of ketolacids the enzyme acetolactate synthase shows an oxygen-consuming side reaction. Partially purified acetolactate synthase from corn (Zea mays L.) and barley (Hordeum vulgare L.) exhibits chemiluminescence in the presence of oxygen, Mn2+ and low concentrations of pyruvate. Light emission is inhibited by azide, but not by catalse or superoxide dismutase. The data suggest the formation of singlet oxygen during the catalytic cycle, and provides a basis for a highly sensitive assay for the oxygenase reaction of acetolactate synthesis. Both synthase activity and chemiluminescence are inhibited by sulfonylurea herbicides. The results add a new aspect to the irreversible inhibition of acetolactate synthase by these herbicides which may be enhanced by the presence of reactive oxygen species.

Cytotoxicity of dental composite components and mercury compounds in pulmonary cells.

The cytotoxic potentials of the dental composite components triethyleneglycoldimethacrylate (TEGDMA) and 2-hydroxy-ethylmethacrylate (HEMA) as well as mercuric chloride (HgCl2) and methyl mercury chloride (MeHgCl) were investigated. Proliferating A549 and L2 cell monolayers were cultured in the absence or presence of composite components or mercurials. Twenty-four hours later the tetrazolium salt XTT (sodium 3'-[1-phenyl-aminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzenesulphonic acid) was added. Formazan formation was quantified using a microtiter plate reader. EC50 values were obtained as half-maximum-effect concentrations from fitted curves. EC50 values were in A549 cells (mean values +/- standard deviation; n = 12; micromol/l); HEMA 8854+/-1882; TEGDMA 1821+/-529; HgCl2 41+/-7 and MeHgCl 27+/-3. EC50 values in L2 cells were: HEMA 191+/-28; TEGDMA 112+/-16; HgCl2 25+/-6 and MeHgCl 8+/-6. All tested substances induced a dose-dependent loss of viability in A549 and L2 cells after 24 h. The EC50 values of both mercurials were significantly (p < 0.05) lower compared to the values of both composite components. TEGDMA was about 5-fold (A549 cells) and about 2-fold (L2 cells) more toxic compared to HEMA. It is to be assumed that the risk of lung cell damage by dental composite components is even more unlikely.

Biological clearance of HEMA in guinea pigs.

No toxicokinetic data are available about the dental composite component 2-hydroxyethylmethacrylate (HEMA) in vivo in the literature. Therefore, the excretion of HEMA in feces and urine in vivo and, using the pendular perfusion technique with segments of jejunum and colon, in the biliary and enteric excretion in situ were investigated in anesthetized guinea pigs. In the in situ experiments, guinea pigs (n = 4) received HEMA (0.02 mmol/kgbw labelled with a tracer dose 14C-HEMA 0.3 kBq/gbw) injected into the jugular vein. In the in vivo experiments, guinea pigs (n = 4) received HEMA (+ 14C-HEMA, same dose as above) via gastric tube. Urine and feces were collected for 24h. In the in situ experiments, organs from guinea pigs were removed 60 min after the beginning of the experiment, and then the 14C-radioactivity was measured. During the 60 min perfusion period the calculated amount of 14C-activity excreted into the total jejunum and colon was 6.0 +/- 1.0% and 2.7 +/- 0.7% of the dose administered, respectively (mean +/- sem). Of the 14C-HEMA dose, 5.3 +/- 0.3% was found in the bile. Significantly (p < 0.05) higher bile/blood concentration ratios were found at 10-40 min after the injection of HEMA, as compared to the ratio at 60 min. The total 14C-recovery in all organs tested was 20.0 +/- 2.6%. During 24h the amounts of 14C-activity excreted in the feces and urine were 1.1 +/- 0.1% or 17.1 +/- 1.50% of the dose administered, respectively (mean +/- sem). The total 14C-recovery in all organs tested was 11.6 +/- 0.6%. In a second series of in vivo experiments, exhaled air from the animals was captured during the 24h experimental period. 14C was exhaled to 63.6 +/- 2.11% of the administered 14C-HEMA dose (mean +/- sem; n = 4) as 14C-carbondioxide. The results indicate a rapid clearance of 14C-HEMA and/or 14C-HEMA metabolite(s) from the organism, exhalation being the major route of elimination.

