Approaches to Investigating Complex Genetic Traits in a Large-Scale Inbred Mouse Aging Study.

Inbred mice are a unique model system for studying aging because of the genetic homogeneity within inbred strains, the short life span of mice relative to humans, and the rich array of analytic tools that are available. A large-scale aging study was conducted on 28 inbred strains representing great genetic diversity to determine, via histopathology, the type and diversity of spontaneous diseases that aging mice develop. A total of 20 885 different diagnoses were made, with an average of 12 diagnoses per mouse in the study. Eighteen inbred strains have had their genomes sequenced, and many others have been partially sequenced to provide large repositories of data on genetic variation among the strains. This vast amount of genomic information can be utilized in genome-wide association studies to find candidate genes that are involved in the pathogenesis of spontaneous diseases. As an illustration, this article presents a genome-wide association study of the genetic associations of age-related intestinal amyloidosis, which implicated 3 candidate genes: translocating chain-associated membrane protein 1 (Tram1); splicing factor 3b, subunit 5 (Sf3b5); and syntaxin 11 (Stx11). Representative photomicrographs are available on the Mouse Tumor Biology Database and Pathbase to serve as a reference when evaluating inbred mice used in other genetic or experimental studies to rule out strain background lesions. Many of the age-related mouse diseases are similar, if not identical, to human diseases; therefore, the genetic discoveries have direct translational benefit.

Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice.

Glaucomas are a major cause of blindness. Visual loss typically involves retinal ganglion cell death and optic nerve atrophy subsequent to a pathologic elevation of intraocular pressure (IOP). Some human glaucomas are associated with anterior segment abnormalities such as pigment dispersion syndrome (PDS) and iris atrophy with associated synechiae. The primary causes of these abnormalities are unknown, and their aetiology is poorly understood. We recently characterized a mouse strain (DBA/2J) that develops glaucoma subsequent to anterior segment changes including pigment dispersion and iris atrophy. Using crosses between mouse strains DBA/2J (D2) and C57BL/6J (B6), we now show there are two chromosomal regions that contribute to the anterior segment changes and glaucoma. Progeny homozygous for the D2 allele of one locus on chromosome 6 (called ipd) develop an iris pigment dispersion phenotype similar to human PDS. ipd resides on a region of mouse chromosome 6 with conserved synteny to a region of human chromosome 7q that is associated with human PDS. Progeny homozygous for the D2 allele of a different locus on chromosome 4 (called isa) develop an iris stromal atrophy phenotype (ISA). The Tyrpl gene is a candidate for isa and likely causes ISA via a mechanism involving pigment production. Progeny homozygous for the D2 alleles of both ipd and isa develop an earlier onset and more severe disease involving pigment dispersion and iris stromal atrophy.

The root meristem is shaped by brassinosteroid control of cell geometry.

Growth extent and direction determine cell and whole-organ architecture. How they are spatio-temporally modulated to control size and shape is not well known. Here we tackled this question by studying the effect of brassinosteroid (BR) signalling on the structure of the root meristem. Quantification of the three-dimensional geometry of thousands of individual meristematic cells across different tissue types showed that the modulation of BR signalling yields distinct changes in growth rate and anisotropy, which affects the time that cells spend in the meristem and has a strong impact on the final root form. By contrast, the hormone effect on cell volume was minor, establishing cell volume as invariant to the effect of BR. Thus, BR has the highest effect on cell shape and growth anisotropy, regulating the overall longitudinal and radial growth of the meristem, while maintaining a coherent distribution of cell sizes. Moving from single-cell quantification to the whole organ, we developed a computational model of radial growth. The simulation demonstrates how differential BR-regulated growth between the inner and outer tissues shapes the meristem and thus explains the non-intuitive outcomes of tissue-specific perturbation of BR signalling. The combined experimental data and simulation suggest that the inner and outer tissues have distinct but coordinated roles in growth regulation.

Detection of a protein-acetaldehyde adduct in the liver of rats fed alcohol chronically.

