Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

Membrane organization of photosystem I complexes in the most abundant phototroph on Earth.

Prochlorococcus is a major contributor to primary production, and globally the most abundant photosynthetic genus of picocyanobacteria because it can adapt to highly stratified low-nutrient conditions that are characteristic of the surface ocean. Here, we examine the structural adaptations of the photosynthetic thylakoid membrane that enable different Prochlorococcus ecotypes to occupy high-light, low-light and nutrient-poor ecological niches. We used atomic force microscopy to image the different photosystem I (PSI) membrane architectures of the MED4 (high-light) Prochlorococcus ecotype grown under high-light and low-light conditions in addition to the MIT9313 (low-light) and SS120 (low-light) Prochlorococcus ecotypes grown under low-light conditions. Mass spectrometry quantified the relative abundance of PSI, photosystem II (PSII) and cytochrome b6f complexes and the various Pcb proteins in the thylakoid membrane. Atomic force microscopy topographs and structural modelling revealed a series of specialized PSI configurations, each adapted to the environmental niche occupied by a particular ecotype. MED4 PSI domains were loosely packed in the thylakoid membrane, whereas PSI in the low-light MIT9313 is organized into a tightly packed pseudo-hexagonal lattice that maximizes harvesting and trapping of light. There are approximately equal levels of PSI and PSII in MED4 and MIT9313, but nearly twofold more PSII than PSI in SS120, which also has a lower content of cytochrome b6f complexes. SS120 has a different tactic to cope with low-light levels, and SS120 thylakoids contained hundreds of closely packed Pcb-PSI supercomplexes that economize on the extra iron and nitrogen required to assemble PSI-only domains. Thus, the abundance and widespread distribution of Prochlorococcus reflect the strategies that various ecotypes employ for adapting to limitations in light and nutrient levels.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity.

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with TAQ polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.

Repression of protein kinase C and stimulation of cyclic AMP response elements by fumonisin, a fungal encoded toxin which is a carcinogen.

Fusarium moniliforme (FM) is a major fungal pathogen of corn and is involved with stalk rot disease. FM is widely spread throughout the world, including the United States. Most strains of FM produce several mycotoxins, the most prominent of which is called fumonisin. Recent epidemiological studies indicated that ingestion of fumonisin correlates with a higher incidence of esophageal cancer in Southern and Northern Africa and China. Furthermore, fumonisin causes a neurodegenerative disease in horses, induces hepatic cancer in rats, and induces pulmonary edema in swine. Considering that high levels of fumonisin have been detected in healthy and diseased corn grown in the United States, fumonisin may pose a health threat to humans and livestock animals. Structurally, fumonisin resembles sphingolipids which are present in the membranes of animal and plant cells. At the present time, very little is known concerning the mechanism by which fumonisin elicits its carcinogenic effect. Our studies indicate that fumonisin represses expression of protein kinase C and AP-1-dependent transcription. In contrast, fumonisin stimulated a simple promoter containing a single cyclic AMP response element. Since fumonisin did not alter protein kinase A activity, it appears that cyclic AMP response element activation was independent of protein kinase A. It is hypothesized that the ability of fumonisin to alter signal transduction pathways plays a role in carcinogenesis.

Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases.

Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.HhaI and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine.

Colletotrichum trifolii mutants disrupted in the catalytic subunit of cAMP-dependent protein kinase are nonpathogenic.

Colletotrichum trifolii is the fungal pathogen of alfalfa that causes anthracnose disease. For successful plant infection, this fungus must undergo a series of morphological transitions following conidial attachment, including germination and subsequent differentiation, resulting in appressorium formation. Our previous studies with pharmacological effectors of signaling pathways have suggested the involvement of cyclic AMP (cAMP)-dependent protein kinase (PKA) during these processes. To more precisely evaluate the role of PKA in C. trifolii morphogenesis, the gene encoding the catalytic (C) subunit of PKA (Ct-PKAC) was isolated, sequenced, and inactivated by gene replacement. Southern blot analysis with C. trifolii genomic DNA suggested that Ct-PKAC is a single-copy gene. Northern (RNA) blot analysis with total RNA from different fungal growth stages indicated that the expression of this gene was developmentally regulated. When Ct-PKAC was insertionally inactivated by gene replacement, the transformants showed a small reduction in growth relative to the wild type and conidiation patterns were altered. Importantly, PKA-deficient strains were unable to infect intact alfalfa (host) plants, though only a slight delay was observed in the timing for conidial germination and appressorial formation in the Ct-PKAC disruption mutants. Moreover, these mutants were able to colonize host tissues following artificial wounding, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggest that the defect in pathogenicity is likely due to a failure in penetration. Our results demonstrate that PKA has an important role in regulating the transition between vegetative growth and conidiation, and is essential for pathogenic development in C. trifolii.

