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A virtual screening method is presented that is grounded on a receptor-derived pharmacophore model termed "virtual ligand" or "pseudo-ligand". The model represents an idealized constellation of potential ligand sites that interact with residues of the binding pocket. For rapid virtual screening of compound libraries the potential pharmacophore points of the virtual ligand are encoded as an alignment-free correlation vector, avoiding spatial alignment of pharmacophore features between the pharmacophore query (i.e., the virtual ligand) and the candidate molecule. The method was successfully applied to retrieving factor Xa inhibitors from a Ugi three-component combinatorial library, and yielded high enrichment of actives in a retrospective search for cyclooxygenase-2 (COX-2) inhibitors. The approach provides a concept for "de-orphanizing" potential drug targets and identifying ligands for hitherto unexplored or allosteric binding pockets.

Most common genetic disorders have a complex inheritance and may result from variants in many genes, each contributing only weak effects to the disease. Pinpointing these disease genes within the myriad of susceptibility loci identified in linkage studies is difficult because these loci may contain hundreds of genes. However, in any disorder, most of the disease genes will be involved in only a few different molecular pathways. If we know something about the relationships between the genes, we can assess whether some genes (which may reside in different loci) functionally interact with each other, indicating a joint basis for the disease etiology. There are various repositories of information on pathway relationships. To consolidate this information, we developed a functional human gene network that integrates information on genes and the functional relationships between genes, based on data from the Kyoto Encyclopedia of Genes and Genomes, the Biomolecular Interaction Network Database, Reactome, the Human Protein Reference Database, the Gene Ontology database, predicted protein-protein interactions, human yeast two-hybrid interactions, and microarray co-expressions. We applied this network to interrelate positional candidate genes from different disease loci and then tested 96 heritable disorders for which the Online Mendelian Inheritance in Man database reported at least three disease genes. Artificial susceptibility loci, each containing 100 genes, were constructed around each disease gene, and we used the network to rank these genes on the basis of their functional interactions. By following up the top five genes per artificial locus, we were able to detect at least one known disease gene in 54% of the loci studied, representing a 2.8-fold increase over random selection. This suggests that our method can significantly reduce the cost and effort of pinpointing true disease genes in analyses of disorders for which numerous loci have been reported but for which most of the genes are unknown.

We studied the midbrain SERT availability in patients with major depression and assessed the relation of SERT occupancy by citalopram to the treatment response. 21 non-medicated patients with major depression and 13 healthy controls were examined by [(123)I]-ADAM SPECT. The midbrain SERT availability (SERT V(3)'') was calculated using individual MRI scans. In 13/21 patients SPECT was repeated 7 days after oral medication with citalopram (10 mg/day). We found no significant difference in the mean midbrain SERT availability between the studied patients with major depression and healthy controls (0.86 +/- 0.27 vs. 0.71 +/- 0.44, p = 0.069). The mean SERT occupancy accounted to 61%. The degree of SERT blockade by citalopram did not correlate with the reduction in HAMD total score. Treatment with low-dosed citalopram caused individually variable occupancy of the midbrain-SERT and a rapid clinical improvement in 54% of the investigated patients.

The identification of the candidate genes for autism through linkage and association studies has proven to be a difficult enterprise. An alternative approach is the analysis of cytogenetic abnormalities associated with autism. We present a review of all studies to date that relate patients with cytogenetic abnormalities to the autism phenotype. A literature survey of the Medline and Pubmed databases was performed, using multiple keyword searches. Additional searches through cited references and abstracts from the major genetic conferences from 2000 onwards completed the search. The quality of the phenotype (i.e. of the autism spectrum diagnosis) was rated for each included case. Available specific probe and marker information was used to define optimally the boundaries of the cytogenetic abnormalities. In case of recurrent deletions or duplications on chromosome 15 and 22, the positions of the low copy repeats that are thought to mediate these rearrangements were used to define the most likely boundaries of the implicated 'Cytogenetic Regions Of Interest' (CROIs). If no molecular data were available, the sequence position of the relevant chromosome bands was used to obtain the approximate molecular boundaries of the CROI. The findings of the current review indicate: (1) several regions of overlap between CROIs and known loci of significant linkage and/or association findings, and (2) additional regions of overlap among multiple CROIs at the same locus. Whereas the first finding confirms previous linkage/association findings, the latter may represent novel, not previously identified regions containing genes that contribute to autism. This analysis not only has confirmed the presence of several known autism risk regions but has also revealed additional previously unidentified loci, including 2q37, 5p15, 11q25, 16q22.3, 17p11.2, 18q21.1, 18q23, 22q11.2, 22q13.3 and Xp22.2-p22.3.

