Biomedical subjects

D Allan Butterfield

Publications and source records attributed to D Allan Butterfield.

At least 91 records · Page 5Linked to original sources

PubMed2004

Striatal damage and oxidative stress induced by the mitochondrial toxin malonate are reduced in clorgyline-treated rats and MAO-A deficient mice.

Intrastriatal administration of the succinate dehydrogenase (SDH) inhibitor malonate produces neuronal injury by a "secondary excitotoxic" mechanism involving the generation of reactive oxygen species (ROS). Recent evidence indicates dopamine may contribute to malonate-induced striatal neurodegeneration; infusion of malonate causes a pronounced increase in extracellular dopamine and dopamine deafferentation attenuates malonate toxicity. Inhibition of the catabolic enzyme monoamine oxidase (MAO) also attenuates striatal lesions induced by malonate. In addition to forming 3,4-dihydroxyphenylacetic acid, metabolism of dopamine by MAO generates H2O2, suggesting that dopamine metabolism may be a source of ROS in malonate toxicity. There are two isoforms of MAO, MAO-A and MAO-B. In this study, we have investigated the role of each isozyme in malonate-induced striatal injury using both pharmacological and genetic approaches. In rats treated with either of the specific MAO-A or -B inhibitors, clorgyline or deprenyl, respectively, malonate lesion volumes were reduced by 30% compared to controls. In knock-out mice lacking the MAO-A isoform, malonate-induced lesions were reduced by 50% and protein carbonyls, an index ROS formation, were reduced by 11%, compared to wild-type animals. In contrast, mice deficient in MAO-B showed highly variable susceptibility to malonate toxicity precluding us from determining the precise role of MAO-B in this form of brain damage. These findings indicate that normal levels of MAO-A participate in expression of malonate toxicity by a mechanism involving oxidative stress.

Animals↗

PubMed2004

Proteomic analysis of brain proteins in the gracile axonal dystrophy (gad) mouse, a syndrome that emanates from dysfunctional ubiquitin carboxyl-terminal hydrolase L-1, reveals oxidation of key proteins.

Ubiquitin carboxyl-terminal hydrolase L-1 (UCH L-1) is a crucial enzyme for proteasomal protein degradation that generates free monomeric ubiquitin. Our previous proteomic study identified UCH L-1 as one specific target of protein oxidation in Alzheimer's disease (AD) brain, establishing a link between the effect of oxidative stress on protein and the proteasomal dysfunction in AD. However, it is unclear how protein oxidation affects function, owing to the different responses of proteins to oxidation. Analysis of systems in which the oxidized protein displays lowered or null activity might be an excellent model for investigating the effect of the protein of interest in cellular metabolism and evaluating how the cell responds to the stress caused by oxidation of a specific protein. The gracile axonal dystrophy (gad) mouse is an autosomal recessive spontaneous mutant with a deletion on chromosome 5 within the gene encoding UCH L-1. The mouse displays axonal degeneration of the gracile tract. The aim of this proteomic study on gad mouse brain, with dysfunctional UCH L-1, was to determine differences in brain protein oxidation levels between control and gad samples. The results showed increased protein oxidation in thioredoxin peroxidase (peroxiredoxin), phosphoglycerate mutase, Rab GDP dissociation inhibitor alpha/ATP synthase and neurofilament-L in the gad mouse brain. These findings are discussed with reference to the effect of specific protein oxidation on potential mechanisms of neurodegeneration that pertain to the gad mouse.

Animals↗

PubMed2004

Protective effect of the xanthate, D609, on Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress in primary neuronal cells.

