Target-directed discovery and production of pharmaceuticals in transgenic mutant plant cells.

Plants are a source of complex bioactive compounds, with value as pharmaceuticals, or leads for synthetic modification. Many of these secondary metabolites have evolved as defenses against competing organisms and their pharmaceutical value is "accidental", resulting from homology between target proteins in these competitors, and human molecular therapeutic targets. Here we show that it is possible to use mutation and selection of plant cells to re-direct their "evolution" toward metabolites that interact with the therapeutic target proteins themselves. This is achieved by expressing the human target protein in plant cells, and selecting mutants for survival based on the interaction of their metabolome with this target. This report describes the successful evolution of hairy root cultures of a Lobelia species toward increased biosynthesis of metabolites that inhibit the human dopamine transporter protein. Many of the resulting selected mutants are overproducing the active metabolite found in the wild-type plant, but others overproduce active metabolites that are not readily detectable in non-mutants. This technology can access the whole genomic capability of a plant species to biosynthesize metabolites with a specific target. It has potential value as a novel platform for plant drug discovery and production, or as a means of optimizing the therapeutic value of medicinal plant extracts.

Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

OBJECTIVE: Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS: The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS: We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Mortality in dry-cleaning workers: an update.

BACKGROUND: A cohort of 1,708 dry-cleaning workers identified from union records was exposed to perchloroethylene (PCE), a known animal carcinogen and probable human carcinogen, for at least 1 year before 1960. Many workers also had exposure to Stoddard solvent, a petroleum-based dry-cleaning solvent. METHODS: Vital status was updated through 1996 and life table analyses conducted. RESULTS: The cohort had excess cancer mortality (271 deaths, standardized mortality ratio [SMR] 1.25, 95% confidence interval [CI] 1.11-1.41). Elevated SMRs for tongue, bladder, esophagus, intestine, lung, and cervical cancer, pneumonia, and diseases of the stomach and duodenum were statistically significant. CONCLUSION: The current study confirms findings of prior updates and other studies that dry-cleaning workers have excess cancer mortality at several sites. Although important lifestyle and socioeconomic risk factors exist for both cervical and esophageal cancer mortality, excesses of these sites in the PCE only subcohort and among workers with longer duration of PCE exposure suggest an association with PCE exposure.

Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia.

The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1. 4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.

Crystallization of the regulatory and effector domains of the key sporulation response regulator Spo0A.

The key response-regulator gene of sporulation, spo0A, has been cloned from Bacillus stearothermophilus and the encoded protein purified. The DNA-binding and phospho-acceptor domains of Spo0A have been prepared by tryptic digestion of the intact protein and subsequently crystallized in forms suitable for X-ray crystallographic studies. The DNA-binding domain has been crystallized in two forms, one of which diffracts X-rays to beyond 2. 5 A spacing. The crystals of the phospho-acceptor domain diffract X-rays beyond 2.0 A spacing using synchrotron radiation.

Expression and immunolocalisation of the snowdrop lectin, GNA in transgenic rice plants.

Transgenic rice (Oryza sativa L.) plants generated through particle bombardment expressed high levels of an insecticidal protein (the snowdrop lectin, GNA) directed against sap-sucking insects. Engineered plants expressed GNA either constitutively or in a tissue specific manner, depending on the nature of the promoter used to drive expression of the gene. We used specific antibodies raised against GNA to localize its expression in phloem tissue in plants engineered with the rice sucrose synthase promoter driving GNA expression. We report here molecular, biochemical and immunological analyses for fifteen independently-derived transformants out of more than 200 plants we generated.

Microbiological and chemical quality of raw milk in New York State.

Between December 1993 and January 1996, samples of raw milk from bulk tanks were collected by licensed milk haulers at the time of pick-up from 855 randomly selected farms in New York State (representing approximately 10% of all dairy farms in the state). The milk was examined for microbial and chemical qualities. Bacterial numbers were determined by standard plate count, laboratory-pasteurized count, coliform count, heat-resistant spore-forming psychrotroph count, aerobic spore count (mesophilic), rapid psychrotrophic count, and preliminary incubation count. The frequency distributions for these counts are presented. Paired correlation analyses between the microbiological parameters showed low correlations between test results; no correlation coefficients were > 0.8. The four highest positive correlation coefficients were found between standard plate count and rapid psychrotrophic count (0.7685), rapid psychrotrophic count and preliminary incubation count (0.6648), standard plate count and preliminary incubation count (0.5800), and aerobic spore count and laboratory-pasteurized count (0.5393). All other correlation coefficients were < 0.5. Milk freezing points and acid degree values were determined for all samples. Frequency distributions for these results are also presented.

Carbon-source regulation of virulence gene expression in Listeria monocytogenes.