Fungal biology and agriculture: revisiting the field.

Plant pathology has made significant progress over the years, a process that involved overcoming a variety of conceptual and technological hurdles. Descriptive mycology and the advent of chemical plant-disease management have been followed by biochemical and physiological studies of fungi and their hosts. The later establishment of biochemical genetics along with the introduction of DNA-mediated transformation have set the stage for dissection of gene function and advances in our understanding of fungal cell biology and plant-fungus interactions. Currently, with the advent of high-throughput technologies, we have the capacity to acquire vast data sets that have direct relevance to the numerous subdisciplines within fungal biology and pathology. These data provide unique opportunities for basic research and for engineering solutions to important agricultural problems. However, we also are faced with the challenge of data organization and mining to analyze the relationships between fungal and plant genomes and to elucidate the physiological function of pertinent DNA sequences. We present our perspective of fungal biology and agriculture, including administrative and political challenges to plant protection research.

A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation.

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.

Isolation and characterization of a beta-tubulin-encoding gene from Colletotrichum gloeosporioides f. sp. aeschynomene.

Colletotrichum gloeosporioides f. sp. aeschynomene (C.g.a.) is a fungal pathogen of legumes and is used as a commercial mycoherbicide for rice and soybeans. As an initial study to potentially improve the utility of this fungus and develop a gene transfer system, a beta-tubulin (beta Tub)-encoding gene (TUB1) was isolated, cloned and sequenced. The coding sequence and deduced amino acid sequence of the C.g.a. TUB1 gene was highly homologous to the TUB1 gene of Colletotrichum graminicola. Southern hybridizations, using the C.g.a. TUB1 and C. graminicola TUB2 genes as probes, suggest that C.g.a. contains two TUB genes. Variation in both the restriction pattern and the number of TUB genes present in different formae specialis of C. gloeosporioides was evident. These observations are relevant for assessing relationships among formae specialis of C. gloeosporioides.

von Economo encephalitis.

In April 1917, Dr Constantin von Economo presented his clinical and pathologic findings of a new disease--soon to be part of a worldwide epidemic--before the Vienna Psychiatric Society. He named it encephalitis lethargica. After years of careful observation, he collected and analyzed thousands of cases and classified them into 3 clinical syndromes: somnolent-ophthalmoplegic, hyperkinetic, and amyostatic-akinetic forms. He described the now legendary postencephalitic Parkinsonism, noting that symptoms could emerge years after the original infection, often without signs of prodromal "flu." He emphasized the neuropathologic findings: inflammatory changes in the tegmentum of the midbrain accounting for the sleep disturbance and ocular signs. After encountering sporadic cases following the epidemic, he concluded that the somnolent-ophthalmoplegic syndrome was the primary expression of encephalitis lethargica. This article outlines the observations and conclusions of Dr von Economo during and after the epidemic through seminal quotations primarily from his published works, as well as from more recent reports.

Differential localization of dopamine D1 and D2 receptors in rat retina.

Dopamine interacts with distinct receptors on different target neurons in the rat retina. Dopamine receptors were labeled in the rat retina by autoradiography using 3H-SCH-23390 and 3H-spiperone binding to retinal sections. The 3H-SCH-23390 binding to D1 receptors was most concentrated in the inner plexiform, inner nuclear, and ganglion cell layers; it was absent from the outer nuclear layer and the photoreceptor inner and outer segments. Competition studies indicated that 3H-SCH-23390 binding to the inner retina was inhibited with high affinities by the D1-specific agonist and antagonist, SKF-38393 and SCH-23390. The D2-specific compounds were ineffective in competing for 3H-SCH-23390 binding. The 3H-spiperone binding to D2 receptors, however, was most concentrated in the photoreceptor inner and outer segments and in the outer nuclear layer. The D2-specific agonist and antagonists, such as quinpirole, sulpiride, and eticlopride, competed for 3H-binding with high affinities; SCH-23390 and SKF-38393 were ineffective. The D2 receptors on the photoreceptors had a high affinity for clozapine but lower affinities for the modified benzamides. This is characteristic of a novel subtype of D2 receptors. Thus, D1 and D2 dopamine receptors are localized differentially in the rat retina to mediate different physiologic effects of dopamine.

