Studies of embryotoxic mechanisms of niridazole: evidence that oxygen depletion plays a role in dysmorphogenicity

Previous study has shown that niridazole (NDZ) is dysmorphogenic to rat embryos between days 10 and 11 under culture conditions including 5% oxygen. Other studies have found that reductive embryonic biotransformation is required but that covalent binding is not a major basis of this embryotoxicity. In research presented here, NDZ exposure of homogenates prepared from day 10 rat embryos resulted in stimulation of oxygen uptake from incubation media. Further studies showed that a large percentage of this increased oxygen uptake was associated with the generation of superoxide anion radical and hydrogen peroxide. These findings led us to hypothesize that redox cycling forms the basis of the in vitro dysmorphogenicity of NDZ. The basic premise of this hypothesis is that as a result of redox cycling, oxygen is depleted from the sensitive tissues of embryos. In order to investigate it, we devised a technique for carefully controlling and monitoring oxygen tensions in embryo cultures. We found that when oxygen concentrations of 4% were established, a highly significant incidence of asymmetric defects resulted. These defects appeared analogous to those induced by NDZ exposure, consisting of asymmetric necrosis of mesenchymal tissue near the cephalic end of the neural tube and thinning of the neuroepithelium on the right. We concluded that the hypoxia induced by redox cycling of NDZ and related nitroheterocycles represents a major embryotoxic principle of action.     

(+)-Arisugacin A--computational evidence of a dual binding site covalent inhibitor of acetylcholinesterase

A computation docking study of the highly potent, non-nitrogen containing, acetylcholinesterase inhibitor (+)-arisugacin A is presented. The model suggests that (+)-arisugacin A is a dual binding site covalent inhibitor of AChE. These findings are examined in the context of Alzheimer’s disease-modifying therapeutic design. (+)-Arisugacin A’s revealed mode of action is unique, and may serve as a basis for the development of AD therapeutics capable of treating the symptomatic aspects of AD, while being neuroprotective with long term efficacy.     

No evidence for a major role of polymorphisms during bupropion treatment

This study evaluated the ability of polymorphisms in five candidate genes to predict weight gain among patients taking bupropion or placebo in a smoking cessation trial. Five hundred fifty-three smokers were enrolled into a randomized double-blind, placebo-controlled trial and followed for 12 months. Five candidate genes [DRD2 Taq1 (rs1800497), DRD2-141 (rs1799732), C957T (rs6277), COMT (rs4818), and SLC6A3] were genotyped. Weights at baseline, at end of treatment, and after 6 and 12 months of follow-up were self-reported. Smoking abstinence at each endpoint was self-reported and confirmed biochemically. A self-reported average weight gain after 12 months of 1.1 +/- 6.0 kg (mean +/- standard deviation) in the bupropion group and 1.8 +/- 4.8 kg in the placebo group was noted. For subjects with biochemically confirmed abstinence from smoking, the HL genotype (alleles coding Val at codon 108 are denoted as H, and those coding Met are denoted as L) at the COMT locus and A1A1 genotype at the DRD2 Taq1 locus were associated with less weight gain at the end of treatment. The TC genotype at the C957T locus was associated with increased weight gain at 6 months of follow-up. However, no polymorphisms or their interactions with bupropion consistently and significantly predicted baseline BMI or weight change during treatment for all study subjects. Overall, our results do not support a major role for these five candidate genes in weight gain after smoking cessation.     

Exercise hyperpnea in birds: evidence against a primary role for pCO2

Ventilatory responses of domestic fowl to graded intensities of treadmill exercise were compared when the birds breathed air, 3% CO2 in air or 4.2% CO2 in air. During exercise in air, increased minute ventilation resulted mainly from increased respiratory rate with little change in tidal volume. This pattern of ventilatory response was not altered when the birds respired CO2. In contrast, the pattern of ventilatory response to CO2, at given work loads, consisted of a primary increase in tidal volume with little change in respiratory rate. It is concluded that intrapulmonary pCO2 does not affect the ventilatory response to exercise.     