Distribution and excretion of TEGDMA in guinea pigs and mice.

The monomer triethyleneglycoldimethacrylate (TEGDMA) is used as a diluent in many resin-based dental materials. It was previously shown in vitro that TEGDMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of 14C-TEGDMA applied via gastric, intradermal, and intravenous administration at dose levels well above those encountered in dental care were examined in vivo in guinea pigs and mice as a test of the hypothesis that TEGDMA reaches cytotoxic levels in mammalian tissues. 14C-TEGDMA was taken up rapidly from the stomach and small intestine after gastric administration in both species and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as 14CO2. The peak equivalent TEGDMA levels in all mouse and guinea pig tissues examined were at least 1000-fold less than known toxic levels. The study therefore did not support the hypothesis.

Cytotoxicity of dental composite components and mercury compounds in lung cells.

OBJECTIVE: The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA), as well as mercuric chloride (HgCl2) and methylmercury chloride (MeHgCl) was investigated on the release of lactatedehydrogenase (LDH) from alveolar epithelial lung cell lines in vitro. METHODS: The confluent cell layers from the A549 (human, malignant) and the L2 cells (rat) were incubated with various concentrations of HEMA, TEGDMA, MeHgCl and HgCl2 at 37 degrees C in 2% (v/v) CO2 atmosphere for 8h. In further experiments the L2 cells were incubated with the same compounds for 6-48 h. LDH release was measured and the values were expressed as percentage of the LDH content. The values were plotted on a concentration log-scale and the substance concentration at the maximum slope was assessed as effective concentration (EC50). RESULTS: A significant (p<0.05) increase in the LDH release was found in the L2 cells after 8-h incubation with HEMA (4 mmol/l), TEGDMA (2 mmol/l), MeHgCl (0.01 mmol/l) and HgCl2 (0.015 mmol/l), and in A549 cells with HEMA (14 mmol/l), TEGDMA (15 mmol/l), MeHgCl (0.15 mmol/l) and HgCl2 (0.05 mmol/l), compared to controls. The EC50 values from compounds in the L2 cells are shown in the following table (mean; sem in parentheses; n=3-6; #n=1): [see text]. SIGNIFICANCE: The toxic effect of HgCl2 and MeHgCl from the L2 cells was about 100-700-fold higher than of the dental composite components. A significant (p<0.05) time dependent increase of toxicity was observed with TEGDMA, HEMA and MeHgCl.

Toxicokinetic of HEMA in guinea pigs.

OBJECTIVE: Unconverted 2-hydroxyethylmethacrylate (HEMA) can be released from dental resin materials and can enter the body in humans. In the present study the uptake, distribution and excretion of 14C-HEMA applied via different routes were examined in vivo in guinea pigs. METHODS: HEMA (0.02 mmol/kg bw labelled with a tracer dose 14C-HEMA 0.3 Bq/g bw) was administered by gastric tube or by subcutaneous injection. Urine, feces, and exhaled carbon dioxide were collected for 24 h after administration. Guinea pigs were killed 24 h after the beginning of the experiment and various organs removed and 14C radioactivity measured. RESULTS: Low fecal 14C levels (about 2% of the dose) and urinary levels of about 15% after 24 h were noted with either route of administration. Direct measurement of exhaled CO(2) showed that about 70% of the dose left the body via the lungs. Two pathways for the metabolism of 14C-HEMA can be described. It is likely that 14C-pyruvate is formed in vivo resulting in the formation of toxic 14C-HEMA intermediates. 14C-HEMA was taken up rapidly from the stomach and small intestine after gastric administration and was widely distributed in the body following administration by each of the routes. CONCLUSIONS: Clearance from most tissues following gastric and intradermal administration was essentially complete within one day. The peak HEMA levels in all tissues examined after 24 h were at least onemillion-fold less than known toxic levels.

