Reduced effect on apoptosis of 4-hydroxyhexenal and oxidized LDL enriched with n-3 fatty acids from postmenopausal women

BACKGROUND: Oxidized low-density lipoprotein (oxLDL) promotes apoptosis in atherosclerotic plaques in the vascular wall, a process mediated through its oxidized lipids. 4-Hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), derived from oxidation of n-6 and n-3 fatty acids, respectively, are among the major oxidized products in oxLDL. HYPOTHESIS: This study hypothesized that eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA)-rich versus linoleic acid-rich oxLDL obtained from postmenopausal women and HNE versus HHE differentially influence apoptosis in U937 cells. EXPERIMENTAL DESIGN: Thirty healthy postmenopausal women were supplemented with 14 g/day safflower oil (SO), 7 g/day of both fish oil and SO (low dose LFO) or 14 g/day fish oil (high dose HFO) for 5 weeks. Low-density lipoprotein, obtained after supplementation, was oxidized with 5 microM CuSO(4) at 37 degrees C for 6 h. The concentration of cholesteryl ester hydroperoxides (CEOOH) and conjugated dienes was measured in the oxidized LDL (oxLDL). U937 cells were incubated with the oxLDL, 10 microM of HHE, 7 muM of HHE plus 3 microM of HNE, 5 microM of both HHE and HNE or 10 microM of HNE and the extent of apoptosis measured three ways. RESULTS: The concentration of CEOOH and conjugated dienes in oxLDL did not differ among the three treatment groups. The percent of apoptotic cells was approximately 40% lower when incubated with oxLDL obtained from the HFO-supplemented group than the SO-supplemented group measured by both the Annexin V and the DNA fragmentation assays (P = .04 and .004, respectively). Apoptosis of U937 cells was significantly lower in cells incubated with 10 microM of HHE, and mixtures of HHE and HNE than the 10 microM HNE when measured by the Annexin V, DNA fragmentation and 4,6-diamidino-2-phenylindole (DAPI) staining. CONCLUSIONS: These data suggest that the cardioprotective properties of n-3 fatty acids may derive in part from their less reactive oxidized lipid metabolites.     

Lactate uptake contributes to the NAD(P)H biphasic response and tissue oxygen response during synaptic stimulation in area CA1 of rat hippocampal slices

Synaptic train stimulation (10 Hz × 25 s) in hippocampal slices results in a biphasic response of NAD(P)H fluorescence indicating a transient oxidation followed by a prolonged reduction. The response is accompanied by a transient tissue PO₂ decrease indicating enhanced oxygen utilization. The activation of mitochondrial metabolism and/or glycolysis may contribute to the secondary NAD(P)H peak. We investigated whether extracellular lactate uptake via monocarboxylate transporters (MCTs) contributes to the generation of the NAD(P)H response during neuronal activation. We measured the effect of lactate uptake inhibition [using the MCT inhibitor α-cyano-4-hydroxycinnamate (4-CIN)] on the NAD(P)H biphasic response, tissue PO₂ response, and field excitatory post-synaptic potential in hippocampal slices during synaptic stimulation in area CA1 (stratum radiatum). The application of 4-CIN (150–250 μmol/L) significantly decreased the reduction phase of the NAD(P)H response. When slices were supplemented with 20 mmol/L lactate in 150–250 μmol/L 4-CIN, the secondary NAD(P)H peak was restored; whereas 20 mmol/L pyruvate supplementation did not produce a recovery. Similarly, the tissue PO₂ response was decreased by MCT inhibition; 20 mmol/L lactate restored this response to control levels at all 4-CIN concentrations. These results indicate that lactate uptake via MCTs contributes significantly to energy metabolism in brain tissue and to the generation of the delayed NAD(P)H peak after synaptic stimulation.     