We report here the formation in vivo of a protein-acetaldehyde adduct (protein-AA) in liver when rats were fed alcohol chronically. This chemically modified protein was demonstrated by electroimmunotransblot technique and with rabbit polyclonal antibodies that recognize acetaldehyde adduct as an epitope (i.e., both anti-hemocyanin-AA IgG and anti-myoglobin-AA IgG). It has a molecular weight of 37,000. It can be detected in the liver of rats fed the alcohol-containing American Institute of Nutrition 1976 liquid diet for only 1 wk. Since the protein profiles of soluble hepatic proteins from alcohol-fed and control rats were identical on SDS-PAGE, the peroxidase-positive band demonstrated by electroimmunotransblot was most likely not a new protein synthesized de novo. Borohydride reduction was not necessary to stabilize this protein-AA. Intraperitoneal injections of ethanol (2 g/kg body wt) at 8-h intervals to rats over a 24-h period did not produce any detectable protein-AA in the liver. Incubation of the liver homogenate from a control liver with acetaldehyde without sodium cyanoborohydride for 4 h also failed to generate any protein-AA. Therefore, the formation of the 37-kD protein-AA in vivo reported here is dependent on chronic alcohol consumption.

Polysaccharide-mediated protection against abscess formation in experimental intra-abdominal sepsis.

Abscess formation is a major complication of intra-abdominal sepsis that causes significant morbidity and mortality. In such cases, Bacteroides fragilis is the predominant anaerobic isolate. In a rat model of intra-abdominal sepsis, the capsular polysaccharide complex (CPC) from B. fragilis promotes abscess formation and when administered sub-cutaneously, protects against this host response by a T cell-dependent immune mechanism. In the present study, the polysaccharide A (PS A) component of CPC protected animals against challenge with live heterologous bacterial species (mixtures of anaerobes and facultative organisms) that are most commonly isolated from intra-abdominal abscesses in humans. Protection against heterologous bacterial challenge was transferred by T cells. Administration of PS A shortly before or even after challenge with B. fragilis protected against this host response. In experiments designed to simulate fecal contamination of the human peritoneal cavity, PS A protected animals against abscess formation induced by a rat cecal contents inoculum. The surprisingly broad protective activity of PS A indicates that this molecule is likely suppressing a nonspecific host tissue reaction that forms in response to a variety of abscess-inducing organisms and that it might be useful in preventing abscess formation associated with intra-abdominal sepsis in the clinical setting.

Familial hypobetalipoproteinemia caused by a mutation in the apolipoprotein B gene that results in a truncated species of apolipoprotein B (B-31). A unique mutation that helps to define the portion of the apolipoprotein B molecule required for the formation of buoyant, triglyceride-rich lipoproteins.

Apolipoprotein B-100 has a crucial structural role in the formation of VLDL and LDL. Familial hypobetalipoproteinemia, a syndrome in which the concentration of LDL cholesterol in plasma is abnormally low, can be caused by mutations in the apo B gene that prevent the translation of a full-length apo B-100 molecule. Prior studies have revealed that truncated species of apo B [e.g., apo B-37 (1728 amino acids), apo B-46 (2057 amino acids)] can occasionally be identified in the plasma of subjects with familial hypobetalipoproteinemia; in each of these cases, the truncated apo B species has been a prominent protein component of VLDL. In this report, we describe a kindred with hypobetalipoproteinemia in which the plasma of four affected heterozygotes contained a unique truncated apo B species, apo B-31. Apolipoprotein B-31 is caused by the deletion of a single nucleotide in the apo B gene, and it is predicted to contain 1425 amino acids. Apolipoprotein B-31 is the shortest of the mutant apo B species to be identified in the plasma of a subject with hypobetalipoproteinemia. In contrast to longer truncated apo B species, apo B-31 was undetectable in the VLDL and the LDL; however, it was present in the HDL fraction and the lipoprotein-deficient fraction of plasma. The density distribution of apo B-31 in the plasma suggests the possibility that the amino-terminal 1425 amino acids of apo B-100 are sufficient to permit the formation and secretion of small, dense lipoproteins but are inadequate to support the formation of the more lipid-rich VLDL and LDL particles.

Structural relationship of human apolipoprotein B48 to apolipoprotein B100.