Quantification of elutable substances from methacrylate based sealers and their cytotoxicity effect on with human gingival fibroblasts.

OBJECTIVES: Previous studies have shown that resin composites may cause persistent inflammation of oral or pulpal tissues as well as cell death through eluted substances. The aim of this study was to investigate the leaching of ingredients from commercial dental fissure sealers as well as their cytotoxic effects on human gingival fibroblast (HGF). METHODS: The sealers tested were: Helioseal(®) F, Helioseal(®) Refill, Fissurit(®) F, Grandio(®) Seal, Ultraseal XT(®) plus and Delton(®) FS. Ten discs of each sealer were respectively immersed in methanol or water and incubated at 37°C. The eluates were analysed by gas chromatography/mass spectrometry at day 1, 3 and 7. In the XTT-test, eight discs of each fissure sealer were immersed into medium. The eluates of the respective sealer were mixed and used undiluted and diluted with medium. HGF were incubated with the dilutions at 37°C for 24h. Then XTT-salt was added and the XTT-formazan formation was quantified. RESULTS: In eluates from polymerized sealers, comonomers (triethylene glycol dimethacrylate (TEGDMA)) and additives were found (e.g. camphorquinone (CQ), butylated hydroxytoluene, triphenylstibane). 7 d after the beginning of the experiments the highest amount of TEGDMA was found in the aqueous eluate from Grandio(®) Seal (9944.31 (2250.56) µmol/l). The most cytotoxic eluate found in the XTT-test was from Fissurit(®) F (EC50 value at 27.13 (7.04)%; (mean(SD)). SIGNIFICANCE: Because of the use of sealers in preventative dental medicine it should be taken into account that substances like TEGDMA or CQ, that are often causing allergic reactions, are elutable. Before using the sealers patients should be asked for allergic reactions to these substances.

Synergistic interaction caused to human gingival fibroblasts from dental monomers.

OBJECTIVES: The toxicity of monomers like bisphenol-A-glycidyldimethacrylate (BisGMA) and urethane-dimethacrylate (UDMA) to cells is well studied. In these studies solubilizers, which have a toxic potential, are used to dissolve the basic monomers in the aqueous medium. In these experiments it is not possible to confidently exclude a synergistic effect of basic monomers and solubilizers in cells. Moreover, less is known about the synergistic interaction between basic- and comonomers (triethyleneglycoldimethacrylate (TEGDMA); 2-hydroxyethylmethacrylate (HEMA)) in cells. We dissolved the basic monomers in the comonomers and incubated human gingival fibroblasts (HGFs) with these binary mixtures in different concentrations. METHODS: Proliferating HGFs monolayers were cultured in the absence or presence of mixtures of BisGMA/TEGDMA, BisGMA/HEMA, UDMA/TEGDMA and UDMA/HEMA. Twenty-four hours later XTT was added and the formazan formation was quantified. EC(50) values were obtained at half-maximum-effect concentrations from fitted curves. RESULTS: EC(50) values were (mmol/l; mean±sem; n=5): 0.01 BisGMA/0.48 TEGDMA; 0.04 BisGMA/4.99 HEMA; 0.04 UDMA/1.60 TEGDMA and 0.02 UDMA/2.26 HEMA. All tested mixtures induced a dose-dependent loss of viability in HGFs after 24h. SIGNIFICANCE: The EC(50) values of binary mixtures were significantly (p<0.05) lower compared to the EC(50) values of the pure substances indicating a synergistic interaction of the mixtures on the HGFs. The widely used (co)monomers BisGMA and TEGDMA have the lowest EC(50) values. The highest decrease of EC(50) values, compared with the pure substances, were found in the mixture UDMA/HEMA. Worst case calculations show that the EC(50) values from binary mixtures are at least 6 fold lower compared with known amounts of elutable (co)monomers from polymerized composites.

Cytokine release from human leukocytes exposed to silorane- and methacrylate-based dental materials.

OBJECTIVES: Silorane-based dental monomers contain an epoxy functional group. Less is known about the toxicological and inflammatory potential of silorane-based composites. Therefore we compared the release of 24 cytokines from human leukocytes after incubation with silorane-based Filtek™ Silorane (Silo) and methacrylate-based TetricEvo Flow® (TC). METHODS: Leukocytes from nine healthy test persons (P) were incubated with Silo or TC for up to 72h. All 24h cytokines were quantified with a magnetic bead assay. RESULTS: Silo stimulates the leukocytes to higher release of cytokines when compared to TC. 72h after beginning the experiment, leukocytes from P6 incubated with Silo secreted more than an 18-fold amount of interleukin (IL)-6 when compared with leukocytes incubated with TC (771.8 vs 42.1pg/ml). Only leukocytes from P8 incubated with Silo release up to 14.4pg/ml IL-2 after 72h. SIGNIFICANCE: The significantly higher induction of cytokines with Silo in comparison to TC is test person independent. This indicates a higher sensitization potential for Silo. Because of the cytokine release pattern (especially the release of T-cell dependent IL-2) from leukocytes from P8 after incubation with Silo it is likely that P8 can develop an allergic Type IV sensitization to Silo. Therefore the cytokine release assay is a helpful tool for providing information about possible immunological reactions to dental resins in individual cases as well as for a general risk assessment and comparison between different dental materials.

[Infrared spectroscopy of urinary calculi before 1900 in votive offerings of the Bavarian pilgrimage church at Grafrath]. / Infrarotspektroskopie von alten Harnsteinen aus den Votivgaben der Wallfahrtskirche Grafrath.

BACKGROUND: The old urinary calculi of the votive offerings in the pilgrimage church at Grafrath offer the possibility of analysing the components by infrared spectroscopy to give insights into factors that might influence their formation. A total of 166 specimens were taken from 139 objects (134 stones, 5 bones), in some stones from different layers. MATERIAL AND METHODS: Spectral analysis showed typical components for urinary calculi in 127 stones. These were compared with a control group of 98 urinary stones from carriers (77 male, 21 female) during 2007/2008 in Bavaria. RESULTS: The percentage of occurrence of ammonium acid urate (NH(4)U) was high in the old stones (68.0%) versus the 2007/2008 group (1.0%). In uric acid (HS) there was no relevant difference between the two groups, whereas the occurrence of the oxalates whewellite (Whe) and weddellite (Wed) was much less in the old stones (Whe 18.1-69.4%, Wed 7.9-26.5 %). The phosphates differ in the components in favour of brushite in the old stones. The high occurrence of NH(4) in the old stones is comparable with (a) the old pre-1900 collection of Norwich (England), especially with the pre-1800 juvenile bladder stones, and (b) urinary stones in endemic areas of stone disease in children such as in North Thailand. Data about the Grafrath stone carriers (name, age, hometown) are not available but can indirectly be derived from the miracle books (1444-1728) of Grafrath with 12,131 reports; 1,165 had urologic disease of which 70% were children with urinary calculi coming from areas of Upper Bavaria and Swabia. CONCLUSION: The finding of a high NH(4)U content indicates that this area might have been a stone belt for bladder stones in children. Under- or malnutrition with low protein and low fluid intake may be the aetiologic factor.

Release of TEGDMA from composite during the chewing situation.

The aim of this study was to investigate the triethylene glycol (TEGDMA) elution kinetics from light-cured composite with and without chewing simulation over a time period of 86 h. An experimental composite with TEGDMA labeled with a tracer dose of 14C-TEGDMA was used. The material parameters were in the range of commercially available composites. The mastification was simulated with the Fatigue-machine and the MUC-3 chewing simulator. 14C was eluted to 2.55% of the applied 14C-TEGDMA dose within 86 h after chewing simulation with the Fatigue-machine and to 2.60% after chewing simulation with the MUC-3. Similar 14C-kinetic data were found for 14C-elution with and without chewing simulation with the Fatigue-machine and with MUC-3. During the first 26 h after the beginning of the experiments a linear 14C-elution kinetic was observed, followed by a second linear 14C-elution kinetic with a lower slope up to 86 h in both apparatus. It could be shown that chewing simulation has no significant (p<0.05) effect on the release of 14C-TEGDMA (and/or 14C-degradation products) from polymerized composites.

The toxicokinetics and distribution of 2-hydroxyethyl methacrylate in mice.

The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized resin-based composites due to the degradation processes or the incomplete polymerisation of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental resin-based composites. It was shown in vitro that HEMA was released into the adjacent biophase from such materials during the first days after placement. In this study uptake, distribution, and excretion of 14C-HEMA applied via gastric tube or subcutaneous administration at dose levels well above those encountered in dental care were examined in mice to test the hypothesis that HEMA can reach cytotoxic levels in mammalian tissues. 14C-HEMA was taken up rapidly from the stomach and intestines after gastric administration and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as (14)CO(2). Two metabolic pathways of 14C-HEMA can be described. The peak HEMA levels in all tissues examined after 24h were lower than known toxic levels. Therefore the study did not support the hypothesis.

Nitric oxide and gene regulation in plants.

There is increasing evidence that nitric oxide (NO), which was first identified as a unique diffusible molecular messenger in animals, plays an important role in diverse physiological processes in plants. Recent progress that has deepened our understanding of NO signalling functions in plants, with special emphasis on defence signalling, is discussed here. Several studies, based on plants with altered NO-levels, have recently provided genetic evidence for the importance of NO in gene induction. For a general overview of which gene expression levels are altered by NO, two studies, involving large-scale transcriptional analyses of Arabidopsis thaliana using custom-made or commercial DNA-microarrays, were performed. Furthermore, a comprehensive transcript profiling by cDNA-amplification fragment length polymorphism (AFLP) revealed a number of Arabidopsis thaliana genes that are involved in signal transduction, disease resistance and stress response, photosynthesis, cellular transport, and basic metabolism. In addition, NO affects the expression of numerous genes in other plant species such as tobacco or soybean. The NO-dependent intracellular signalling pathway(s) that lead to the activation or suppression of these genes have not yet been defined. Several lines of evidence point to an interrelationship between NO and salicylic acid (SA) in plant defence. Recent evidence suggests that NO also plays a role in the wounding/jasmonic acid (JA) signalling pathway. NO donors affect both wounding-induced H2O2 synthesis and wounding- or JA-induced expression of defence genes. One of the major challenges ahead is to determine how the correct specific response is evoked, despite shared use of the NO signal and, in some cases, its downstream second messengers.

Ubiquitin in the prokaryote Anabaena variabilis.

The ubiquitin-dependent pathway for protein degradation has been found to play a major role in controlling protein turnover in the cell. Ubiquitin is one of the most conserved proteins yet identified, and up until now it has been thought to be present only in eukaryotes and archaebacteria. This is the first report on the detection and purification of ubiquitin from a eubacterium, the cyanobacterium Anabaena variabilis. The purification procedure included a heat denaturing step, fractionated ammonium sulfate precipitation, two gel filtration runs (Sephadex G-50 and Superose 12), and a final hydroxylapatite chromatography. Comparisons with bovine ubiquitin showed a high similarity with respect to antigenicity to anti-ubiquitin (bovine), molecular mass (M(r) = 6,000), isoelectric point (pI 6.5), and NH2-terminal sequence. The existence of ubiquitin in A. variabilis was confirmed by Southern hybridization. In in vitro experiments both cyanobacterial and bovine ubiquitin were covalently attached to several target proteins from A. variabilis, respectively. Data are presented which suggest ubiquitination of dinitrogenase reductase, the Fe-protein subunit of nitrogenase. Our findings imply that ubiquitination equivalent to the eukaryotic system is instrumental in this organism.