A pharmaco-EEG study on antipsychotic drugs in healthy volunteers

RATIONALE: Both psychotropic drugs and mental disorders have typical signatures in quantitative electroencephalography (EEG). Previous studies found that some psychotropic drugs had EEG effects opposite to the EEG effects of the mental disorders treated with these drugs (key-lock principle). OBJECTIVES: We performed a placebo-controlled pharmaco-EEG study on two conventional antipsychotics (chlorpromazine and haloperidol) and four atypical antipsychotics (olanzapine, perospirone, quetiapine, and risperidone) in healthy volunteers. We investigated differences between conventional and atypical drug effects and whether the drug effects were compatible with the key-lock principle. METHODS: Fourteen subjects underwent seven EEG recording sessions, one for each drug (dosage equivalent of 1 mg haloperidol). In a time-domain analysis, we quantified the EEG by identifying clusters of transiently stable EEG topographies (microstates). Frequency-domain analysis used absolute power across electrodes and the location of the center of gravity (centroid) of the spatial distribution of power in different frequency bands. RESULTS: Perospirone increased duration of a microstate class typically shortened in schizophrenics. Haloperidol increased mean microstate duration of all classes, increased alpha 1 and beta 1 power, and tended to shift the beta 1 centroid posterior. Quetiapine decreased alpha 1 power and shifted the centroid anterior in both alpha bands. Olanzapine shifted the centroid anterior in alpha 2 and beta 1. CONCLUSIONS: The increased microstate duration under perospirone and haloperidol was opposite to effects previously reported in schizophrenic patients, suggesting a key-lock mechanism. The opposite centroid changes induced by olanzapine and quetiapine compared to haloperidol might characterize the difference between conventional and atypical antipsychotics.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Multilead quantitative electroencephalogram profile and cognitive evoked potentials (P300) in healthy subjects after a single dose of olanzapine

RATIONALE: Olanzapine is an atypical antipsychotic drug with a more favourable safety profile than typical antipsychotics with a hitherto unknown topographic quantitative electroencephalogram (QEEG) profile. OBJECTIVES: We investigated electrical brain activity (QEEG and cognitive event related potentials, ERPs) in healthy subjects who received olanzapine. METHODS: Vigilance-controlled, 19-channel EEG and ERP in an auditory odd-ball paradigm were recorded before and 3 h, 6 h and 9 h after administration of either a single dose of placebo or olanzapine (2.5 mg and 5 mg) in ten healthy subjects. QEEG was analysed by spectral analysis and evaluated in nine frequency bands. For the P300 component in the odd-ball ERP, the amplitude and latency was analysed. Statistical effects were tested using a repeated-measurement analysis of variance. RESULTS: For the interaction between time and treatment, significant effects were observed for theta, alpha-2, beta-2 and beta-4 frequency bands. The amplitude of the activity in the theta band increased most significantly 6 h after the 5-mg administration of olanzapine. A pronounced decrease of the alpha-2 activity especially 9 h after 5 mg olanzapine administration could be observed. In most beta frequency bands, and most significantly in the beta-4 band, a dose-dependent decrease of the activity beginning 6 h after drug administration was demonstrated. Topographic effects could be observed for the beta-2 band (occipital decrease) and a tendency for the alpha-2 band (frontal increase and occipital decrease), both indicating a frontal shift of brain electrical activity. There were no significant changes in P300 amplitude or latency after drug administration. Conclusion: QEEG alterations after olanzapine administration were similar to EEG effects gained by other atypical antipsychotic drugs, such as clozapine. The increase of theta activity is comparable to the frequency distribution observed for thymoleptics or antipsychotics for which treatment-emergent somnolence is commonly observed, whereas the decrease of beta activity observed after olanzapine administration is not characteristic for these drugs. There were no clear signs for an increased cerebral excitability after a single-dose administration of 2.5 mg and 5 mg olanzapine in healthy controls.