Analysis of fumonisin B1-induced apoptosis.

Fumonisins are mycotoxins produced by Fusarium moniliforme, a prevalent fungus that infects corn and other cereal grains. Fumonisin B1(FB1 is the most common mycotoxin produced by F. moniliforme, suggesting it has toxicologic significance. The structure of FB1 resembles sphingoid bases, and it inhibits ceramide synthase. Because sphingoid bases regulate cell growth, differentiation, transformation, and apoptosis, it is not surprising to find that FB1 can alter growth of certain mammalian cells. Previous studies concluded FB1-induced apoptosis, or cell cycle arrest, in African green monkey kidney fibroblasts (CV-1). In this study we have identified genes that inhibit FB1 induced apoptosis in CV-1 cells and two mouse embryo fibroblasts (MEF). A baculovirus gene, inhibitor of apoptosis (CpIAP), protected these cells from apoptosis. CpIAP blocks apoptosis induced by the tumor necrosis factor (TNF) pathway as well as other mechanisms. Further support for the involvement of the TNF signal transduction pathway in FB1 induced apoptosis was the cleavage of caspase 8. Inhibition of caspases by the baculovirus gene (italic)p35 also inhibited FB1-induced apoptosis. The tumor suppressor gene p53 was not required for FB1 induced apoptosis because p53-/- MEF undergo apoptosis following FB1 treatment. Furthermore, Bcl-2 was not an effective inhibitor of FB1-induced apoptosis in CV-1 cells or p53+/+ MEF. In summary, these results provide new information to help understand the mechanism by which FB1 induces apoptosis.

Antisense expression of the calmodulin gene from Colletotrichum trifolii impairs prepenetration development(1).

Calmodulin is a ubiquitous highly conserved calcium binding protein involved in cell signalling. Previous studies in our laboratory suggested a role for calmodulin in prepenetration morphogenesis in Colletotrichum trifolii, the causal agent of alfalfa anthracnose. In this report, we describe the cloning, sequencing and partial characterization of the calmodulin gene from C. trifolii. The gene is present as a single copy in the genome of C. trifolii and its predicted amino acid sequence shows considerable homology to other fungal calmodulins. The gene is most highly expressed during conidial germination and appressorial development. Using a Neurospora crassa inducible promoter driving the calmodulin gene in antisense orientation, transformants were obtained with constitutive levels of antisense calmodulin expression. Upon induction, transformants did not develop appressoria and were not pathogenic on alfalfa plants.

Identification of differentially expressed genes following treatment of monkey kidney cells with the mycotoxin fumonisin B(1).

Fumonisin B(1) (FB(1)) is a mycotoxin produced by the phytopathogenic fungus Fusarium moniliforme, which structurally resembles sphingoid bases. FB(1) perturbs sphingolipid synthesis by inhibiting the activity of ceramide synthase. Depending on the host, ingestion of FB(1) causes equine leukoencephalomalacia or porcine pulmonary edema. It is also carcinogenic to rats and may play a role in certain human cancers. Previous studies showed that FB(1) repressed specific isoforms of protein kinase C and cyclin-dependent kinase 2 (CDK2) activity. Conversely, FB(1) induced expression of CDK inhibitors, p21(Waf1/Cip1), p27(Kip1), and p57(Kip2) in monkey kidney cells (CV-1). Consequently, FB(1) treatment of CV-1 cells leads to cell-cycle arrest and apoptosis. The baculovirus IAP gene (inhibitor of apoptosis), which blocks tumor necrosis factor (TNF)-induced apoptosis, protects several fibroblast cell types from apoptosis, suggesting the TNF pathway is important for FB(1)-induced apoptosis. To identify genes that are induced by FB(1), we used a PCR-based subtraction approach. Eight genes that showed high similarity (> 90%) to known mammalian genes were identified. These genes included: tumor necrosis factor type 1 receptor associated protein 2 (TRAP2), human leukemia virus receptor (GLVR1), human Scaffold attachment factor A (SAF-A) also called heterogeneous nuclear ribonucleoprotein U (hnRNP-U), human protein kinase C-binding protein (RACK7), human oligosaccharyl transferase STT3 subunit, mouse WW-domain binding protein 2 (WBP2), human fibronectin, and an unknown human clone. The ability of FB(1) to alter gene expression and signal transduction pathways may be necessary for its carcinogenic and toxic effects.

Hong Kong male subfertility links to mercury in human hair and fish.