A comparative 'bottom up' proteomics strategy for the site-specific identification and quantification of protein modifications by electrophilic lipids

We report a mass spectrometry-based comparative "bottom up" proteomics approach that combines d(0)/d(4)-succinic anhydride labeling with commercially available hydrazine (Hz)-functionalized beads (Affi-gel Hz beads) for detection, identification and relative quantification of site-specific oxylipid modifications in biological matrices. We evaluated and applied this robust and simple method for the quantitative analysis of oxylipid protein conjugates in cardiac mitochondrial proteome samples isolated from 3- and 24-month-old rat hearts. The use of d(0)/d(4)-succinic anhydride labeling, Hz-bead based affinity enrichment, nanoLC fractionation and MALDI-ToF/ToF tandem mass spectrometry yielded relative quantification of oxylipid conjugates with residue-specific modification information. Conjugation of acrolein (ACR), 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-noneal (ONE) to cysteine, histidine and lysine residues were identified. HHE conjugates were the predominant subset of Michael-type adducts detected in this study. The HHE conjugates showed higher levels in mitochondrial preparations from young heart congruent with previous findings by others that the n-3/n-6 PUFA ratio is higher in young heart mitochondrial membranes. Although this study focuses on protein adducts of reactive oxylipids, the method might be equally applicable to protein carbonyl modifications caused by metal catalyzed oxidation reactions.     

Docosahexaenoic acid synthesis from n-3 fatty acid precursors in rat hippocampal neurons

Docosahexaenoic acid (DHA), the most abundant n-3 polyunsaturated fatty acid in the brain, has important functions in the hippocampus. To better understand essential fatty acid homeostasis in this region of the brain, we investigated the contributions of n-3 fatty acid precursors in supplying hippocampal neurons with DHA. Primary cultures of rat hippocampal neurons incorporated radiolabeled 18-, 20-, 22-, and 24-carbon n-3 fatty acid and converted some of the uptake to DHA, but the amounts produced from either [1-¹⁴C]α-linolenic or [1-¹⁴C]eicosapentaenoic acid were considerably less than the amounts incorporated when the cultures were incubated with [1-¹⁴C]22:6n-3. Most of the [1-¹⁴C]22:6n-3 uptake was incorporated into phospholipids, primarily ethanolamine phosphoglycerides. Additional studies demonstrated that the neurons converted [1-¹⁴C]linoleic acid to arachidonic acid, the main n-6 fatty acid in the brain. These findings differ from previous results indicating that cerebral and cerebellar neurons cannot convert polyunsaturated fatty acid precursors to DHA or arachidonic acid. Fatty acid compositional analysis demonstrated that the hippocampal neurons contained only 1.1–2.5 mol% DHA under the usual low-DHA culture conditions. The relatively low-DHA content suggests that some responses obtained with these cultures may not be representative of neuronal function in the brain.     

Release-enhancing pre-synaptic muscarinic and nicotinic receptors co-exist and interact on dopaminergic nerve endings of rat nucleus accumbens

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [³H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR–mAChR agonists acetylcholine (ACh) and carbachol provoked [³H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 μmol/L, together with 3, 10, or 100 μmol/L (−)nicotine provoked synergistic effect on [³H]DA overflow. The [³H]DA overflow elicited by 100 μmol/L (−)nicotine plus 30 μmol/L oxotremorine was reduced by atropine down to the release produced by (−)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (−)nicotine/oxotremorine evoked [³H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (−)nicotine/oxotremorine. Similarly to (−)nicotine, the selective α4β2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, α4β2 nAChRs exert a permissive role on the releasing function of reportedly M₅ mAChRs co-existing on the same dopaminergic nerve endings.     

Light activates H2 15O flow in rice: Detailed monitoring using a positron-emitting tracer imaging system (PETIS)

Water (H₂ ¹⁵O) translocation from the roots to the top of rice plants ( Oryza saliva L. cv. Nipponbare) was visualized over time by a positron-emitting tracer imaging system (PETIS). H₂ ¹⁵O flow was activated 8 min after plants were exposed to bright light (1 500 μmol m⁻² s⁻¹). When the light was subsequently removed, the flow gradually slowed and completely stopped after 12 min. In plants exposed to low light (500 μmol m⁻² s⁻¹), H₂ ¹⁵O flow was activated more slowly, and a higher translocation rate of H₂ ¹⁵O was observed in the same low light at the end of the next dark period. NaCl (80 m M ) and methylmercury (1 m M ) directly suppressed absorption of H₂ ¹⁵O by the roots, while methionine sulfoximine (1 m M ), abscisic acid (10 μ M ) and carbonyl cyanide m -chlorophenylhydrazone (10 m M ) were transported to the leaves and enhanced stomatal closure, reducing H₂ ¹⁵O translocation.     