A homologue of aliB is found in the capsule region of nonencapsulated Streptococcus pneumoniae

The epidemiology, phylogeny, and biology of nonencapsulated Streptococcus pneumoniae are largely unknown. Increased colonization capacity and transformability are, however, intriguing features of these pneumococci and play an important role. Twenty-seven nonencapsulated pneumococci were identified in a nationwide collection of 1,980 nasopharyngeal samples and 215 blood samples obtained between 1998 and 2002. On the basis of multilocus sequence typing and capsule region analysis we divided the nonencapsulated pneumococci into two groups. Group I was closely related to encapsulated strains. Group II had a clonal population structure, including two geographically widespread clones able to cause epidemic conjunctivitis and invasive diseases. Group II strains also carried a 1,959-bp homologue of aliB (aliB-like ORF 2) in the capsule region, which was highly homologous to a sequence in the capsule region of Streptococcus mitis. In addition, strains of the two major clones in group II had an additional sequence, aliB-like ORF 1 (1,968 to 2,004 bp), upstream of aliB-like ORF 2. Expression of aliB-like ORF 1 was detected by reverse transcription-PCR, and the corresponding RNA was visualized by Northern blotting. A gene fragment homologous to capN of serotypes 33 and 37 suggests that group II strains were derived from encapsulated pneumococci some time ago. Therefore, loss of capsule expression in vivo was found to be associated with the importation of one or two aliB homologues in some nonencapsulated pneumococci.

Prognostic value of the modified American College of Cardiology/American Heart Association lesion morphology classification for clinical outcome after sirolimus-eluting stent placement (results of the prospective multicenter German Cypher Registry)

The modified American College of Cardiology/American Heart Association (ACC/AHA) lesion morphology classification scheme has prognostic impact for early and late outcomes when bare-metal stents are used. Its value after drug-eluting stent placement is unknown. The predictive value of this lesion morphology classification system in patients treated using sirolimus-eluting stents included in the German Cypher Registry was prospectively examined. The study population included 6,755 patients treated for 7,960 lesions using sirolimus-eluting stents. Lesions were classified as type A, B1, B2, or C. Lesion type A or B1 was considered simple (35.1%), and type B2 or C, complex (64.9%). The combined end point of all deaths, myocardial infarction, or target vessel revascularization was seen in 2.6% versus 2.4% in the complex and simple groups, respectively (p = 0.62) at initial hospital discharge, with a trend for higher rates of myocardial infarction in the complex group. At the 6-month clinical follow-up and after adjusting for other independent factors, the composite of cumulative death, myocardial infarction, and target vessel revascularization was nonsignificantly different between groups (11.4% vs 11.2% in the complex and simple groups, respectively; odds ratio 1.08, 95% confidence interval 0.8 to 1.46). This was also true for target vessel revascularization alone (8.3% of the complex group, 9.0% of the simple group; odds ratio 0.87, 95% confidence interval 0.72 to 1.05). In conclusion, the modified ACC/AHA lesion morphology classification system has some value in determining early complications after sirolimus-eluting stent implantation. Clinical follow-up results at 6 months were generally favorable and cannot be adequately differentiated on the basis of this lesion morphology classification scheme.

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.

Long-term estrogen exposure of whitefish Coregonus lavaretus induces intersex but not Lake Thun-typical gonad malformations

A high prevalence of gonad morphological variations has been observed in whitefish Coregonus lavaretus from Lake Thun (Switzerland). To clarify the role of endocrine disruption as a possible cause of the gonad alterations, whitefish were reared in a long-term laboratory experiment under exposure to 17 beta-estradiol (E2). Fish were fed from first-feeding until 3 yr of age at a daily rate of 0 (control), 0.5 or 50 microg E2 kg(-1) fish. E2 exposure resulted in a time- and concentration-dependent increase of prevalence and intensity of intersex gonads, i.e. gonads that macroscopically appeared as either testis or ovary but microscopically contained both male and female germ cells. Four types of intersex could be distinguished: Types 1 and 2 were composed of mainly male tissue, with Type 1 containing single oocytes and Type 2 displaying an ovary-like lamellar structure of the tissue. In Type 3, an increased percentage of the tissue was occupied by female germ cells, while in Type 4, the majority of the gonad tissue consisted of female germ cells. Chronic E2 exposure additionally resulted in a concentration-dependent shift of the sex ratio towards females, a reduced condition factor, retarded gonad growth together with delayed maturation of germ cells, and elevated levels of hepatic vitellogenin mRNA. However, Lake Thun-typical alterations of gonad morphology were not induced by chronic E2 exposure. The results provide evidence that estrogen-active compounds unlikely play a role in the etiology of gonad malformations in Lake Thun whitefish.