The focus of the present study was on the relationship between Hong Kong male subfertility and fish consumption. Mercury concentrations found in the hair of 159 Hong Kong males aged 25-72 (mean age = 37 years) was positively correlated with age and was significantly higher in Hong Kong subjects than in European and Finnish subjects (1.2 and 2.1 ppm, respectively). Mercury in the hair of 117 subfertile Hong Kong males (4.5 ppm, P < 0.05) was significantly higher than mercury levels found in hair collected from 42 fertile Hong Kong males (3.9 ppm). Subfertile males had approx. 40% more mercury in their hair than fertile males of similar age. Although there were only 35 female subjects, they had significantly lower levels of hair mercury than males in similar age groups. Overall, males had mercury levels that were 60% higher than females. Hair samples collected from 16 vegetarians living in Hong Kong (vegans that had consumed no fish, shellfish or meat for at least the last 5 years) had very low levels of mercury. Their mean hair mercury concentration was only 0.38 ppm.

Downcore sulphur isotope ratios and diatom inferred pH in an artificially acidified Canadian shield lake.

Three gravity cores were removed from near the deepest point in Lake 223 on 9 June 1984, eight years after the Experimental Lakes Area (ELA) staff began the artificial acidification of the lake with sulphuric acid. The first of these cores was analysed for diatoms and pollen stratigraphy while the second and third were analysed for downcore sulphur isotope ratios (H. Thode) and downcore changes in sulphur reducing bacterial densities (S. Rao). Sediment core chronologies were based on lead-210 and cesium-137 data (R. Anderson) and the Ambrosia pollen rise (M. Dickman). Analysis of the first core to the depth of the Ambrosia pollen rise (9 cm) indicated that diatom inferred pH in Lake 223 at the time of the Ambrosia rise (circa 1890) was 6.8-7.0. At a sediment depth of 3 cm the diatom inferred pH was 6.7. Thereafter diatom inferred pH began a decline culminating in the present day (observed) pH range for 1984 (5.3-5.5). At a sediment depth of 1 cm, an increase in the abundance of two benthic alkalophilic diatoms occurred. The increase in the abundance of these diatoms was ascribed to an increase in hypolimnetic alkalinity following the artificial acidification of Lake 223. This is the first time that lake acidification has been linked to an increase in benthic alkalophilic diatoms associated with hypolimnetic alkalinity production following sulphate reduction. Sulphur in the anaerobic (black) sediment layers (0-1.5 cm) was isotopically light relative to the sulphur in the deeper layers. This was due to sulphur isotope fractionation resulting from the bacterial reduction of sulphate to hydrogen sulphide in the anaerobic portion of the water column. A jet black FeS-rich layer in the uppermost 1.5 cm of the lake's sediments was associated with an increase in the abundance of sulphate reducing bacteria (e.g. Desulfovibrio spp.).

Impacts of a mosquito selective pesticide, Bti, on the macroinvertebrates of a subtropical stream in Hong Kong.

To test the hypothesis that Bti was specific to mosquito larvae, the granular form of Bti was tested on commonly found Hong Kong species from four naturally occuring aquatic insect orders and one species of decapod crustacean (Neocardina serra). Because data on Bti impacts on species in tropical and subtropical countries is relatively rare, the present study was conducted in Hong Kong's New Territories. Using static acute toxicity bioassays, all mosquito larvae exposed to Bti at the recommended dosage were dead except for some dark pigmented fourth instar individuals that had stopped feeding prior to emergence and as a result, did not ingest the Bti toxin. The only non-target species killed by the Bti were some chironomid species. In 1998 Bti inoculated pools along the Tai Tan River in the New Territories of Hong Kong had significantly fewer chironomids and mosquito larvae than control pools. By January, 1999, these same Bti inoculated pools still had no mosquito larvae in them, however, chironomids were no longer significantly rarer when compared to the control ponds. Thus, it would appear, that during the 1998-99 study in which Bti was added to the study sites at weekly intervals, Bti resistant chironomid species replaced Bti sensitive species in the Bti inoculated pools.

Mercury and organochlorine exposure from fish consumption in Hong Kong.