The embryotoxicity of phenytoin: an update on possible mechanisms

PHT has multiple effects in adult humans and animals, and there is no reason to assume that it will not have multiple effects in embryos and fetuses. Although one of the first associations between anticonvulsant therapy and an adverse development effect in humans was noted in 1964 (104), the mechanism(s) whereby these adverse effects occur has thus far eluded research efforts. In this review, I have focused on three possible mechanisms. Overall, the evidence does not appear to implicate folate deficiency in PHT-induced embryotoxicity. A role for glucocorticoids or interaction between PHT and the glucocorticoid receptor has not been ruled out. However, a significant amount of work remains to be done to examine the involvement of the arachidonic acid cascade in PHT-induced embryotoxicity in vivo. The bulk of the experimental evidence would seem to favor a role for the generation of a reactive intermediate and its subsequent binding to embryonic macromolecules. This metabolite(s) has not been identified. Additionally, the association between covalent binding of metabolites and embryotoxicity remains simply an association; a causal relationship has not been established. Much work remains to be done to determine whether any of these possibilities, some other possibility, or a combination of several mechanisms will explain the adverse development effects of this very important, therapeutically useful anticonvulsant.     

Studies on the DT-diaphorase-catalysed reaction employing quinones as substrates: evidence for a covalent modification of DT-diaphorase by tetrachloro-p-benzoquinone

In this study, the kinetic parameters, V(max) and K(m), of rat liver DT-diaphorase were determined for a series of p-benzoquinones, with methyl, methoxy, cyano, hydroxy and halo substituents. The results show that there is no correlation between the experimentally determined rates of p-benzoquinone reduction by DT-diaphorase and the calculated chemical reactivity of the examined substrates as expressed by the energy of the lowest unoccupied molecular orbital, E(LUMO). However, a reasonable correlation was found between the natural logarithm of V(max)/K(m) and the partition coefficient of the p-benzoquinones (r=0.81). Furthermore, tetrachloro-p-benzoquinone, one of the tested quinones is shown to be an inhibitor of rat DT-diaphorase. The presence of bovine serum albumin (BSA) in the incubation mixture protects DT-diaphorase against the inactivation by tetrachloro-p-benzoquinone, probably by interacting with the quinone. Maldi-Tof analysis of the incubation mixture of the purified DT-diaphorase and tetrachloro-p-benzoquinone showed that every subunit of the enzyme shifted about +414 amu, whereas the dimer shifted about +849 amu relative to control values. This indicates a covalent modification of the rat liver DT-diaphorase by tetrachloro-p-benzoquinone.     

Aspartate 120 of Escherichia coli methylenetetrahydrofolate reductase: evidence for major roles in folate binding and catalysis and a minor role in flavin reactivity

Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADH-linked reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as cofactor. MTHFR is unusual among flavin oxidoreductases because it contains a conserved, negatively rather than positively charged amino acid (aspartate 120) near the N1-C2=O position of the flavin. At this location, Asp 120 is expected to influence the redox properties of the enzyme-bound FAD. Modeling of the CH(3)-H(4)folate product into the enzyme active site suggests that Asp 120 may also play crucial roles in folate binding and catalysis. We have replaced Asp 120 with Asn, Ser, Ala, Val, and Lys and have characterized the mutant enzymes. Consistent with a loss of negative charge near the flavin, the midpoint potentials of the mutants increased from 17 to 30 mV. A small kinetic effect on the NADH reductive half-reaction was also observed as the mutants exhibited a 1.2-1.5-fold faster reduction rate than the wild-type enzyme. Catalytic efficiency (k(cat)/K(m)) in the CH(2)-H(4)folate oxidative half-reaction was decreased significantly (up to 70000-fold) and in a manner generally consistent with the negative charge density of position 120, supporting a major role for Asp 120 in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. Asp 120 is also intimately involved in folate binding as increases in the apparent K(d) of up to 15-fold were obtained for the mutants. Examining the E(red) + CH(2)-H(4)folate reaction at 4 degrees C, we obtained, for the first time, evidence for the rapid formation of a reduced enzyme-folate complex with wild-type MTHFR. The more active Asp120Ala mutant, but not the severely impaired Asp120Lys mutant, demonstrated the species, suggesting a connection between the extent of complex formation and catalytic efficiency.     

Studies of the inhibition of aldose reductase: evidence for multiple site binding

1. Comparison of structure-inhibition relationships and kinetic data between the N-[(4-benzoylamino)phenyl]sulfonyl]amino acids (BAPS-amino acids) and phenylsulfonylamino acids (PS-amino acids) suggests that the additional benzoyl moiety present in the BAPS-amino acids enhances inhibition by direct interaction with aldose reductase (EC 1.1.1.21) without altering the mode of interaction with the enzyme. 2. Also the 2-, 3- and 4-nitro regioisomers of BAPS-glycine (NBAPSG) display parallel structure- inhibition relationships with the 2-, 3- and 4-nitrobenzaldehyde substrates and the 2-, 3- and 4-nitroacetophenone competitive inhibitors. 3. Competition studies and multiple inhibition analyses demonstrate that the 4-nitrobenzoyl group of 4-NBAPSG binds at the substrate site of aldose reductase, while the PS-glycine moiety of 4-NBAPSG binds cooperatively at a distinct site.     