Low resolution brain electromagnetic tomography (LORETA) functional imaging in acute, neuroleptic-naive, first-episode, productive schizophrenia

Functional imaging of brain electrical activity was performed in nine acute, neuroleptic-naive, first-episode, productive patients with schizophrenia and 36 control subjects. Low-resolution electromagnetic tomography (LORETA, three-dimensional images of cortical current density) was computed from 19-channel of electroencephalographic (EEG) activity obtained under resting conditions, separately for the different EEG frequencies. Three patterns of activity were evident in the patients: (1) an anterior, near-bilateral excess of delta frequency activity; (2) an anterior-inferior deficit of theta frequency activity coupled with an anterior-inferior left-sided deficit of alpha-1 and alpha-2 frequency activity; and (3) a posterior-superior right-sided excess of beta-1, beta-2 and beta-3 frequency activity. Patients showed deviations from normal brain activity as evidenced by LORETA along an anterior-left-to-posterior-right spatial axis. The high temporal resolution of EEG makes it possible to specify the deviations not only as excess or deficit, but also as inhibitory, normal and excitatory. The patients showed a dis-coordinated brain functional state consisting of inhibited prefrontal/frontal areas and simultaneously overexcited right parietal areas, while left anterior, left temporal and left central areas lacked normal routine activity. Since all information processing is brain-state dependent, this dis-coordinated state must result in inadequate treatment of (externally or internally generated) information.

Smell and taste of chewing gum affect frequency domain EEG source localizations

We investigated brain electric field signatures of subjective feelings after chewing regular gum or gum base without flavor. 19-channel eyes-closed EEG from 20 healthy males before and after 5 minutes of chewing the two gum types in random sequence was source modeled in the frequency domain using the FFT-Dipole-Approximation. 3-dimensional brain locations and strengths (Global Field Power, GFP) of the equivalent sources of five frequency bands were computed as changes from pre-chewing baseline. Gum types differed (ANOVA) in pre-post changes of source locations for the alpha-2 band (to anterior and right after regular gum, opposite after gum base) and beta-2 band (to anterior and inferior after regular gum, opposite after gum base), and of GFP for delta-theta, alpha-2 and beta-1 (regular gum: increase, gum base: decrease). Subjective feeling changed to more positive values after regular gum than gum base (ANOVA).—Thus, chewing gum with and without taste-smell activates different brain neuronal populations.

A COL11A2 mutation in Labrador retrievers with mild disproportionate dwarfism.

We describe a mild form of disproportionate dwarfism in Labrador Retrievers, which is not associated with any obvious health problems such as secondary arthrosis. We designate this phenotype as skeletal dysplasia 2 (SD2). It is inherited as a monogenic autosomal recessive trait with incomplete penetrance primarily in working lines of the Labrador Retriever breed. Using 23 cases and 37 controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 4.44 Mb interval on chromosome 12. We re-sequenced the genome of one affected dog at 30x coverage and detected 92 non-synonymous variants in the critical interval. Only two of these variants, located in the lymphotoxin A (LTA) and collagen alpha-2(XI) chain gene (COL11A2), respectively, were perfectly associated with the trait. Previously described COL11A2 variants in humans or mice lead to skeletal dysplasias and/or deafness. The dog variant associated with disproportionate dwarfism, COL11A2:c.143G>C or p.R48P, probably has only a minor effect on collagen XI function, which might explain the comparatively mild phenotype seen in our study. The identification of this candidate causative mutation thus widens the known phenotypic spectrum of COL11A2 mutations. We speculate that non-pathogenic COL11A2 variants might even contribute to the heritable variation in height.