Tricyclodecan-9-yl-xanthogenate (D609) is an inhibitor of phosphatidylcholine-specific phospholipase C, and this agent also has been reported to protect rodents against oxidative damage induced by ionizing radiation. Previously, we showed that D609 mimics glutathione (GSH) functions and that a disulfide is formed upon oxidation of D609 and the resulting dixanthate is a substrate for GSH reductase, regenerating D609. Considerable attention has been focused on increasing the intracellular GSH levels in many diseases, including Alzheimer's disease (AD). Amyloid beta-peptide [Abeta(1-42)], elevated in AD brain, is associated with oxidative stress and toxicity. The present study aimed to investigate the protective effects of D609 on Abeta(1-42)-induced oxidative cell toxicity in cultured neurons. Decreased cell survival in neuronal cultures treated with Abeta(1-42) correlated with increased free radical production measured by dichlorofluorescein fluorescence and an increase in protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (4-hydroxy-2-nonenal) formation. Pretreatment of primary hippocampal cultures with D609 significantly attenuated Abeta(1-42)-induced cytotoxicity, intracellular ROS accumulation, protein oxidation, lipid peroxidation and apoptosis. Methylated D609, with the thiol functionality no longer able to form the disulfide upon oxidation, did not protect neuronal cells against Abeta(1-42)-induced oxidative stress. Our results suggest that D609 exerts protective effects against Abeta(1-42) toxicity by modulating oxidative stress. These results may be of importance for the treatment of AD and other oxidative stress-related diseases.

Alzheimer Disease↗

PubMed2004

Ethyl ferulate, a lipophilic polyphenol, induces HO-1 and protects rat neurons against oxidative stress.

In the CNS, the heme oxygenase (HO) system has been reported to be active and to operate as a fundamental defensive mechanism for neurons exposed to an oxidant challenge. We have recently shown that both curcumin and caffeic acid phenethyl ester, two phenolic natural compounds, potently induce HO-1 expression and activity in rat astrocytes. We have extended our previous findings examining the effects of two other plant-derived phenolic compounds, with analogous chemical structures, in rat astrocytes and neurons. Ethyl ferulate (ethyl 4-hydroxy-3-methoxycinnamate) (EFE), the naturally occurring ester of ferulic acid, was able to induce HO-1 protein expression. Maximal expression of HO-1 mRNA and protein and a significant increase in HO activity were detected after 6 h of incubation with 15 microM EFE in astrocytes and 5 microM EFE in neurons. Higher concentrations of EFE (50 microM) caused a substantial cytotoxic effect with no change in HO-1 protein expression and activity. Exposure of astrocytes to resveratrol, a phytoalexin derived from grapes, resulted in an increase of HO-1 mRNA, but it was not able to induce HO-1 protein expression and activity. Interestingly, preincubation (12 h) of neurons with EFE resulted in an enhanced cellular resistance to glucose oxidase-mediated oxidative damage; this cytoprotective effect was considerably attenuated by zinc protoporphyrin IX, an inhibitor of HO activity. This study identifies a novel natural compound that could be used for therapeutic purposes as a potent inducer of HO-1 for the protection of brain cells against oxidative and neurodegenerative conditions.

Animals↗

PubMed2004

Redox regulation in neurodegeneration and longevity: role of the heme oxygenase and HSP70 systems in brain stress tolerance.

Efficient functioning of maintenance and repair processes seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of several genes termed "vitagenes," among these, the heat shock system, a highly conserved mechanism responsible for the preservation and repair of cellular macromolecules, such as proteins, RNAs, and DNA. Recent studies have shown that the heat shock response contributes to establishing a cytoprotective state in a wide variety of human diseases, including ischemia and reperfusion damage, inflammation, cancer, as well as metabolic and neurodegenerative disorders. Recently, the involvement of the heme oxygenase (HO) pathway in antidegenerative mechanisms has received considerable attention, as it has been demonstrated that the expression of HO is closely related to that of amyloid precursor protein. HO induction occurs together with the induction of other heat shock proteins during various physiopathological conditions. The vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, products of HO-catalyzed reaction, represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly, manipulation of endogenous cellular defense mechanisms, via the heat shock response, through nutritional antioxidants or pharmacological compounds, may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. Consistently, by maintaining or recovering the activity of vitagenes, it is feasible to delay the aging process and decrease the occurrence of age-related diseases with resulting prolongation of a healthy life span.