Celiac disease is probably the best-understood immune-related disorder. The disease presents in the small intestine and results from the interplay between multiple genes and gluten, the triggering environmental factor. Although HLA class II genes explain 40% of the heritable risk, non-HLA genes accounting for most of the familial clustering have not yet been identified. Here we report significant and replicable association (P = 2.1 x 10(-6)) to a common variant located in intron 28 of the gene myosin IXB (MYO9B), which encodes an unconventional myosin molecule that has a role in actin remodeling of epithelial enterocytes. Individuals homozygous with respect to the at-risk allele have a 2.3-times higher risk of celiac disease (P = 1.55 x 10(-5)). This result is suggestive of a primary impairment of the intestinal barrier in the etiology of celiac disease, which may explain why immunogenic gluten peptides are able to pass through the epithelial barrier.

Support vector machines (SVM) were trained to predict cyclooxygenase 2 (COX-2) and thrombin inhibitors. The classifiers were obtained using sets of known COX-2 and thrombin inhibitors as "positive examples" and a large collection of screening compounds as "negative examples". Molecules were encoded by topological pharmacophore-point triangles. In retrospective virtual screening, 50-90% of the known active compounds were listed within the first 0.1% of the ranked database. To check the validity of the constructed classifiers, we developed a method for feature extraction and visualization using SVM. As a result, potential pharmacophore points were weighted according to their importance for COX-2 and thrombin inhibition. Known thrombin and COX-2 pharmacophore points were correctly recognized by the machine learning system. In a prospective virtual screening study, several potential COX-2 inhibitors were predicted and tested in a cellular activity assay. A benzimidazole derivative exhibited significant inhibitory activity with an IC(50) of 0.2 microM, which is better than Celecoxib in our assay. It was demonstrated that the SVM machine-learning method can be used in virtual screening and be analyzed in a human-interpretable way that results in a set of rules for designing novel molecules.

This work was conducted on Pinctada maxima nacre (mother of pearl) in order to understand its multiscale ordering and the role of the organic matrix in its structure. Intermittent-contact atomic force microscopy with phase detection imaging reveals a nanostructure within the tablet. A continuous organic framework divides each tablet into nanograins. Their shape is supposed to be flat with a mean extension of 45nm. TEM performed in the darkfield mode evidences that at least part of the intracrystalline matrix is crystallized and responds like a 'single crystal'. The tablet is a 'hybrid composite'. The organic matrix is continuous. The mineral phase is thus finely divided still behaving as a single crystal. It is proposed that each tablet results from the coherent aggregation of nanograins keeping strictly the same crystallographic orientation thanks to a hetero-epitaxy mechanism. Finally, high-resolution TEM performed on bridges from one tablet to the next, in the overlying row, did not permit to evidence a mineral lattice but crystallized organic bridges. The same organic bridges were evidenced by SEM in the interlaminar sequence.

Myrtucommulone (MC) and semimyrtucommulone (S-MC) are unique oligomeric, nonprenylated acylphloroglucinols contained in the leaves of myrtle (Myrtus communis). Although extracts of myrtle have been traditionally used in folk medicine for the treatment of various disorders, studies addressing select cellular or molecular pharmacological properties of these extracts or specific ingredients thereof are rare. Here, we show for the first time that MC and S-MC potently suppress the biosynthesis of eicosanoids by direct inhibiting cyclooxygenase-1 and 5-lipoxygenase in vitro and in vivo at IC50 values in the range of 1.8 to 29 microM. Moreover, we show that MC and S-MC prevent the mobilization of Ca2+ in polymorphonuclear leukocytes, mediated by G protein signaling pathways at IC50 values of 0.55 and 4.5 microM, respectively, and suppress the formation of reactive oxygen species and the release of elastase at comparable concentrations. The isobutyrophenone core of MC as well as S-MC was much less potent or even not active at all. In addition, MC or S-MC only partially inhibited peroxide formation or failed to block Ca2+ mobilization and elastase release when polymorphonuclear leukocytes were challenged with ionomycin that circumvents G protein signaling for cell activation. We conclude that, in view of their ability to suppress typical proinflammatory cellular responses, the unique acylphloroglucinols MC and S-MC from myrtle may possess an anti-inflammatory potential, suggesting their therapeutic use for the treatment of diseases related to inflammation and allergy.