All known virulence genes of Listeria monocytogenes are under positive regulation by the transcription factor PrfA. Previous work employing the L. monocytogenes strain NCTC7973 suggested that the disaccharide cellobiose might serve as a specific "signature molecule' which functions to prevent activation of the PrfA-controlled regulon in a soil environment. We have examined three other L. monocytogenes strains, 10403S, LO28 and EGD, all commonly regarded as wild-type isolates, and find that NCTC7973 is anomalous with respect to the effect of carbohydrates on the expression of PrfA-controlled gene expression. In the case of 10403S, LO28 and EGD, several other readily metabolized mono- and disaccharides are as effective as cellobiose in repressing expression of the PrfA-controlled gene hly, indicating that the cellobiose effect is not specific, and suggesting that NCTC7973 may be a partially deregulated variant. Moreover, concentrations of cellobiose and other sugars required for repression of hly expression (> 1 mM) were found to significantly enhance growth of L. monocytogenes cultures, suggesting that the repression phenomenon probably results from a metabolic effect of sugar utilization rather than a signal-sensing response. Thus the previously reported cellobiose effect may reflect an aspect of a more global mechanism of catabolite repression in L. monocytogenes. Although cellobiose represses expression of hly and plcA at the level of transcript accumulation, quantitative Western blot analysis indicates that cellobiose has no effect on PrfA levels. These results are consistent with a model in which PrfA activity is controlled by interaction with a hypothetical cofactor, the synthesis or depletion of which is responsive to the presence of readily metabolized carbohydrates.

Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements.

New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21delta2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn9l7, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpermissive temperature (45 degrees C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21delta2TetM, a tetracycline-resistant and ampicillin-sensitive Tn9l7-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp.fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn9l7 mutagenesis in the genus Streptococcus.

Structure and function of the Bacillus SpoIIE protein and its localization to sites of sporulation septum assembly.

Functioning of the spoIIE locus of Bacillus subtilis is required for formation of a normal polar septum during sporulation and for activation of the transcription factor sigma F, which directs early forespore-specific gene expression. We have determined the DNA sequence of the wild type and several mutant alleles of the spoIIE gene of B. subtilis and sequenced a substantial portion of its presumptive homologue in Bacillus megaterium. We show that the spoIIE locus encodes a single large protein with a predicted molecular mass of 92 kDa. Each of five point-mutation alleles, which have traditionally defined the locus, and two transposon-generated mutations were shown to fall within the coding sequence for the 92 kDa gene product or within sequences expected to be required for its expression. The amino-terminal portion of the predicted SpoIIE gene product, comprising approximately 40% of the protein, is extremely hydrophobic and is expected to contain up to 12 membrane-spanning segments. The remainder of the protein contains no hydrophobic segments long enough to span a lipid bilayer and is therefore presumed to comprise one or more globular, aqueous-phase exposed domains. An in-frame fusion joining the 3' end of the B. megaterium spoIIE coding sequence to the 5' end of gfp, a gene encoding the green fluorescent protein (GFP) of Aquorea victoria, resulted in a strong, sporulation-specific fluorescent signal localized to the sites of sporulation septum assembly. We speculate that SpoIIE plays a role in assembling the sporulation septum, perhaps determining the special properties of the structure that permit intercompartment signalling during development.

Characterization of the macromolecular synthesis (MMS) operon from Listeria monocytogenes.

The macromolecular synthesis (MMS) operon consists of three genes: rpsU, which encodes the S21 ribosomal protein in Bacillus subtilis (Bs), rpsU is replaced by orfP23 which encodes a protein of unknown function), dnaG, encoding the DNA primase involved in the initiation of chromosome replication, and rpoD, which encodes the principal sigma subunit of RNA polymerase. The operon was cloned in three segments from Listeria monocytogenes (Lm), initially using a probe designed from a highly conserved region of RpoD. Analysis of the nucleotide sequence revealed three genes: orfP17 (whose product, P17, is homologous to Bs P23), dnaG and rpoD. The Lm DnaG resembles the primase from Escherichia coli through the first two-thirds of the sequence. C-terminal similarity was observed between DnaG from Lm and Bs. Lm RpoD is similar to Bs SigA, shares identical DNA-binding domains with SigA, and is a member of the sigma 43 subgroup of the sigma 70 family.

Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a probable DNA-binding domain.

Spo0A is a phosphorylation-activated transcription factor of Bacillus subtilis. It is a member of the response regulator superfamily of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of sporulation. To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera. An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain. We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix-turn-helix motif, and, therefore, represents the actual DNA-containing surface of the protein. In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene-disruption analysis that the homologue actually is required for initiation of sporulation. Apparent homologues of the B. subtilis spoIVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined. The discovery of homologues of B. subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation-specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences. Exhaustive efforts to detect a spo0A-like gene in non-endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A-like proteins may be exclusive to the endospore-forming bacteria.