Aging↗

PubMed2004

Free radicals: key to brain aging and heme oxygenase as a cellular response to oxidative stress.

Aging is one of the unique features in all organisms. The impaired functional capacity of many systems characterizes aging. When such impairments occur in the brain, the susceptibility to neurodegenerative diseases amplifies considerably. The free radical theory of aging posits that the functional impairments in brains are due to the attack on critical cellular components by free radicals, reactive oxygen species, and reactive nitrogen species produced during normal metabolism. In this review, we examine this concept based on the parameters of oxidative stress in correlation to aging. The parameters for lipid peroxidation are phospholipid composition, reactive aldehydes, and isoprostanes. The parameters for protein oxidation are protein carbonyl levels, protein 3-nitrotyrosine levels, electron paramagnetic resonance, and oxidative stress-sensitive enzyme activities. We conclude that free radicals are, at least partially, responsible for the functional impairment in aged brains. The aging brain, under oxidative stress, responds by induction of various protective genes, among which is heme oxygenase. The products of the reaction catalyzed by heme oxygenase, carbon monoxide, iron, and biliverdin (later to bilirubin) each have profound effects on neurons. Although there may be other factors contributing to brain aging, free radicals are involved in the damaging processes associated with brain aging, and cellular stress response genes are induced under free radical oxidative stress. Therefore, this review supports the proposition that free radicals are, indeed, a key to brain aging.

Aging↗

PubMed2004

Amyloid beta-peptide(1-42) contributes to the oxidative stress and neurodegeneration found in Alzheimer disease brain.

Oxidative stress is extensive in Alzheimer disease (AD) brain. Amyloid beta-peptide (1-42) has been shown to induce oxidative stress and neurotoxicity in vitro and in vivo. Genetic mutations that result in increased production of Abeta1-42 from amyloid precursor protein are associated with an early onset and accelerated pathology of AD. Consequently, Abeta1-42 has been proposed to play a central role in the pathogenesis of AD as a mediator of oxidative stress. In this review, we discuss the role of Abeta1-42 in the lipid peroxidation and protein oxidation evident in AD brain and the implications of such oxidative stress for the function of various proteins that we have identified as specifically oxidized in AD brain compared to control, using proteomics methods. Additionally, we discuss the critical role of methionine 35 in the oxidative stress and neurotoxic properties exhibited by Abeta1-42.

Alzheimer Disease↗

PubMed2004

Rodent Abeta(1-42) exhibits oxidative stress properties similar to those of human Abeta(1-42): Implications for proposed mechanisms of toxicity.

Alzheimer's disease is a neurodegenerative disorder associated with aging and cognitive decline. Amyloid beta peptide (1-42) [Abeta(1-42)] is a primary constituent of senile plaques - a hallmark of Alzheimer's disease - and has been implicated in the pathogenesis of the disease. Previous studies have shown that methionine residue 35 of beta(1-42) may play a critical role in Abeta(1-42)-mediated oxidative stress and neurotoxicity. Several additional mechanisms of neurotoxicity have been proposed, including the role of Cu(II) binding and reduction to produce hydrogen peroxide and the role of peptide aggregation. It has been reported that rodent Abeta is less likely to form larger beta-sheet structures, and consequently, large aggregates. As a consequence of the lack of deposition of the peptide in rodent brain, rodent Abeta has been proposed to be non-toxic. Additionally, the sequence of the rodent variety of Abeta(1-42) contains three amino acid substitutions compared to the human sequence. These substitutions include the shift of arginine 5, trysosine 10, and histidine 13 to glycine, phenylalanine, and arginine, respectively. This shift in sequence within the Cu(II) binding region of the peptide results in a decrease in the ability of the rodent Abeta peptide to reduce Cu(II) to Cu(I) compared to the human Abeta peptide. As a result of the effect of the amino acid variations on the ability of the rodent peptide to reduce Cu(II) to Cu(I) compared to the human peptide, the rodent beta has been proposed to lack oxidative stress properties. In this study, the oxidative stress and neurotoxic properties of rodent beta(1-42) [Abeta(1-42)Rat] were evaluated and compared to those of human Abeta(1-42). Both human Abeta(1-42) and beta(1-42)Rat were found to have a significant effect on neuronal DNA fragmentation, loss of neuritic networks, and cell viability. beta(1-42) Rat was found to cause a significant increase in both protein oxidation and lipid peroxidation, similar to Abeta(1-42), both of which were inhibited by the lipid-soluble, chain breaking antioxidant vitamin E, suggesting that reactive oxygen species play a role in the Abeta-mediated toxicity. Taken together, these results suggest that Cu(II) reduction may not play a critical role inbeta(1-42)Rat-induced oxidative stress, and that the oxidative stress exhibited by this peptide may be a consequence of the presence of methionine 35, similar to the findings associated with the native human beta(1-42) peptide.