Phenytoin embryotoxicity: role of enzymatic bioactivation in a murine embryo culture model

A murine embryo culture model was developed to study the potential contribution of enzymatic bioactivation to the teratogenicity of phenytoin. To assess the relative embryonic and maternal contributions to bioactivation, embryos were cultured respectively alone or in the presence of an exogenous source of cytochromes P-450 (P-450), which are thought to bioactivate phenytoin to a teratogenic reactive intermediate. Embryological development from gestational day 9 to day 10 was assessed, and bioactivation was quantified by the irreversible binding of radiolabeled phenytoin to embryonic protein. Embryos cultured with phenytoin and an exogenous P-450 bioactivating system showed a significant decrease in the incidence of turning and closure of the anterior neuropore, yolk sac diameter, and protein content as well as growth retardation. In the absence of an exogenous P-450 system, phenytoin did not decrease the incidence of turning or anterior neuropore closure but did cause growth retardation and a lesser but significant reduction in yolk sac diameter and embryonic protein content. An exogenous P-450 system enhanced the bioactivation of phenytoin, although significant activity also was detectable in embryos cultured without an exogenous bioactivating system. These results suggest that the embryo itself can enzymatically bioactivate embryotoxically significant amounts of phenytoin, and that bioactivation and embryotoxicity can be further enhanced, qualitatively and quantitatively, by an exogenous P-450 system, implicating a possible maternal contribution to phenytoin teratogenicity.     

New evidence for a multi-functional role of herbivore-induced plant volatiles in defense against herbivores