Although the complete amino acid sequence of human apolipoprotein (apo) B100 is known (4536 amino acids), the structure of apo B48 has not been defined. The objective of our study was to define the structure of apo B48 and its relationship to apo B100. Antibodies were produced against 22 synthetic peptides corresponding to sequences in human apo B100. The levels of immunoreactivity of the antipeptides to apo B100 and apo B48 were used to define the structural relationship between these two species of apo B. Six antibodies from sequences in the amino-terminal half of apo B100, including antipeptide 2110-2129, bound to both apo B100 and apo B48. 15 other apo B-specific antipeptides from sequences carboxyl-terminal to residue 2152 bound to apo B100, but not to apo B48. Immunoblots of cyanogen bromide digests of apo B100 and apo B48 with antipeptides 2068-2091 and 2110-2129 detected a 16-KD fragment (residues 2016-2151) in the apo B100 digest and a fragment of identical size in the apo B48 digest. Because apo B48 appears to contain the apo B100 cyanogen bromide fragment 2016-2151 and because an antiserum specific for the peptide 2152-2168 does not bind to apo B48, we conclude that apo B48 represents the amino-terminal 47% of apo B100 and that the carboxyl terminus of apo B48 is in the vicinity of residue 2151 of apo B100.

Epstein-Barr virus-induced autoimmune responses. I. Immunoglobulin M autoantibodies to proteins mimicking and not mimicking Epstein-Barr virus nuclear antigen-1.

In previous studies of infectious mononucleosis, we found IgM autoantibodies which react with hematopoietic cell antigens. Many of these were inhibited by synthetic glycine/alanine peptides representing the glycine/alanine repeat of Epstein-Barr virus nuclear antigen-1. We have cloned and expressed fragments of genes encoding two of these autoantigens. One gene (p542) encodes a protein containing a glycine-rich 28-mer, which is its chief autoantigenic epitope and which represents a newly identified class of evolutionarily conserved autoepitopes. The other gene (p554) encodes a protein that is not demonstrably cross-reactive with Epstein-Barr virus nuclear antigen-1 or with any other EBV protein, but forms complexes with other proteins. Immunoaffinity-purified anti-p542 and anti-p554 have relatively high binding affinities, as evidenced by inhibition at 10(6)-10(8) M-1, and neither autoantibody showed polyreactivity with other common antigens. The data thus suggest that neither autoantibody is simply an expression of polyclonal B cell activation. We conclude that the two autoantigens stimulate autoantibody synthesis by different mechanisms. One autoantigen shares homology to a viral protein which generates cross-reacting antibodies to the autoantigenic epitope. The other has no recognizable cross-reaction with the infecting pathogen and may become immunogenic through complexing with other proteins.

Targeted Disruption of the Myocilin Gene (Myoc) Suggests that Human Glaucoma-Causing Mutations Are Gain of Function.

Glaucoma is a heterogeneous eye disease and a major cause of blindness worldwide. Recently, primary open angle glaucoma (POAG)-associated mutations have been found in the trabecular meshwork inducible glucocorticoid response gene (TIGR), also known as the myocilin gene (MYOC), at the GLC1A locus on chromosome 1q21-q31. These mutations occurred in a subset of patients with juvenile- and adult-onset POAG and exhibited autosomal dominant inheritance. Ocular expression and its involvement in POAG suggest that TIGR/MYOC may have a role(s) in regulating intraocular pressure (IOP). Here, we report the generation and analysis of mice heterozygous and homozygous for a targeted null mutation in Myoc. Our study shows that Myoc mutant mice are both viable and fertile. Our in vivo findings further demonstrate that Myoc is not required for normal IOP or normal ocular morphology. The lack of a discernable phenotype in both Myoc-heterozygous and Myoc-null mice suggests that haploinsufficiency is not a critical mechanism for POAG in individuals with mutations in MYOC. Instead, disease-causing mutations in humans likely act by gain of function.

G-protein involvement in matrix-mediated motility and invasion of high and low experimental metastatic B16 melanoma clones.

Membranes from a B16 murine melanoma clone of high experimental metastatic capacity show increased amounts of pertussis toxin (PT) substrate when compared to a low metastatic counterpart. Using specific antibodies, we identified Gi2 as the PT-sensitive G-protein uniquely abundant in highly metastatic cells. ADP ribosylation of a G-protein alpha subunit by PT decreased both the migration of tumor cells through Matrigel (Collaborative Research, Bedford, MA) and the fibronectin-, laminin-, and collagen type IV-mediated motility of a high metastatic clone. Treatment of cells from a low metastatic clone with PT did not alter either the relatively low invasive capacity or lower motility of these cells. While cholera toxin treatment of cells resulted in decreased invasion and motility of both high and low metastatic clones, there were significant qualitative and quantitative differences, when compared to the PT effects, which indicated that the two toxins were acting on different second messenger systems. PT treatment of B16 clones of high or low experimental metastatic capacity does not result in any alteration in cellular cyclic AMP accumulation suggesting that the PT substrate is not linked with the adenylyl cyclase enzyme complex. The data suggest that a PT-sensitive G-protein, possibly Gi2, regulates second messenger pathways that contribute to the metastatic capacity of B16 melanoma cells.