Alzheimer Disease↗

PubMed2003

Elevation of mitochondrial glutathione by gamma-glutamylcysteine ethyl ester protects mitochondria against peroxynitrite-induced oxidative stress.

Mitochondria under oxidative stress are thought to play a key role in various neurodegenerative disorders by directing neurons to cell death. Protection by antioxidants against oxidative stress to mitochondria may prove to be beneficial in delaying onset or progression of these diseases. We have investigated the ability of gamma-glutamylcysteine ethyl ester (GCEE) to upregulate mitochondrial glutathione (GSH) in vivo or in vitro and protect against subsequent in vitro peroxynitrite (ONOO-) damage. Mitochondria pretreated in vitro with GCEE were protected against oxidative damage induced by peroxynitrite, as assessed by mitochondrial swelling, changes in mitochondrial membrane potential, 3-nitrotyrosine formation, protein carbonyl formation, and cytochrome c release. Loss of mitochondrial function in neuronal cell cultures by the oxidants 2,2,'Azobis(2-amidino-propane)dihydrochloride (AAPH) and ONOO- was ameliorated by treatment with GCEE. In vivo studies showed that mitochondria isolated from animals injected intraperitoneally with GCEE were protected partially against oxidative modifications induced by ONOO-. Taken together, these results suggest that GCEE may be effective in increasing mitochondrial GSH and may be prove to have therapeutic relevance in neurodegenerative disorders associated with oxidative stress and mitochondrial dysfunction.

Animals↗

PubMed2003

Antioxidant activity of X-34 in synaptosomal and neuronal systems.

Inhibiting aggregation and deposition of amyloid beta-peptide (Abeta) in brain is a therapeutic strategy for Alzheimer's disease (AD). A Congo-red-like molecule, X-34, is reported to bind to Abeta deposits. Oxidative stress associated with Abeta is hypothesized to be critical for the neurotoxic properties of this peptide. The present study was undertaken to test the hypothesis that X-34, with its salicylate groups, would act as an antioxidant. When challenged by hydroxyl or peroxyl free radicals or Abeta(1-42), oxidative stress and neurotoxicity occurred in neural systems as assessed by several indices. However, pretreatment of synaptosomes and primary neuronal cell culture with X-34 greatly ameliorated lipid peroxidation induced by these free radicals and Abeta(1-42). Protein oxidation was not prevented by X-34. These results are discussed in terms of potential therapeutic use of X-34 and related compounds in AD.

Alkenes↗

PubMed2003

Role of the proteasome in protein oxidation and neural viability following low-level oxidative stress.

Numerous studies suggest that proteasome inhibition may play a causal role in mediating the increased levels of protein oxidation and neuron death observed in conditions associated with oxidative stress. In the present study we demonstrate that administration of non-toxic levels of oxidative stress does not result in impairment of 20S/26S proteasome activity, and actually increases the expression of specific proteasome subunits. Non-toxic levels of oxidative stress were observed to elevate the amount of protein oxidation in the presence of preserved proteasomal function, suggesting that proteasome inhibition may not mediate increases in protein oxidation following low-level oxidative stress. Preserving basal proteasome function appears to be critical to preventing the neurotoxicity of low-level oxidative stress, based on the ability of proteasome inhibitor treatment to exacerbate oxidative stress toxicity. Taken together, these data indicate that maintaining neural proteasome function may be critical to preventing neurotoxicity, but not the increase in protein oxidation, following low-level oxidative stress.