A Role for 4-Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, in Disruption of Ion Homeostasis and Neuronal Death Induced by Amyloid β-Peptide

Abstract: Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid β-peptide (Aβ) can promote free radical production, we tested the hypothesis that HNE mediates Aβ25-35-induced disruption of neuronal ion homeostasis and cell death. Aβ induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na⁺,K⁺-ATPase activity and induced an increase of neuronal intracellular free Ca²⁺ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both Aβ and HNE. The antioxidant propyl gallate protected neurons against Aβ toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates Aβ-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.     

Understanding your inhibitions: effects of GABA and GABAA receptor modulation on brain cortical metabolism

A targeted neuropharmacological, ¹H/¹³C NMR spectroscopy and multivariate statistical approach was used to examine the effects of exogenous GABA and ligands at the GABA_A receptor family on brain metabolism in the Guinea pig cortical tissue slice. All ligands at GABA_A receptors generated metabolic patterns which were distinct from one another with the major variance in the data arising because of metabolic work (shown by net flux into Krebs cycle byproducts and increased metabolic pool sizes). Three major clusters of metabolic signatures were identified which corresponded to: (i) activity at phasic (synaptic) GABA_A receptors, dominated by α1-containing receptors and responsive to GABA at 10 μmol/L; (ii) activity at perisynaptic receptors, dominated by response to high (40 μmol/L) GABA and the superagonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride, and C, activity at extrasynaptic receptors, dominated by response to low (0.1–1.0 μmol/L) GABA, zolpidem (400 nmol/L) and the non-specific allosteric modulator RO19-4603 (1 nmol/L). These results highlight the utility of a different but robust approach to study of the GABAergic system using metabolic systems analysis.     

Mitochondrial oxidation of 4-hydroxy-2-nonenal in rat cerebral cortex

4-hydroxy-trans-2-nonenal (HNE) is a neurotoxic product of lipid peroxidation whose levels are elevated in multiple neurodegenerative diseases and CNS trauma. The detoxification of HNE may take the route of glutathione conjugation to the C3 carbon and the oxidation or reduction of the C1 aldehyde. In this work, we examined whether the oxidation of HNE to its corresponding carboxylic acid, 4-hydroxy-trans-2-nonenoate (HNEAcid) was detoxifying event, if it occurred in rat cerebral cortex, and in which subcellular compartments. Our results show that HNEAcid did not form protein adducts and was non-toxic to Neuro 2A cells. HNEAcid formation occurred in rat cerebral cortex slices following exposure to HNE in a time-dependent and dose-dependent fashion. Homogenate studies indicated that HNEAcid formation was NAD+ dependent. Subcellular fractionation demonstrated that mitochondria had the highest specific activity for HNEAcid formation with a KM of 21 micro m HNE. These data indicate that oxidation of HNE to its corresponding acid is a major detoxification pathway of HNE in the CNS and that mitochondria play a role in this process.     

Serotoninergic modulation of GABAergic synaptic transmission in developing rat CA3 pyramidal neurons

Serotoninergic modulation of GABAergic mIPSCs was investigated in immature (postnatal 12–16-days old) rat CA3 pyramidal neurons using a conventional whole-cell patch clamp technique. Serotonin or 5-hydroxytryptamine (5-HT) (10 μmol/L) transiently and explosively increased mIPSC frequency with a small increase in the current amplitude. However, 5-HT did not affect the GABA-induced postsynaptic currents, indicating that 5-HT acts presynaptically to facilitate the probability of spontaneous GABA release. The 5-HT action on GABAergic mIPSC frequency was completely blocked by 100 nmol/L MDL72222, a selective 5-HT₃ receptor antagonist, and mimicked by mCPBG, a selective 5-HT₃ receptor agonist. The 5-HT action on GABAergic mIPSC frequency was completely occluded either in the presence of 200 μmol/L Cd²⁺ or in the Na⁺-free external solution, suggesting that the 5-HT₃ receptor-mediated facilitation of mIPSC frequency requires a Ca²⁺influx passing through voltage-dependent Ca²⁺channels from the extracellular space, and that presynaptic 5-HT₃ receptors are less permeable to Ca²⁺. The 5-HT action on mIPSC frequency in the absence or presence of extracellular Na⁺ gradually increased with postnatal development. Such a developmental change in the 5-HT₃ receptor-mediated facilitation of GABAergic transmission would play important roles in the regulation of excitability as well as development in CA3 pyramidal neurons.     