A diverse, often species-specific, array of herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack. Although research in the last 3 decades indicates a multi-functional role of these HIPVs, the evolutionary rationale underpinning HIPV emissions remains an open question. Many studies have documented that HIPVs can attract natural enemies, and some studies indicate that neighboring plants may eavesdrop their undamaged neighbors and induce or prime their own defenses prior to herbivore attack. Both of these ecological roles for HIPVs are risky strategies for the emitting plant. In a recent paper, we reported that most branches within a blueberry bush share limited vascular connectivity, which restricts the systemic movement of internal signals. Blueberry branches circumvent this limitation by responding to HIPVs emitted from neighboring branches of the same plant: exposure to HIPVs increases levels of defensive signaling hormones, changes their defensive status, and makes undamaged branches more resistant to herbivores. Similar findings have been reported recently for sagebrush, poplar and lima beans, where intra-plant communication played a role in activating or priming defenses against herbivores. Thus, there is increasing evidence that intra-plant communication occurs in a wide range of taxonomically unrelated plant species. While the degree to which this phenomenon increases a plant’s fitness remains to be determined in most cases, we here argue that withinplant signaling provides more adaptive benefit for HIPV emissions than does between-plant signaling or attraction of predators. That is, the emission of HIPVs might have evolved primarily to protect undamaged parts of the plant against potential enemies, and neighboring plants and predators of herbivores later co-opted such HIPV signals for their own benefit.Key words: intra-plant signaling, plantplant communication, eavesdropping, systemic wound signals, plant defense, tri-trophic interactionsPlants often emit a unique blend of volatiles in response to herbivore attack. The emission of these herbivore-induced plant volatiles (HIPVs) is an active response to herbivore feeding, producing a blend of volatiles that is distinct from those emitted following mechanical injury alone.¹ Their emission can be variable; while some compounds follow a diurnal pattern with increasing amounts during the time of high photosynthesis,^2,3 others are emitted primarily at night.⁴ In some cases, the HIPV blend produced also differs depending on the species of herbivore feeding on the plant.⁵ This specificity is thought to be due to chemicals in the herbivore’s regurgitant, such as the fatty-acid amino-acid conjugate volicitin, that activate the emission of volatiles in plants.^6,7 Furthermore, HIPVs are emitted not only from the site of damage, but also at times from systemically undamaged parts of the plant.⁸ This and other systemic responses are, however, restricted within a plant such that only parts of the plant that share vascular connections with the damaged tissue receive wound signals and have the potential to respond.^9,10The ecological role of HIPVs has been a subject of fascination and the evolutionary advantage gained for plants by emitting HIPVs remains an unresolved topic of discussion. While some HIPV compounds, and some of their precursors, have sufficient volatility that their release is essentially inevitable after synthesis,¹¹ most tend to be tightly regulated. Assuming that HIPV emissions evolved as a result of trophic interactions among plants, herbivores, and natural enemies, there are four general ecological roles that HIPVs may play: (1) a direct negative effect on the herbivore, (2) a signal to alert natural enemies of the herbivore, (3) a warning signal to nearby undamaged plants, and (4) a systemic warning signal within the damaged plant (Fig. 1). The first two potential roles involve the manipulation of animal behavior, while the last two may alter plant “behavior”.Open in a separate windowFigure 1Herbivore-induced plant volatiles (HIPVs) play multiple roles in interactions among plants, herbivores, and natural enemies (possible interactions are depicted by arrows). Some of them benefit the HIPV-emitting plant (Emitter); these positive interactions include repellent effects on herbivores, attraction of natural enemies of herbivores, activation or priming of defenses in unwounded parts within the emitting plant (within-plant signaling), and growth inhibitory effects on neighboring plants (Receiver) through allelopathy. On the other hand, HIPVs may negatively affect the emitting plant by attracting herbivores or natural enemies (e.g., certain parasitoids) that result in increased damage. Finally, neighboring plants may “eavesdrop” from the emitting plant by responding to HIPVs (between-plant signaling). This latter interaction may be negative to the emitter if it is outcompeted by neighbors who receive wound signals, but beneficial to the receiving plant. Drawing by Robert Holdcraft.Scents can have a demonstrable effect on animal behavior. With respect to plant-herbivore interactions, scents can provide information about the status of a plant to herbivores and their natural enemies. For example, HIPVs may repel adults moths searching for oviposition sites,³ which has been interpreted from the perspective of either a plant minimizing damage or, perhaps more realistically, an adult moth searching for an undamaged, high quality resource for her offspring. Conversely, HIPV-emitting plants may increase their chance of being injured if herbivores are attracted to these volatiles.¹² The more commonly accepted role of HIPVs in manipulating animal behavior is to attract natural enemies of the herbivores. This tri-trophic “cry for help”¹³ has a potential evolutionary benefit for both the plant emitting the volatiles and the natural enemies responding to this emission.^14–16 Although this idea makes sense in an evolutionary perspective, only a few studies have documented the occurrence of this phenomenon in natural systems.¹⁷ Indeed, the effectiveness of a cry for help depends on the presence of a helper and, equally importantly, the ability of the helper to increase plant fitness. In the case of predator attraction, the herbivore may be removed from the plant and consumed, thereby reducing damage for the emitting plant.¹⁸ However, insect herbivores infected by parasitoids, which also use HIPV cues to locate hosts,¹⁹ may also consume less plant material²⁰ but may also in some cases consume more plant material than unparasitized insect herbivores.²¹ Since there is currently no evidence that plants can modify HIPV blends to attract selectively predators versus parasitoids, an answered cry for help may not reliably decrease the total amount of damage to an emitting plant. Thus, the fact that natural enemies respond to HIPVs does not imply that these volatiles evolved for this purpose or that there is an adaptive advantage for a plant to use HIPVs to attract natural enemies. Rather, natural enemies of insect herbivores may have learned to co-opt the HIPV signal emitted by plants and, by doing so, increased their fitness irrespective of the ultimate fitness outcome to the plant.Though more controversial, scents can also have an effect on plant behavior.²² Early work suggested that HIPVs from wounded willows,²³ poplars²⁴ and sugar maples²⁴ could trigger defense responses from other neighboring conspecifics. More recent studies have shown that this signaling can occur between different species of plants.²⁵ While these results are intriguing, they appear to have little adaptive function from the perspective of an emitting plant, which could be facilitating the fitness of potential resource competitors. Further, unless the individual within the same plant species shared some degree of kinship,²⁶ an emitting plant would also be at a disadvantage by providing an HIPV wound signal to a conspecific that, in theory, occupies the same competitive niche space. On the other hand, unwounded conspecific should benefit from being able to ‘eavesdrop’ by detecting HIPVs from wounded plants as they share the same herbivore complex and thus are vulnerable to attack. Moreover, from a heterospecific receiver’s perspective, the benefits of eavesdropping can be confounded by the potential of mounting defenses against a signal generated by incompatible herbivores feeding on a different plant species.²⁷ So, eavesdropping may be adaptive for a receiving plant if it realizes increased fitness relative to a conspecific that did not receive the signal. The emitting plant derives no apparent adaptive benefit of using HIPVs to warn neighboring plants. However, the emitting plant may benefit if their HIPVs have inhibitory allelopathic activity on neighboring plants.²⁸Our recent work¹ highlighted another scenario by which an HIPV-emitting plant would derive a direct benefit from the emissions: when HIPVs act as systemic wound signals within damaged plants. We showed that branches of blueberry shrubs lack effective vascular connections and thus cannot transmit wound signals among branches via the vasculature. To compensate, HIPVs can be transmitted among branches and, in so doing, overcome the vascular constraints of the branching life history strategy. Exposure to HIPVs increased levels of defensive signaling hormones in undamaged branches, changed their defensive chemical status, and made them more resistant to herbivores.¹ This idea that HIPVs may function in intra-plant communication to activate or prime defenses in other parts of the emitting plant against future attack was first suggested separately by Farmer²⁹ and Orians.⁹ The hypothesis was first tested with mechanically clipped wild sagebrush,³⁰ and it was further tested with insect herbivores of wild lima bean³¹ and hybrid poplar.³² Under this scenario, the emitting plant derives a direct benefit from the HIPVs, providing an unambiguous fitness advantage.So, what is the most beneficial factor to a plant for emitting volatiles in response to herbivore feeding? In terms of maximizing the potential benefit and minimizing the potential risk to the emitting plant, the function of HIPVs in mediating systemic wound signaling clearly provides the greatest potential adaptive advantage. Thus, we propose that the primary adaptive benefit for the evolution of HIPVs is to signal and protect unwounded parts of the attacked plant with high risk of infestation against herbivores. Later, these volatiles provided cues that led to adaptive fitness advantages for neighboring plants and natural enemies of herbivores, which may or may not benefit the HIPV-emitting plant. Indeed, ecologically adaptive advantages have emerged and contribute to a diverse, multi-functional chemical ecology mediated by HIPVs.     