Transcription factor SEF: Purification and functional characterization

Cytokine-induced transcription of the serum amyloid A3 (SAA3) gene promoter requires a transcriptional enhancer that contains three functional elements: two C/EBP-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa cell nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA-affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing, oligonucleotide competition and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression plasmid with SAA3-luciferase reporter resulted in 3- to 5-fold activation of SAA3 promoter. Interestingly, when SEF-transfected cells were treated with either conditioned medium (CM) or interleukin (IL) 1, the SAA3 promoter was synergistically activated in a dose-dependent manner. Furthermore, when SEF-binding site was mutated, the response of SAA3 promoter to IL-1 or CM stimulation was abolished or drastically decreased, suggesting that SEF may functionally cooperate with an IL-1-inducible transcription factor. Indeed, our functional studies showed that NFκB is a key transcription factor that mediates the IL-1-induced expression of SAA3 gene, and that SEF can synergize with NFκBp65 to activate SAA3 promoter. By coimmunoprecipitation experiments, we found that SEF could specifically interact with NFκBp65, and that the association of these two factors was enhanced upon IL-1 and CM stimulation. This suggests that the molecular basis for the functional synergy between SEF and NFκB may be due to the ability of SEF to physically interact with NPκB. In addition to its interaction with SEF, NFκB-dependent activation also requires the weak κB site in the C element and its interaction with C/EBP. Besides its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of α2-macroglobulin, Aα fibrinogen, and 6–16 genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes. By coimmunoprecipitation experiments, we determined that SEF could specifically associate with both Stat3 and Stat2 upon cytokine stimulation. To examine the functional roles of such interactions, we evaluated the effects of SEF on the transcriptional regulation of two reporter genes: Aα fibrinogen and 6–16, which are IL-6- and interferon-α-responsive, respectively. Our results showed that cotransfection of SEF expression plasmid can activate the expression of Aα fibrinogen gene and 6–16 gene. Moreover, SEF can dramatically enhance the interferon-α-induced expression of 6–16 gene and IL-6-induced expression of Aα fibrinogen gene, suggesting that SEF may functionally cooperate with ISGF3 and Stat3 to mediate interferon-α and IL-6 signaling. ^ Our findings that SEF can interact with multiple cytokine-inducible transcription factors to mediate the expression of target genes open a new avenue of investigation of cooperative transcriptional regulation of gene expression, and should further our understanding of differential gene expression in response to a specific stimulus. In summary, our data provide evidence that SEF can mediate the signaling of different cytokines by interacting with various cytokine-inducible transcription factors. ^

The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Zα. Here we report the solution structure of free Zα and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Zα)2/Z-DNA complex shows that most Z-DNA contacting residues in free Zα are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (α+β)helix–turn–helix/B-DNA complexes suggests that binding of Zα to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B- and Z-DNA.

Functional Domains of Rep2, a Transcriptional Activator Subunit for Res2–Cdc10, Controlling the Cell Cycle “Start”

In the fission yeast Schizosaccharomyces pombe, passage from G1 to S-phase requires the execution of the transcriptional factor complex that consists of the Cdc10 and Res1/2 molecules. This complex activates the MluI cell cycle box cis-element contained in genes essential for S-phase onset and progression. The rep2+ gene, isolated as a multicopy suppressor of a temperature-sensitive cdc10 mutant, has been postulated to encode a putative transcriptional activator subunit for the Res2–Cdc10 complex. To identify the rep2+ function and molecularly define its domain organization, we reconstituted the Res2–Cdc10 complex-dependent transcriptional activation in Saccharomyces cerevisiae. Reconstitution experiments, deletion analyses using one and two hybrid systems, and in vivo Res2 coimmunoprecipitation assays show that the Res2–Cdc10 complex itself can recognize but cannot activate MluI cell cycle box without Rep2, and that consistent with its postulated function, Rep2 contains 45-amino acid Res2 binding and 22-amino acid transcriptional activation domains in the middle and C terminus of the molecule, respectively. The functional essentiality of these domains is also demonstrated by their requirement for rescue of the cold-sensitive rep2 deletion mutant of fission yeast.

Induced fit DNA recognition by a minor groove binding analogue of Hoechst 33258: fluctuations in DNA A tract structure investigated by NMR and molecular dynamics simulations

NMR analysis and molecular dynamics simulations of d(GGTAATTACC)2 and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar–phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving ‘lock and key’ recognition.

Microtubules mediate mitochondrial distribution in fission yeast.

The Schizosaccharomyces pombe mutant, ban5-4, displays aberrant mitochondrial distribution. Incubation of this conditional-lethal mutant at the nonpermissive temperature led to aggregated mitochondria that were distributed asymmetrically within the cell. Development of this mitochondrial asymmetry but not mitochondrial aggregation required progression through the cell division cycle. Genetic analysis revealed that ban5-4 is an allele of atb2 encoding alpha 2-tubulin. Consistent with this finding, cells with the cold-sensitive nda3 mutation in beta-tubulin displayed aggregated and asymmetrically distributed mitochondria after incubation at lowered temperatures. These results indicate that microtubules mediate mitochondrial distribution in fission yeast and provide the first genetic evidence for the role of microtubules in mitochondrial movement.