The average person in Hong Kong consumes fish or shellfish four or more times a week averaging about 60 Kg of fish per year. Even though the mean mercury level in store-bought Hong Kong fish was only 0.12 mg/kg, corroborating evidence is presented from numerous studies to support the view that mercury bioaccumulates. By the time a typical Hong Kong male reaches 30 years of age he will have accumulated approximately 4 mg/kg mercury in his hair. By age 60, his hair mercury levels will have increased to about 7.5 mg/kg. Hair is a useful indicator of mercury exposure. In the U.S. over a million hair samples have been examined for mercury (mean, 1.5 mg/kg). The mean hair mercury concentration for over 200 Hong Kong residents was 3.3 mg/kg which is more than double the U.S. mean (well over one standard deviation above the U.S. mean). Two lines of evidence support the hypothesis that fish is the major source of methyl mercury in the diet of Hong Kong residents. 1. Individuals consuming 4 or more meals of fish per week had a hair mercury of 4.07 mg/kg dry weight of hair while those consuming fish less frequently had significantly lower levels (2.56 mg/kg). 2. Hong Kong residents that consume no fish had only 0.38 mg/kg hair mercury. The World Health Organization has adopted the U.S. EPA levels for mercury and recommends that food with mercury concentrations of 0.5 mg/kg or more should not be sold for human consumption. Data presented in this paper are consistent with the notion that adoption of a 0.3 mg/kg mercury guideline would benefit residents in countries where rates of fish consumption are significantly higher than in the U.S. Japan, for example, has already adopted a 0.3 mg/kg mercury guideline. In Hong Kong there is a significant correlation (p < 0.05) between male subfertility and the level of mercury in the hair of males between the ages of 25 and 75. Our study of individuals who have been eating mercury contaminated fish steadily for many years suggests that a daily mercury intake of only 0.3 to 0.7 mg/kg body weight may be sufficient to inhibit spermatogenesis in some Hong Kong males. Male subfertility has been correlated with both elevated mercury and the presence of various organochlorines in the diet. To determine whether fish sold in Hong Kong with elevated levels of mercury also had elevated levels of organochlorines we analyzed fish for both mercury and organochlorine content of their dorsal muscle tissue. Because analysis of fish tissue for lipids and a wide range of organochlorines is both very time consuming and expensive, only 15 different species of fish were tested. Organochlorine concentrations were low and there was no correlation between mercury and organochlorine in the 15 fish tested for both organochlorines and mercury. As a result of these tests we concluded that mercury could not be ruled out as the principal causal factor associated with the lack of fertility in Hong Kong males.

Chemistry and biological activity of AAL toxins.

AAL toxins and fumonisins comprise a family of highly reactive, chemically related mycotoxins that disrupt cellular homeostasis in both plant and animal tissues. Two critical issues to resolve are the detection of the entire family in food matricies and the mode of cellular disruption. Analysis of the entire set of chemical congeners in food matrices is difficult but has been achieved by a combination of different HPLC and mass spectrometry strategies. The mode of cellular disruption is unknown but likely involves changes associated with the inhibition of ceramide synthase in both plants and animals. Toxin treated cells exhibit morphological and biochemical changes characteristic of apoptosis. Further evaluation of the specific genetic and biochemical changes that occur during toxin-induced cell death may aid in understanding the mole of the action of these mycotoxins.

Electro-encephalography spectral analysis of heroin addicts compared with abstainers and normal controls.

UNLABELLED: Previous studies have shown that opiates slow the EEG and, in high doses, reduce the threshold of seizure activity. The present work looks at computerized EEG analyses (in the Fast Fourier Transform-FFT-method) of heroin addicts, recent abstainers and normal controls, with the aim of comparison and delineation of group characteristics. Examinations of 60 taped EEG recordings were performed: 20 subjects were current heroin users, 20 were recent abstainers and 20 were normal controls. Statistical analysis was performed for the relative frequency of wave bands. To amplify the known findings of slowing in heroin users, specific ratios were calculated: the alpha ratio (namely, the 8.0-9.5 Hz to 9.5-12.0 Hz ratio) and the delta to low alpha ratio. The specificity and sensitivity of the FFT method were evaluated through the use of discriminant analysis. The EEG was also recorded on conventional paper and evaluated by a neurologist. RESULTS: The addicts had a higher alpha ratio. The abstainers had a slowing of their alpha waves, a high incidence of delta waves and a delta to low alpha ratio that was relatively high. As a function of time from the beginning of abstinence, this ratio decreased. Abstainers for more than 80 days and controls had identical EEGs. The FFT method had 50% sensitivity for the addicts and 70% for the abstainers, while the specificity was 55%. In contrast, the neurologist's reading of the EEG had a very low sensitivity (25% for the addicts, 30% for the abstainers and 20% for the controls) and a 70% specificity.