Follow-up study of chrysotile asbestos textile workers: cohort mortality and case-control analyses.

Previous studies of mortality among white males employed in a Charleston, South Carolina asbestos textile plant using chrysotile demonstrated significant excess mortality due to asbestos-related disease and a steep exposure-response relationship for lung cancer. This cohort was further studied by adding 15 years of follow-up and including mortality among white female and black male workers. Nested case-control analyses were undertaken to further explore possible differences in lung cancer risk by textile operation as well as possible confounding by mineral oil exposures. Preliminary data for white males have been previously published. White males experienced statistically significant excess mortality due to lung cancer (standardized mortality ratio [SMR] = 2.30; confidence interval [CI] = 1.88-2.79), all causes (SMR = 1.48; CI = 1.38-158), all cancers (SMR = 1.50; CI = 1.29-1.72), diabetes mellitus (SMR = 2.05; CI = 1.18-3.33), heart disease (SMR = 1.41; CI = 1.26-1.58), cerebrovascular disease (SMR = 1.50; CI = 1.08-2.02), pneumoconiosis and other respiratory diseases (SMR = 4.10; CI = 3.10-5.31), and accidents (SMR = 1.49; CI = 1.15-1.91). Among white females, statistically significant excesses occurred for lung cancer (SMR = 2.75; CI = 2.06-3.61), all causes (SMR = 1.21; CI = 1.11-1.32), pneumoconiosis and other respiratory diseases (SMR = 2.40; CI = 1.53-3.60), and other respiratory cancers (SMR = 14.98; CI = 4.08-38.7). Among the total cohort of black males, the only statistically significant excess observed was for pneumoconiosis (SMR = 2.19; CI = 1.23-3.62). Based on historical exposure measurements at the plant, there was a positive exposure-response relationship for both lung cancer and pneumoconiosis. Data for the entire cohort demonstrate an increase in the lung cancer relative risk of 2-3% for each fiber/cc-year of cumulative chrysotile exposure. This relationship was more consistent for the white male workers. The excess risk for lung cancer among white males and females appeared to occur at cumulative exposures lower than those for black males. Possible reasons for the lesser lung cancer risk among black males include less smoking and differences in airborne fiber characteristics experienced by black males as a result of plant job placement patterns. The case-control analysis found employment in preparation and carding operations (where most of the black males worked) to be associated with a slightly reduced lung cancer risk, although not statistically significant, whereas spinning and twisting employment was associated with a statistically significant increased lung cancer risk compared to other plant operations.(ABSTRACT TRUNCATED AT 400 WORDS)

Cancer mortality in female and male dry-cleaning workers.

A cohort study of dry-cleaning workers (1109 women, 592 men) in the mid-1980s revealed significant excess bladder cancer mortality. This article updates vital status through 1990. Significant excesses were seen for bladder cancer (nine deaths, standardized mortality ratio [SMR] = 2.54, 95% confidence interval [CI] = 1.16-4.82), esophageal cancer (10 deaths, SMR = 2.14, 95% CI = 1.02-3.94), and intestinal cancer (26 deaths, SMR = 1.56, 95% CI = 1.02-2.29). In a subcohort exposed only to perchloroethylene (PCE), those with 5 or more years of employment and 20 or more years since first exposure had a significant increased risk of esophageal cancer (four deaths, SMR = 7.17, 95% CI = 1.92-19.82). Women had significant excess esophageal cancer (five deaths, SMR = 3.24, 95% CI = 1.05-7.58) and elevated SMRs for intestinal, pancreatic, and bladder cancer mortality. This study confirms the esophageal cancer risk among dry-cleaning workers seen in another study and suggests an association with PCE. It further documents the risks for intestinal, pancreatic, and bladder cancers in this industry.

Mortality patterns among female and male chrysotile asbestos textile workers.

This study updates a retrospective cohort mortality analysis of workers from a South Carolina textile plant where chrysotile asbestos was the primary exposure. The update adds 15 years of observation to the original study, adds analyses of white women and black men, and allows comparison of mortality risks between race/gender groups. The total cohort includes 3,022 workers: 1,229 white women (363 deaths), 1,247 white men (607 deaths), and 546 black men (289 deaths). Statistically significant risks for lung cancer were observed among white women (standardized mortality ratio [SMR] = 2.07; 90% confidence interval [CI] = 1.55-2.71) and white men (SMR = 2.24; 90% CI = 1.83-2.72); both of these groups exhibited positive exposure-response trends. Although the lung cancer risk among black men was lower than expected (SMR = 0.70; 90% CI = 0.42-1.08), a statistically significant increase was observed at high levels of exposure. Statistically significant excess risk for pneumoconiosis and other respiratory diseases were observed for all race/gender groups. Despite the relatively high percentage of white women lost to follow-up and missing death certificates, both of which allow underestimation of the true relative risk, statistically significant excess risks were observed for lung cancer and pneumoconiosis among this group.