Potassium dependence and phytoplankton ecology: an experimental study

SUMMARY 1. Unialgal cultures of three species common in the freshwater phytoplankton were used to test limitation of specific growth rate and final yield in defined media of low K⁺ concentration (range <0.3–6 μmol L⁻¹ or mmol m⁻³).
2. Growth rate of the diatom Asterionella formosa was independent of K⁺ concentration above 0.7 μmol L⁻¹. Final yield was dependent on initial concentration when accompanied by K⁺ depletion below this concentration, but not by lesser depletion with more residual K⁺. Analyses of particulate K in the biomass indicated a mean final cell content of 2.8 μmol K 10⁻⁸ cells, approximately 1.0% of the organic dry weight.
3. Less detailed work with the diatom Diatoma elongatum showed no dependence of growth rate or final yield upon the initial K⁺ concentration in the range 0.8–3.2 μmol L⁻¹. The phytoflagellate Plagioselmis nannoplanctica suffered net mortality in the lowest concentration tested, 0.8 μmol L⁻¹.
4. Comparison with the range of K⁺ concentration in natural fresh waters, including a depletion induced by an aquatic macrophyte, suggests that K⁺ is unlikely to limit growth of phytoplankton. Nevertheless, there can be correlation of K⁺ with lake trophy.     

Assessing mortality changes from size‐frequency curves

The relationship between the instantaneous mortality rate ( Z ) and the instantaneous change in length‐frequency distribution of organisms per unit of animal size (μ _L ) takes the following form: Z  = μ _L k ( L _∞ −  L ) +  k , where k and L _∞ are coefficients of the von Bertalanffy equation, and L is organism size (length). Z and μ_L change coherently when they are measured for a specific size or age class. Therefore, observations of μ _L can provide information on the relative changes in mortality. This is useful when no precise information about animal growth is available and growth curve is assumed to be invariable. This method was tested on a heavily exploited population of St Peter's fish (mango tilapia) Sarotherodon galilaeus in Lake Kinneret, Israel, where large fluctuations in the size structure of the catch have occurred over the past few years. Analysis of the changes in the length‐frequency distributions showed that the changes in μ _L over multiple years estimated for fully exploited fish reflected the respective changes in Z .     

Insecticidal and synergistic effects of Majorana hortensis essential oil and some of its major constituents

The essential oil from leaves of Majorana hortensis Moench (Lamiaceae) was isolated by hydrodistillation with a yield of 1.6% (wt/wt). The insecticidal activity of the oil was evaluated against fourth instars of Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) and adults of Aphis fabae L. (Hemiptera: Aphididae). The oil showed a remarkable toxic effect against S. littoralis in a topical application assay (LD₅₀ = 2.48 μg per larva) and in a residual film assay (LC₅₀ = 3.14 g/l). The oil of M. hortensis also exhibited a pronounced toxic effect against A. fabae adults with LC₅₀ values of 1.86 and 2.27 g/l in rapid dipping and residual film assays, respectively. Gas chromatography-mass spectrometry analyses of M. hortensis essential oils revealed the presence of 31 compounds and the main components were terpinen-4-ol (30.0%), γ-terpinene (11.3%), and trans -sabinene hydrate (10.8%). Repeated column chromatography of M. hortensis oil on silica gel led to the isolation of two major constituents, which were characterized based on ¹H-nuclear magnetic resonance and mass spectrometric data, as terpinen-4-ol and γ-terpinene. These two components were examined for their insecticidal and synergistic activities towards S. littoralis and A. fabae . Terpinen-4-ol and γ-terpinene exhibited a significant insecticidal activity against both insects, but γ-terpinene was more toxic than terpinen-4-ol. When tested in a binary mixture with the synthetic insecticides profenofos and methomyl, it was found that both compounds enhanced the insecticidal activity of these insecticides by two- to threefold. These results show that terpinen-4-ol and γ-terpinene have a synergistic effect on the insecticidal activities of synthetic insecticides profenofos and methomyl.     