Activity of 2-fluoro-5-methylarabinofuranosyluracil against mouse leukemias sensitive to and resistant to 1-beta-D-arabinofuranosylcytosine.

A new pyrimidine nucleoside, 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil, previously has been shown to be active against the herpes group of viruses in vitro and in vivo. It is also active against mouse and human leukemic cells in culture and against mouse leukemias L1210, P388, and P815 in vivo. In contrast to other 1-beta-D-arabinofuranosylcytosine (ara-C) derivatives, 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil, when given either i.p. or p.o., is highly active against lines of leukemias P815 and L1210 made resistant to ara-C. Against P815/ara-C and L1210/araC, it is more effective than is 5-azacytidine, a drug which has shown definite effectiveness in patients with acute leukemia whose disease has become resistant to ara-C. For these reasons, 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil would seem to merit clinical trial in patients with acute nonlymphocytic leukemia whose disease has become resistant to ara-C.

Autoregulation of the human N-myc oncogene is disrupted in amplified but not single-copy neuroblastoma cell lines.

Amplification of the N-myc gene is a significant adverse prognostic factor in neuroblastoma, a common childhood tumor. In non-transformed cells, myc expression is controlled through an autoregulatory circuit, through which elevated Myc protein levels lead to down-regulation of myc transcription. The precise mechanism of myc gene autoregulation is unknown. Loss of c-myc autoregulation has been documented in transformed cells from a number of different lineages, but N-myc autoregulation has not yet been investigated. In neuroblastoma, the increased N-Myc protein produced by amplified tumors would be expected to silence N-myc transcription if the autoregulatory loop were intact. To determine whether N-myc autoregulation is operative in human neuroblastoma, and to localize cis-acting elements which mediate N-myc autosuppression, we transfected a series of N-myc 5' promoter constructs into a panel of human neuroblastoma cell lines carrying one or multiple copies of N-myc. The transfected promoter was equally active in single-copy and amplified lines. Significant promoter activity in the presence of abundant Myc protein in amplified neuroblastoma lines indicates that autoregulation is disabled in this subset of tumors. To investigate whether single-copy lines produce insufficient N-Myc protein to trigger autosuppression yet retain an intact autoregulatory circuit, we transfected neuroblastoma lines with 5' promoter constructs in the presence of a c- or N-myc expression vector. Overexpression of c- or N-Myc resulted in diminution of activity of both the transfected promoter and the endogenous N-myc gene in single-copy, but not amplified lines. Using a series of 5' promoter-deletion minigenes, we localized a cis-acting element required for autoregulation close to the transcription start sites. While the precise mechanism of autosuppression remains unknown, we demonstrated that Myc is incapable of silencing the adenovirus major late promoter (AdMLP) in neuroblastoma cells, indicating that Myc suppression of its own promoter and the AdMLP involve distinct components. These studies provide the first systematic investigation of autoregulation in neuroblastoma, and indicate that single-copy neuroblastoma lines produce insufficient N-Myc protein to activate downstream effector(s) of autosuppression; the autoregulatory circuit is otherwise intact. Amplified lines, in contrast, have lost autoregulation.

Modification of liver fatty acid metabolism in mice by n-3 and n-6 delta 6-desaturase substrates and products.