Animals↗

PubMed2003

Intracellular human immunodeficiency virus Tat expression in astrocytes promotes astrocyte survival but induces potent neurotoxicity at distant sites via axonal transport.

The human immunodeficiency virus (HIV)-Tat protein has been implicated in the neuropathogenesis of HIV infection. However, its role in modulating astroglial-neuronal relationships is poorly understood. Astrocyte infection with HIV has been associated with rapid progression of dementia. We thus initially transfected astrocytes with HIV proviral DNA and confirmed Tat production in these cells. Subsequently, using stably Tat-producing asytocyte cell lines, we observed that Tat promoted astrocyte survival by causing a prominent antioxidant effect and resistance to cell injury in these cells. Tat was released extracellularly where it could be taken up by other cells. Tat remained functionally active following uptake and caused long terminal repeat (LTR) transactivation in lymphocytic and astrocytic cell lines. Tat released from astrocytes caused mitochondrial dysfunction, trimming of neurites, and cell death in neurons. Tat neurotoxicity was attenuated by anti-Tat antibodies, kynurenate or heparan sulfate. The neurotoxic effects of Tat were caused at concentrations lower than that needed to cause LTR transactivation. When Tat-expressing cells were injected into the rat dentate gyrus, Tat was taken up by granule cells and transported along neuronal pathways to the CA3 region where it caused glial cell activation and neurotoxicity. The arginine-rich domain of Tat was essential for both the LTR transactivation and the neurotoxic properties of Tat. Thus HIV-Tat is a potent neurotoxin that may act at distant sites while at the same time it assures its production by preventing cell death in astrocytes where it is produced.

Astrocytes↗

PubMed2003

Acrolein inhibits NADH-linked mitochondrial enzyme activity: implications for Alzheimer's disease.

In Alzheimer's disease (AD) brain increased lipid peroxidation and decreased energy utilization are found. Mitochondria membranes contain a significant amount of arachidonic and linoleic acids, precursors of lipid peroxidation products, 4-hydroxynonenal (HNE) and 2-propen-1-al (acrolein), that are extremely reactive. Both alkenals are increased in AD brain. In this study, we examined the effects of nanomolar levels of acrolein on the activities of pyruvate dehydrogenase (PDH) and Alpha-ketoglutarate dehydrogenase (KGDH), both reduced nicotinamide adenine dinucleotide (NADH)-linked mitochondrial enzymes. Acrolein decreased PDH and KGDH activities significantly in a dose-dependent manner. Using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS), acrolein was found to bind lipoic acid, a component in both the PDH and KGDH complexes, most likely explaining the loss of enzyme activity. Acrolein also interacted with oxidized nicotinamide adenine dinucleotide (NAD(+)) in such a way as to decrease the production of NADH. Acrolein, which is increased in AD brain, may be partially responsible for the dysfunction of mitochondria and loss of energy found in AD brain by inhibition of PDH and KGDH activities, potentially contributing to the neurodegeneration in this disorder.

Acrolein↗

PubMed2003

The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice.

Oxidative stress may play a crucial role in age-related neurodegenerative disorders. Here, we examined the ability of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), to reverse the cognitive deficits found in the SAMP8 mouse. By 12 months of age, this strain develops elevated levels of Abeta and severe deficits in learning and memory. We found that 12-month-old SAMP8 mice, in comparison with 4-month-old mice, had increased levels of protein carbonyls (an index of protein oxidation), increased TBARS (an index of lipid peroxidation) and a decrease in the weakly immobilized/strongly immobilized (W/S) ratio of the protein-specific spin label MAL-6 (an index of oxidation-induced conformational changes in synaptosomal membrane proteins). Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood-brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence for a therapeutic role for antioxidants.

Acetylcysteine↗

PubMed2003

Proteomic identification of nitrated proteins in Alzheimer's disease brain.

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O2- leads to formation of peroxynitrite ONOO-, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration.

Aged↗

PubMed2003

Proteomics in Alzheimer's disease: insights into potential mechanisms of neurodegeneration.

Proteomics involves the identification of unknown proteins following their separation, often using two-dimensional electrophoresis, digestion of particular proteins of interest by trypsin, determination of the molecular weight of the resulting peptides, and database searching to make the identification of the proteins. Application of proteomics to Alzheimer's disease (AD), the major dementing disorder of the elderly, has just begun. Differences in protein expression and post-translational modification (mostly oxidative modification) of proteins from AD brain and peripheral tissue, as well as in brain from rodent models of AD, have yielded insights into potential molecular mechanisms of neurodegeneration in this dementing disorder. This review surveys the proteomics studies relevant to AD, from which new understandings of the pathology, biochemistry, and physiology of AD are beginning to emerge.

Alzheimer Disease↗

PubMed2003

Derivatives of xanthic acid are novel antioxidants: application to synaptosomes.

Xanthic acids have long been known to act as reducing agents. Recently, D609, a tricyclodecanol derivative of xanthic acid, has been reported to have anti-apoptotic and anti-inflammatory properties that are attributed to specific inhibition of phosphatidyl choline phospholipase C (PC-PLC). However, because oxidative stress is involved in both of these cellular responses, the possibility that xanthates may act as antioxidants was investigated in the current study. Finding that xanthates efficiently scavenge hydroxyl radicals, the mechanism by which D609 and other xanthate derivatives may protect against oxidative damage was further examined. The xanthates studied, especially D609, mimic glutathione (GSH). Xanthates scavenge hydroxyl radicals and hydrogen peroxide, form disulfide bonds (dixanthogens), and react with electrophilic products of lipid oxidation (acrolein) in a manner similar to GSH. Further, upon disulfide formation, dixanthogens are reduced by glutathione reductase to a redox active xanthate. Supporting its role as an antioxidant, D609 significantly (p < 0.01) reduces free radical-induced changes in synaptosomal lipid peroxidation (TBARs), protein oxidation (protein carbonyls), and protein conformation. Thus, in addition to inhibitory effects on PC-PLC, D609 may prevent cellular apoptotic and inflammatory cascades by acting as antioxidants and novel GSH mimics. These results are discussed with reference to potential therapeutic application of D609 in oxidative stress conditions.

Anti-Inflammatory Agents↗

PubMed2003

Communication between the maternal testa and the embryo and/or endosperm affect testa attributes in tomato.

Two tomato (Lycopersicon esculentum) mutants with dark testae displaying poor germination rate and percentage on both water and 100 microM gibberellin(4 + 7) were recovered. The mutants were allelic (black seed1-1; bks1-1 and bks1-2), inherited in Mendelian fashion as a recessive gene residing on chromosome 11. They are not allelic to bs (brown seed) -1, -2, or -4, which impair seed germination and possess dark testae. The bks/bs mutants accumulated dark pigment in the cell layers of the testa above the endothelium, which itself accumulated proanthocyanidins similar to wild type. The poor germination performance of bks mutant seeds was because of impediment of the mutant testae to radicle egress. Imbibition on gibberellin(4 + 7) did not ameliorate germination percentage or rate. The toughening of the bks testa and associated poor germination were partially overcome when seeds were not dried before germination or were dried under N(2). The seeds of the bks mutant have elevated activity of at least one enzyme responsible for the detoxification of reactive oxygen species. The bks mutant is epistatic to 12 anthocyaninless mutants of tomato. Bio- and physicochemical analysis of the bks testa determined that it accumulated a melanic substance. Inheritance of bks/bs mutations contrasts with that of the anthocyaninless mutants, which are inherited according to the genotype of the maternally derived testa. This suggests that the testa manufactures components before its demise that can maximize testa strength, whereas the endosperm/embryo produces factors that are conveyed to the testa, mitigating this process.

Anthocyanins↗