5-Lipoxygenase (5-LO) is a tightly regulated enzyme in the synthesis of bioactive lipids from arachidonic acid. Here, we demonstrate that 5-LO is regulated by caspases, which are signaling molecules that control critical biological processes by means of specific limited proteolysis. Cell splitting of the Epstein-Barr virus-transformed B lymphocytic cell line BL41-E95-A caused a pronounced, but transient, reduction of functional 5-LO protein, accompanied by the appearance of a 62-kDa 5-LO cleavage product. In parallel, splitting of BL41-E95-A cells induced activation of caspase-6 (casp-6) and casp-8. Caspase activation and 5-LO degradation were blocked by the protein-synthesis inhibitor cycloheximide, and cell-permeable peptide inhibitors of casp-6 and casp-8 prevented 5-LO cleavage. Activation of casp-6 and casp-8 was connected to subsequent enhancement of cell proliferation, whereas selective caspase inhibition blocked cell growth. Last, isolated human 5-LO was cleaved by recombinant casp-6 in vitro to a 58-kDa fragment. Based on site-directed mutagenesis studies, 5-LO is cleaved by casp-6 after Asp-170, which in a homology-based 3D model of 5-LO is located on the enzyme periphery. We suggest that splitting of BL41-E95-A cells induces de novo synthesis of a protein involved in the activation of casp-6, which cleaves 5-LO.

Celiac disease (CD) is a complex genetic disorder of the small intestine. The DQ2/DQ8 human leucocyte antigen (HLA) genes explain approximately 40% of the genetic component of the disease, but the remaining non-HLA genes have not yet been identified. The key environmental factor known to be involved in the disease is gluten, a major protein present in wheat, barley, and rye. Integrating microarray data and linkage data from chromosome 6q21-22 revealed the prolyl endopeptidase (PREP) gene as a potential CD candidate in the Dutch population. Interestingly, this gene encodes for the only enzyme that is able to cleave the proline-rich gluten peptides. To investigate the role of the human PREP gene as a primary genetic factor in CD, we conducted gene expression, sequence analysis, and genetic association studies of the PREP gene and determined PREP enzyme activity in biopsies from CD patients and controls. Sequence analysis of the coding region of the PREP gene revealed two novel polymorphisms. Genetic association studies using two novel polymorphisms and three known PREP variants excluded a genetic association between PREP and CD. Determination of PREP activity revealed weak but significant differences between treated and untreated CD biopsies (P < 0.05). Our results from the association study indicate that PREP is not a causative gene for CD in the Dutch population. These are further supported by the activity determinations in which we observed no differences in PREP activity between CD patients and controls.

Introduction: CYP2D6 gene duplication causing ultrafast metabolism is one reason for failure in responding to CYP2D6-metabolized antidepressants. We studied the effect of the CYP2D6 duplication genotype on doxepin pharmacokinetics and platelet serotonin uptake and concentrations., Methods: Pharmacokinetics of trans (E)- and cis (Z)-doxepin and N-desmethyldoxepin were analyzed after a single dose of 75 mg doxepin in 11 ultrafast metabolizers (UM), 11 extensive metabolizers (EM) and 3 poor metabolizers (PM), identified by genotyping for CYP2D6 alleles *2, *3, *4, *5, *6, *9, *10, *35, *41 and specific analyses to characterize gene duplication. Platelet serotonin concentrations were measured by HPLC., Results: A trend for lower AUC of the active principle (sum of doxepin and N-desmethyldoxepin) in UMs versus EMs was detected (575 versus 1,000 nmol h/l, P=0.07), mainly due to the differences in desmethyldoxepin concentrations (P=0.003). Stereoselective analysis showed a significant effect of the UM genotype on (E)-doxepin pharmacokinetic parameters whereas those of (Z)-doxepin did not differ between the CYP2D6 genotype groups. The 75-mg doxepin dose had no effect on platelet serotonin concentration and uptake, but serotonin concentrations in platelets were significantly higher in UM in comparison to the EM and PM groups. At baseline, these concentrations were 462, 399, and 292 ng/10 platelets in UM, EM and PM (P<0.0001 for trend)., Conclusions: At the same dose, internal exposure to doxepin differed by more than ten-fold between the CYP2D6 genotype groups. CYP2D6 may have an effect on platelet serotonin explained by salvage pathways of 5-methoxytryptamine to serotonin mediated by CYP2D6; however, this finding requires further confirmatory experiments.

The identification of genes primarily responsible for complex genetic disorders is a daunting task. Despite the assignment of many susceptibility loci, there has only been limited success in identifying disease genes based solely on positional information from genome-wide screens. The incorporation of several complementary strategies in a single integrated approach should facilitate and further enhance the efficacy of this search for genes. To permit the integration of linkage, association and expression data, together with functional annotations, we have developed a Java-based software tool: TEAM (tool for the integration of expression, and linkage and association maps). TEAM includes a genome viewer, capable of overlaying karyobands, genes, markers, linkage graphs, association data, gene expression levels and functional annotations in one composite view. Data management, analysis and filtering functionality was implemented and extended with links to the Ensembl, