Amyloid β Protein Primes Cultured Rat Microglial Cells for an Enhanced Phorbol 12-Myristate 13-Acetate-Induced Respiratory Burst Activity

Abstract: Activated microglia, often associated with neuritic amyloid plaques in the Alzheimer's disease brain, are likely to contribute to the progression of the disease process, e.g., by releasing neurotoxic reactive oxygen and/or nitrogen intermediates. In the present study, whether the amyloid β peptide (Aβ), the principal constituent of amyloid plaques, can stimulate microglial respiratory burst activity and/or microglial production of nitric oxide was examined. Using neonatal rat microglial cultures as a model, it was found that neither the spontaneous release of nitric oxide nor the lipopolysaccharide-induced production of nitric oxide was altered in cultures previously incubated with synthetic Aβ(1–40). for 24 h. In addition, no direct stimulatory effect of Aβ(1–40) on the respiratory burst activity was observed. Nevertheless, concomitant with an increase in the number of responsive cells, a profound priming of the phorbol 12-myristate 13-acetate-evoked production of superoxide anion was observed in Aβ(1–40)-treated cultures. Thus, both the maximal rate and the total phorbol 12-myristate 13-acetate-induced production of superoxide appeared to be statistically significantly higher as compared with untreated cultures. It is concluded that, as far as activation of the microglial respiratory burst is concerned, Aβ(1–40) may merely act as a priming rather than a triggering stimulus.     

Oligochaetes and eutrophication; an experiment over four years in outdoor mesocosms

Eight experimental ditch mesocosms were used to study the effect of eutrophication over four years. The experimental ditches had a sand or clay bottom. The ditches were treated with additions of phosphorus, phosphorus and nitrogen, or without additions (controls). Oligochaetes were sampled by deploying trays with substratum for colonization over twenty weeks. Both the important variables phosphorus, nitrogen and oxygen as well as the oligochaete species and numbers are presented. The effects of nutrient additions on phosphorus, nitrogen and oxygen concentrations were described together with changes in oligochaete species composition and numbers. The results were further analyzed by redundancy analysis (RDA). In the clay-lined ditches nutrient addition coincided with fluctuation in oxygen concentration. The higher the nutrient addition levels the longer the period of oxygen depletion became. During oxygen depletion the number of oligochaetes was strongly reduced or even became zero. The low nutrient status of the sandy bed in the sand-lined ditches slowed down the rate of colonization. Only a few tubificids were collected. Eutrophication effects were only observed at the highest nutrient addition level. Considerable variation is attributed to stochastic factors in the sand-lined ditches. Whether oligochaete species were present was related to the length of the colonization period. The substratum composition and food together with oxygen regime decided whether they become more or less abundant in ditches. Large-scale mesocosm experiments require time to develop. Only after the first colonization period variables of species presences and abundances can be employed to detect changes associated with eutrophication. Oligochaetes can be used to measure colonization as well as eutrophication processes.     

Causal connection between detoxification enzyme activity and consumption of a toxic plant compound

Insect herbivores can increase their detoxification activities against a particular plant poison in response to prolonged ingestion of the same compound. For example, larval tobacco hornworms (Manduca sexta) experience a dramatic increase in cytochrome P450 activity against nicotine after ingesting nicotine. While it is generally assumed that this induction process permits increased consumption of toxic plant tissues, we are not aware of any direct experimental support for this assumption. Using a two-tiered approach, we examined the functional significance of P450 induction to M. sexta larvae ingesting a toxic but non-deterrent concentration of nicotine. First, we related the time-course of P450 induction in midgut microsomes to changes in nicotine consumption. When offered a nicotine diet, larvae failed to show a significant increase in consumption before 36 h, which was coincident with the time-course of the induction of midgut P450 activities against aldrin and nicotine. Second, we determined whether inhibiting the induced P450 activities affected nicotine consumption. We found that the increase in nicotine consumption following the induction of nicotine metabolism could be strongly inhibited by treatment with piperonyl butoxide, which by itself did not inhibit consumption. These results provide direct evidence for a causal connection between P450-mediated detoxification activity and consumption of a toxic plant compound.Abbreviation PB piperonyl butoxide     

The other kind of biological NMR—Studies of enzyme-substrate interactions

NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism. Cytochromes P₄₅₀ play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism. Special issue dedicated to Dr. Herman Bachelard.     

Temporal and ontogenetic aspects of protein induction in foliage of the tomato, Lycopersicon esculentum

Damage to foliage of the tomato, Lycopersicon esculentum, causes the induction of proteinase inhibitors and of the oxidative enzymes polyphenol oxidase, peroxidase, and lipoxygenase. The time courses of induction of these proteins by feeding of two caterpillar species (Manduca sexta and Helicoverpa zea) were studied in a series of experiments. In another series of experiments, the effects of plant age on the inducibility of these proteins were studied. In the time course experiments, induction of proteinase inhibitors and oxidative enzymes in the damaged leaflet was rapid, with higher protein activities evident in damaged leaflets within 12–24 h of damage, depending on the enzyme and the species of insect used to damage the plant. Systemic induction of proteinase inhibitors was also rapid, but systemic induction of polyphenol oxidase was delayed relative to systemic induction of proteinase inhibitors, possibly because high constitutive polyphenol oxidase activities obscured expression of systemic induction at earlier time points. Lipoxygenase and peroxidase were not induced systemically. Induction of all proteins persisted for at least 21 days. In the phenology experiments, inducibility of all proteins decreased in magnitude and was less consistent as plants aged. The results of these experiments exemplify the numerous constraints on induction in tomato plants. Knowledge of these physiological constraints is important to an understanding of the ecological role and causal basis of induced resistance.     

Ethanol-dependent oxygen consumption and acetaldehyde formation during vanadyl oxidation by H2O2

Sequential addition of vanadyl sulfate to a phosphate-buffered solution of H₂O₂ released oxygen only after the second batch of vanadyl. Ethanol added to such reaction mixtures progressively decreased oxygen release and increased oxygen consumption during oxidation of vanadyl by H₂O₂. Inclusion of ethanol after any of the three batches of vanadyl resulted in varying amounts of oxygen consumption, a property also shared by other alcohols (methanol, propanol and octanol). On increasing the concentration of ethanol, vanadyl sulfate or H₂O₂, both oxygen consumption and acetaldehyde formation increased progressively. Formation of acetaldehyde decreased with increase in the ratio of vanadyl:H₂O₂ above 2:1 and was undetectable with ethanol at 0.1 mM. The reaction mixture which was acidic in the absence of phosphate buffer (pH 7.0), released oxygen immediately after the first addition of vanadyl and also in presence of ethanol soon after initial rapid consumption of oxygen, with no accompanying acetaldehyde formation. The results underscore the importance of some vanadium complexes formed during vanadyl oxidation in the accompanying oxygen-transfer reactions.     

Distribution and activity of microsomal NADPH-dependent monooxygenases and amino acid decarboxylases in cruciferous and non-cruciferous plants, and their relationship to foliar glucosinolate content

The NADPH-dependent conversion of amino acids to their aldoximes is an initial step in glucosinolate biosynthesis. A number of microsomal aldoxime-forming monooxygenase activities were detected in leaves from a variety of glucosi-nolate-containing species, whereas barley, bean and tobacco leaves did not contain any such activities. The substrates for these monooxygenases in each species largely correlated with the spectrum of glucosinolates found in that species. No activity was detected that metabolized homomethionine (supposed precursor of 2-propenylglucosinolate [sinigrin]), even in species where sinigrin was the major glucosinolate. In Sinapis species containing hydroxybenzylglucosinolate (sinalbin), activity with L-Tyr was detected, whereas Brassica species containing sinalbin had no such activity. However, these Brassicas did contain an L-Phe monooxygenase activity. Partial characterization of the monooxygenases indicated that in Brassica species, Nasturtium officinalis and Raphanus sativus these resembled the flavin-linked monooxygenases previously found in oilseed rape (Brassica napus) and Chinese cabbage (Brassica campestris). The L-Tyr-dependent activity in Sinapis species, and the L-Phe-dependent activity in Tropacolum majus, had characteristics of cytochrome P450-type enzymes. No similarity was found with any other known amino acid metabolizing enzymes (including decarboxylases, amino acid oxidases and diamine/polyamine oxidases).     

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10^–10 cm²/s, and 1.8±0.2 × 10^–10 cm²/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10^–10 cm²/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10^–10 cm²/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT Catalase - D Lateral diffusion coefficient - EDTA Ethylenediaminetetraacetic acid - EGF Epidermal growth factor - E-MEM Eagle's minimum essential medium - FCS Fetal calf serum - FRAP Fluorescence recovery after photobleaching - KRG Krebs-Ringer phosphate buffer - PBS Phosphate-buffered saline - R Mobile fraction - ROS Reactive oxygen species - SEM Standard error of the mean - SOD Superoxide dismutase - UVA Ultraviolet light-A (315-400 nm) - UVB Ultraviolet light-B (280-315 nm)     

Cyclic electron flow around Photosystem II in vivo

The oxygen flash yield (Y_O2) and photochemical yield of PS II (_{PS II}) were simultaneously detected in intact Chlorella cells on a bare platinum oxygen rate electrode. The two yields were measured as a function of background irradiance in the steady-state and following a transition from light to darkness. During steady-state illumination at moderate irradiance levels, Y_O2 and _{PS II} followed each other, suggesting a close coupling between the oxidation of water and Q_A reduction (Falkowski et al. (1988) Biochim. Biophys. Acta 933: 432–443). Following a light-to-dark transition, however, the relationship between Q_A reduction and the fraction of PS II reaction centers capable of evolving O₂ became temporarily uncoupled. _{PS II} recovered to the preillumination levels within 5–10 s, while the Y_O2 required up to 60 s to recover under aerobic conditions. The recovery of Y_O2 was independent of the redox state of Q_A, but was accompanied by a 30% increase in the functional absorption cross-section of PS II (_{PS II}). The hysteresis between Y_O2 and the reduction of Q_A during the light-to-dark transition was dependent upon the reduction level of the plastoquinone pool and does not appear to be due to a direct radiative charge back-reaction, but rather is a consequence of a transient cyclic electron flow around PS II. The cycle is engaged in vivo only when the plastoquinone pool is reduced. Hence, the plastoquinone pool can act as a clutch that disconnects the oxygen evolution from photochemical charge separation in PS II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting enzyme (agents) - Chl chlorophyll - cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_O minimum fluorescence yield in the dark-adapted state - F_I minimum fluorescence yield under ambient irradiance or during transition from the light-adapted state - F_M maximum fluorescence yield in the dark-adapted state - F_M maximum fluorescence yield under ambient irradiance or during transition from light-adapted state - F_V, F_V variable fluorescence (F_V=F_M–F_O ; F_V=F_M–F_I) - FRR fast repetition rate (fluorometer) - _{PS II} quantum yield of Q_A reduction (_{PS II}=(F_M – F_O)/F_M or _{PS II})=(F_M= – F_I=)/F_M=) - LHCII Chl a/b light harvesting complexes of Photosystem II - OEC oxygen evolving complex of PS II - P680 reaction center chlorophyll of PS II - PQ plastoquinone - POH₂ plastoquinol - PS I Photosystem I - PS II Photosystem II - RC II reaction centers of Photosystem II - PS II the effective absorption cross-section of PHotosystem II - TL thermoluminescence - Y_O2 oxygen flash yield The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, fromArabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the -glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.