The effects of dietary supplementation of either alpha-linolenic acid (18:3(n-3)) or stearidonic acid (18:4(n-3)) in combination with either linoleic acid (18:2(n-6)) or gamma-linolenic acid (18:3(n-6)) on liver fatty acid composition in mice were examined. Essential fatty acid deficient male C57BL/6 mice were separated into four groups of seven each and were fed a fat-free semi-purified diet supplemented with 1% (w/w) fatty acid methyl ester mixture (1:1), 18:2(n-6)/18:3(n-3), 18:2(n-6)/18:4(n-3), 18:3(n-6)/18:3(n-3), or 18:3(n-6)/18:4(n-3). After 7 days on the diets, fatty acid compositions in liver phosphatidylcholine and phosphatidylethanolamine fractions were analyzed. In groups fed 18:4(n-3) (18:2(n-6)/18:4(n-3) or 18:3(n-6)/18:4(n-3)) as compared to those fed 18:3(n-3) (18:2(n-6)/18:3(n-3) or 18:3(n-6)/18:3(n-3)), the levels of 20:4(n-3), 20:5(n-3) and 22:5(n-3) were increased, whereas those of 20:3(n-6) and 20:4(n-6) were decreased. When 18:3(n-6) replaced 18:2(n-6) as the source of n-6 acids, the levels of 18:3(n-6), 20:3(n-6), 20:4(n-6) and 22:5(n-6) were increased, whereas those of 20:4(n-3) and 20:5(n-3) were reduced. Replacing 18:3(n-3) by 18:4(n-3) reduced the (n-6)/(n-3) ratio by approx. 30%, whereas replacing 18:2(n-6) by 18:3(n-6) increased the (n-6)/(n-3) ratio by approx. 2-fold. These findings indicated that delta 6-desaturase products were metabolized more readily than their precursors. Both products also competed for the subsequent metabolic enzymes. However, the n-6 fatty acids derived from 18:3(n-6) were incorporated more favourably into liver phospholipids than n-3 fatty acids derived from 18:4(n-3).

Direct current shocks to the heart generate free radicals: an electron paramagnetic resonance study.

OBJECTIVES: We sought to demonstrate that direct current (DC) shocks to the heart generate free radicals. BACKGROUND: Although it is a lifesaving maneuver, defibrillation is known to have myocardial toxicity. The mechanism of this toxicity is unknown. If DC shocks generate free radicals, free radicals could be a mechanism of myocardial injury. METHODS: In a canine model, DC shocks of 10 to 100 J were delivered to the epicardium of both beating and fibrillating hearts, and 200-J transthoracic shocks were administered in dogs with beating hearts. Ascorbate free radical (AFR) concentration was measured in arterial blood and blood continuously withdrawn from the coronary sinus. In some dogs, the antioxidant enzymes superoxide dismutase (15,000 U/kg) and catalase (55,000 U/kg) (SOD/Cat) were administered before shocks. RESULTS: Ascorbate free radicals were generated by DC shocks. A peak AFR increase of 14 +/- 2% (mean +/- SEM) was seen 5 to 6 min after 100-J epicardial shocks. A peak AFR increase of 7 +/- 5% occurred after transthoracic shocks. There was a significant linear relation between the shock energy and peak percent AFR increase: %AFR increase = 0.18 (Shock energy) + 2.9 (r = 0.73, p < 0.0001). Shocks delivered to hearts in ventricular fibrillation (30 s) resulted in generation of AFR equal to but not greater than that observed during similar shocks delivered to beating hearts in sinus rhythm. Multiple successive shocks (100 J delivered twice or five times) did not result in a greater AFR increase than single 100-J shocks, indicating that peak, not cumulative, energy is the principal determinant of AFR increase. Animals receiving SOD/Cat before shock administration showed significant attenuation of the AFR increase. CONCLUSIONS: Direct current epicardial and transthoracic shocks generate free radicals; antioxidant enzymes reduce the free radical generation by shocks.

Magnesium reduces free radicals in an in vivo coronary occlusion-reperfusion model.

OBJECTIVE: This study demonstrated that magnesium (Mg) reduces free radicals after a brief coronary occlusion-reperfusion sequence. BACKGROUND: Magnesium has been shown to reduce infarct size in patients with acute myocardial infarction. We hypothesized that this action of Mg occurs through its action on free radicals. METHODS: Eighteen mongrel dogs were studied (nine control, nine receiving Mg). Catheters were placed into the coronary sinus for continuous blood withdrawal. A Varian E-4 electron paramagnetic resonance spectrometer was used to monitor the ascorbate free radical (AFR) signal in the coronary sinus blood; AFR is a measure of total oxidative stress. Occlusion of the left anterior descending coronary artery for 20 min was followed by reperfusion. The study animals received 4 g Mg intravenously starting at 15 min of occlusion (5 min before reperfusion) and continuing during reperfusion. RESULTS: Results are presented as percent change from baseline +/- SEM. Magnesium blunted the peak AFR increase: at 4 min of reperfusion there was a 4.7 +/- 3.3% increase in AFR signal in the dogs receiving Mg versus an 18.2 +/- 3.3% increase in the control animals (p < 0.05). Total radical flux was reduced during reperfusion by 53% in the Mg dogs compared with controls (p < 0.05). CONCLUSIONS: Magnesium attenuates AFR increase after an occlusion-reperfusion sequence. To our knowledge this is the first in vivo real-time demonstration of Mg's impact on free radicals.