Lung cancer mortality among asbestos textile workers: a review and update.

In an update of the mortality of the cohort of 1200 South Carolina textile workers, of whom almost half died, there were 185 excess deaths (SMR = 1.44), which included 71 cardiovascular diseases (SMR = 1.37), 43 non-malignant respiratory diseases (SMR = 2.25) and 41 lung cancers (SMR = 2.25). Only two definite mesotheliomas were observed. Other possible cases may have occurred but no confirmatory pathology was available. Strong exposure-response relationships have been found for lung cancer and for non-malignant respiratory diseases. The data suggest a doubling of the lung cancer risk at an exposure of approximately 30 fibre years. Mortality from pneumoconiosis and other respiratory diseases was elevated at even the lowest cumulative exposure category (< 2 f ml-1 years). A nested case-control analysis failed to demonstrate a significant role for mineral oil exposure in the etiology of lung cancer. Differences in airborne fibre sizes may be important in explaining different lung cancer and pneumoconiosis risks in various industries. In particular, the data on airborne fibres in textile manufacturing industries suggested 11-27% were longer than 5 microns compared to 2-5% for mining and milling.

Characterization of the genes and attachment sites for site-specific integration of plasmid pSE101 in Saccharopolyspora erythraea and Streptomyces lividans.

The 11.3 kb plasmid pSE101 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site and into the chromosome of Streptomyces lividans at many sites. Multisite integration in S. lividans was also observed when a 1.9 kb segment of pSE101 containing attP and adjacent plasmid sequence was used to transform a pSE101- S. lividans host. Nucleotide sequencing of this segment revealed the presence of a complete open reading frame (ORF) designated int, encoding a putative polypeptide of 448 amino acids that shows similarities to site-specific recombinases of the integrase family. Sequencing of the 1.3 kb segment upstream of int revealed the presence of three additional ORFs: the one most distal to int encodes a putative 76 amino acid basic polypeptide analogous to the Xis proteins of a number of bacteriophages. Nucleotide sequencing of attP, and the attB, attL and attR sites from Sac. erythraea revealed a 46 bp sequence common to all sites with no duplications of chromosomal sequences in the integrated state. A putative structural gene for a tRNA(Thr) was found to overlap the 46 bp common sequence at attB. Sequencing of four pSE101 integration sites (attB') and corresponding attL' and attR' sites in S. lividans showed that the 46 bp sequence was present at each attR' site, whereas only the first three bases, CTT, were retained at each attL' and attB' site. A feature common to the four attB' sites and to attB is a highly conserved 21 bp segment with inverted repeats flanking the CTT sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Speech recognition in recurrent otitis media: results in a set of identical twins.

Performance-intensity functions for Pediatric Speech Intelligibility Test (PSI) word and sentence materials presented in competition were obtained for a set of identical female twins, aged 4 years, 2 months. Twin B had a medical history significant for recurrent otitis media, while Twin A had only one reported occurrence of the disease. Twin A yielded normal functions for both word and sentence materials. Twin B, however, yielded an essentially normal function for sentences, but the function for monosyllabic words was grossly depressed. These findings support the hypothesis that transient conductive hearing loss and auditory deprivation resulting from recurrent otitis media can affect the processing of phonetic information essential to the development of word recognition skills.

Auditory P300 abnormalities and leukocyte activation in HIV infection.

To evaluate whether P300 testing might serve as a screening modality for the early detection of HIV-related neuropathology, we tested 26 HIV-infected men (23 without neurologic symptoms, 2 with peripheral neuropathy, 1 with AIDS-associated dementia) and 15 controls. Although they had no overt neurologic symptoms, the P300 latency was delayed or undetectable in 30% of patients without clinically evident neurologic disease. P300 latencies did not correlate with peripheral blood CD4 T-cell count, serum quinolinic acid or p24 antigen levels, or the numbers of activated peripheral blood monocytes. Three individuals with abnormal P300 latencies had been HIV-seropositive for < or = 1 year, suggesting that delayed evoked responses detect early neurologic dysfunction. P300 responses do not predict imminent dementia. In only one previously asymptomatic individual with abnormal P300 waveforms have overt neurologic symptoms developed during a 2-year followup. Extended longitudinal studies will be necessary to define the predictive value of P300 latencies in the development of AIDS-related dementia. However, the sensitivity, quantitative nature, and speed of administration of this test suggest that it may be useful for identification of early neurologic involvement in HIV infection.