Oxygen binding to Arabidopsis thaliana AHb2 nonsymbiotic hemoglobin: evidence for a role in oxygen transport

Nonsymbiotic hemoglobins AHb1 and AHb2 discovered in Arabidopsis thaliana are likely to carry out distinct physiological roles, in consideration of their differences in sequence, structure, expression pattern, and tissue localization. Despite a relatively fast autoxidation in the presence of O(2) , we were able to collect O(2) -binding curves for AHb2 in the presence of a reduction enzymatic system. AHb2 binds O(2) noncooperatively with a p50 of 0.021 ± 0.003 Torr, a value consistent with a recently proposed role in O(2) transport. The analysis of the internal cavities derived from the structures sampled in molecular dynamics simulations confirms strong differences with AHb1, proposed to work as a NO deoxygenase in vivo. Overall, our results are consistent with a role for AHb2 as an oxygen carrier, as recently proposed on the basis of experiments on AHb2-overexpressing mutants of A. thaliana.     

Protective mechanisms against homocysteine toxicity: the role of bleomycin hydrolase

Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone.     

The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum

ChitO (chito-oligosaccharide oxidase) from Fusarium graminearum catalyses the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His94 and Cys154. The functional role of this unusual bi-covalent flavin-protein linkage was studied by site-directed mutagenesis. The double mutant (H94A/C154A) was not expressed, which suggests that a covalent flavin-protein bond is needed for protein stability. The single mutants H94A and C154A were expressed as FAD-containing enzymes in which one of the covalent FAD-protein bonds was disrupted relative to the wild-type enzyme. Both mutants were poorly active, as the k(cat) decreased (8.3- and 3-fold respectively) and the K(m) increased drastically (34- and 75-fold respectively) when using GlcNac as the substrate. Pre-steady-state analysis revealed that the rate of reduction in the mutant enzymes is decreased by 3 orders of magnitude when compared with wild-type ChitO (k(red)=750 s(-1)) and thereby limits the turnover rate. Spectroelectrochemical titrations revealed that wild-type ChitO exhibits a relatively high redox potential (+131 mV) and the C154A mutant displays a lower potential (+70 mV), while the H94A mutant displays a relatively high potential of approximately +164 mV. The results show that a high redox potential is not the only prerequisite to ensure efficient catalysis and that removal of either of the covalent bonds may perturb the geometry of the Michaelis complex. Besides tuning the redox properties, the bi-covalent binding of the FAD cofactor in ChitO is essential for a catalytically competent conformation of the active site.     