Glycinergic nerve endings in hippocampus and spinal cord release glycine by different mechanisms in response to identical depolarizing stimuli

Studies on hippocampal glycine release are extremely rare. We here investigated release from mouse hippocampus glycinergic terminals selectively pre-labelled with [³H]glycine through transporters of the GLYT2 type. Purified synaptosomes were incubated with [³H]glycine in the presence of the GLYT1 blocker NFPS to abolish uptake (∼ 30%) through GLYT1. The non-GLYT1-mediated uptake was entirely sensitive to the GLYT2 blocker Org25543. Depolarization during superfusion with high-K⁺ (15–50 mmol/L) provoked overflows totally dependent on external Ca²⁺, whereas in the spinal cord the 35 or 50 mmol/L KCl-evoked overflow (higher than that in hippocampus) was only partly dependent on extraterminal Ca²⁺. In the hippocampus, the Ca²⁺-dependent 4-aminopyridine (1 mmol/L)-evoked overflow was five-fold lower than that in spinal cord. The component of the 10 μmol/L veratridine-induced overflow dependent on external Ca²⁺ was higher in the hippocampus than that in spinal cord, although the total overflow in the hippocampus was only half of that in the spinal cord. Part of the veratridine-evoked hippocampal overflow occurred by GLYT2 reversal and part by bafilomycin A₁-sensitive exocytosis dependent on cytosolic Ca²⁺ generated through the mitochondrial Na⁺/Ca²⁺ exchanger. As glycine sites on NMDA receptors are normally not saturated, understanding mechanisms of glycine release should facilitate pharmacological modulation of NMDA receptor function.     

Inhibition of heat shock proteins (HSP) expression by quercetin and differential doxorubicin sensitization in neuroblastoma and Ewing's sarcoma cell lines

Neuroblastoma (NB) and Ewing's sarcoma (ES) represent the most common extracranial solid tumors of childhood. Heat shock proteins (HSP) are elevated in cancer cells and their over-expression was correlated to drug-resistance. In this work we identified the HSP by a sensitive proteomic analysis of NB and ES cell lines, then, we studied the HSP response to doxorubicin. Some identified HSP were constitutively more expressed in NB than in ES cells. Doxorubicin-stimulated HSP response only in NB cells. Quercetin was found to inhibit HSP expression depleting heat shock factor 1 (HSF1) cellular stores. Quercetin caused a higher anti-proliferative effect in NB (IC₅₀: 6.9 ± 5.8 μmol/L) than in ES cells (IC₅₀: 85.5 ± 53.1 μmol/L). Moreover, quercetin caused a very pronounced doxorubicin sensitizing effect in NB cells (241 fold IC₅₀ decrease) and a moderate effect in ES cells. HSP involvement in NB cells sensitization was confirmed by the silencing of HSF1. Quercetin treatment and HSF1 silencing increased the pro-apoptotic effect of doxorubicin. In conclusion, the higher HSP levels, observed in NB cells, did not confer increased resistance to doxorubicin; on the contrary, HSP inhibition by quercetin or gene silencing caused higher sensitization to doxorubicin. These results may have a potential application in the treatment of NB.     

Ca2+ release from intracellular stores by thapsigargin in sea urchin eggs: Relationship to larval development and relevance in egg activation

Thapsigargin (Tg), an inhibitor of microsomal Ca²⁺ ATPase, is used as a tool to study the changes in Ca²⁺ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca²⁺ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC_5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca²⁺ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca²⁺ release from ⁴⁵Ca pre-loaded cortices but leads to a loss of 25% of the total Ca²⁺ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca²⁺ activity as compared with the fertilization-induced Ca²⁺ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca²⁺ sequestration.     

Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal

Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 degrees C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.     

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-β (1–42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid-β (Aβ) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ₄₂ fibrillization and initiate formation of non-fibrillar Aβ₄₂ aggregates, and that the inhibitory effect of Zn(II) (IC₅₀ = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Aβ₄₂ aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ₄₂ fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ₄₂. H13A and H14A mutations in Aβ₄₂ reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-β core structure within region 10–23 of the amyloid fibril. Cu(II)-Aβ₄₂ aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Aβ₄₂ aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.