Effect of acute oral beta adrenergic blockade on muscle blood flow in man.

We have determined the effect of beta blocking drugs in man to ascertain whether cardioselectivity or intrinsic sympathomimetic activity influence muscle blood flow response to exercise. Exercise muscle blood flow was determined using a miniaturised lightweight cadmium telluride detector attached to the skin surface. Mean exercise muscle blood flow for the six subjects, during a constant work load, did not differ significantly either for predosing values on the six separate study days or at 2, 4 and 6 h after dosing with placebo. All the beta blocking agents caused a reduction in exercise muscle blood flow. Propranolol caused significant reduction in muscle blood flow at 2 h only. Metoprolol and atenolol produced the greatest and most prolonged effect and oxprenolol the smallest effect on exercise muscle blood flow. Intrinsic sympathomimetic activity may protect against reduction in exercise muscle blood flow in man.

Assessment of cadmium telluride detector system for measurement of muscle blood flow during dynamic exercise in man.

We have used a miniature cadmium telluride detector in the assessment of muscle blood flow during dynamic exercise in man following local injection of 133Xenon. The results obtained were compared with those using a conventional sodium iodide detector. There was no significant difference between the results obtained with the two detectors. The reproducibility of results was greater with cadmium telluride than with sodium iodide. The cadmium telluride system has the advantage of being small, lightweight and portable and enables measurements to be made during dynamic exercise.

The mouse anterior chamber angle and trabecular meshwork develop without cell death.

BACKGROUND: The iridocorneal angle forms in the mammalian eye from undifferentiated mesenchyme between the root of the iris and cornea. A major component is the trabecular meshwork, consisting of extracellular matrix organized into a network of beams, covered in trabecular endothelial cells. Between the beams, channels lead to Schlemm's canal for the drainage of aqueous humor from the eye into the blood stream. Abnormal development of the iridocorneal angle that interferes with ocular fluid drainage can lead to glaucoma in humans. Little is known about the precise mechanisms underlying angle development. There are two main hypotheses. The first proposes that morphogenesis involves mainly cell differentiation, matrix deposition and assembly of the originally continuous mesenchymal mass into beams, channels and Schlemm's canal. The second, based primarily on rat studies, proposes that cell death and macrophages play an important role in forming channels and beams. Mice provide a potentially useful model to understand the origin and development of angle structures and how defective development leads to glaucoma. Few studies have assessed the normal structure and development of the mouse angle. We used light and electron microscopy and a cell death assay to define the sequence of events underlying formation of the angle structures in mice. RESULTS: The mouse angle structures and developmental sequence are similar to those in humans. Cell death was not detectable during the period of trabecular channel and beam formation. CONCLUSIONS: These results support morphogenic mechanisms involving organization of cellular and extracellular matrix components without cell death or atrophy.

The auditory N2 component in schizophrenia: relationship to MRI temporal lobe gray matter and to other ERP abnormalities.

The N2 component of the auditory event-related potential (ERP) indexes cognitive processes involved in the categorization of deviant stimuli. Although N2 amplitude and latency abnormalities have been reported in schizophrenia, their relationship to MRI structural changes, clinical status, and P3 abnormalities has not been defined. We therefore studied the auditory N2 and P3 components elicited by an oddball paradigm in 15 right-handed male subjects with schizophrenia and 14 control subjects who had quantitative MRI measures of temporal lobe gray-matter structures. To provide a methodological comparison, we measured the auditory N2 from both the target ERP (N2t) and the target-minus-frequent ERP difference (N2d) waveforms. Both N2t and N2d amplitude were bilaterally reduced in schizophrenics, with N2d showing a more pronounced reduction. Within the schizophrenic group, N2 amplitude reduction was associated with reduction in gray-matter volume of the left superior temporal gyrus (STG) and of medial temporal lobe structures bilaterally, and clinically, with greater chronicity. P3 amplitude, in contrast, correlated only with left posterior STG volume, and was more prominently associated with delusions and thought disorder. These findings suggest that the N2 and P3 components, though occurring sequentially in the ERP, tap separable anatomic and behavioral abnormalities in schizophrenia.