Little evidence for a major role of Ca2+ in cold-induced injury of liver cells

We have previously shown that cold-induced injury to hepatocytes and liver endothelial cells occurs predominantly via an iron-dependent pathway. However, other groups have reported evidences suggesting that Ca²⁺ ions could be involved in the process of cold-induced injury of liver cells. We here assessed the relative importance and potential interaction of both pathways in cultured primary hepatocytes and cultured liver endothelial cells. The sequence cold incubation/rewarming of hepatocytes and endothelial cells led to an increase in the cytosolic calcium concentration during the early rewarming phase, but the increased cytosolic calcium concentration did not correlate with cell injury. A partial protection from cold-induced cell injury was achieved by the intracellular calcium chelators Quin-2 and BAPTA. However, additional experiments showed that the ability of these chelators to bind iron was probably responsible for a major part of this protection. Incubation in calcium-free media led to an increased cell injury and a physiological calcium concentration (2.5 mM) was protective. In addition, targeting suggested downstream pathways of calcium-dependent cold-induced injury, i.e. by the addition of Ruthenium Red, an inhibitor of mitochondrial Ca²⁺ uniporter, or by inhibiting Bax translocation to the mitochondria, did not provide protection from cold-induced injury in both cell types. Taken together, our data suggest that calcium increases but does not play a major role in cold-induced cell injury to hepatocytes and liver endothelial cells.     

Unraveling a hotspot for TCR recognition on HLA-A2: evidence against the existence of peptide-independent TCR binding determinants

T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 alpha1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.     

The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro

Covalent binding of the reactive metabolite of acetaminophen has been investigated in hepatic microsomal preparations from phenobarbital-pretreated mice. Low molecular weight thiols (cysteine and glutathione) were found to inhibit this binding, whereas several other amino acids which were tested did not. Bovine serum albumin (BSA), which contains a single free sulfhydryl group per molecule and which thus represents a macromolecular thiol compound, inhibited covalent binding of the reactive acetaminophen metabolite to microsomal protein in a concentration-dependent manner. The acetaminophen metabolite also became irreversibly bound to BSA in these experiments, although this binding was reduced by approx. 47% when the thiol function of BSA was selectively blocked prior to incubation. Covalent binding of the acetaminophen metabolite to bovine alpha s1-casein, a soluble protein which does not contain any cysteine residues, was found to occur to an extent of 37% of that which became bound to native BSA. These results were taken to indicate that protein thiol groups are major sites of covalent binding of the reactive metabolite of acetaminophen in vitro. The covalent binding characteristics of synthetic N-acetyl-p-benzoquinoneimine (NAPQI), the putative electrophilic intermediate produced during oxidative metabolism of acetaminophen, paralleled closely those of the reactive species generated metabolically. These findings support the contention that NAPQI is indeed the reactive arylating metabolite of acetaminophen which binds irreversibly to protein.     

Development of new structural alerts suitable for chemical category formation for assigning covalent and non-covalent mechanisms relevant to DNA binding

The need to assess the ability of a chemical to act as a mutagen is one of the primary requirements in regulatory toxicology. Several pieces of legislation have led to an increased interest in the use of in silico methods, specifically the formation of chemical categories and read-across for the assessment of toxicological endpoints. One of the key steps in the development of chemical categories for mutagenicity is defining the mechanistic organic chemistry associated with the formation of a covalent bond between DNA and an exogenous chemical. To this end this study has analysed, by use of a large set of mutagenicity data (Ames test), the mechanistic coverage of a recently published set of in silico structural alerts developed for category formation. The results show that the majority of chemicals with a positive result in the Ames test were assigned at least one covalent binding mechanism related to the formation of a DNA adduct. The remaining chemicals with positive data in the Ames assay were subjected to a detailed mechanistic analysis from which 26 new structural alerts relating to covalent binding mechanisms were developed. In addition, structural alerts for radical and non-covalent intercalation mechanisms were also defined. The structural alerts outlined in this study are not intended to predict mutagenicity but rather to identify mechanisms associated with covalent and non-covalent DNA binding. This mechanistic profiling information can then be used to form chemical categories suitable for filling data gaps via read-across. A strategy for chemical category formation for mutagenicity is also presented.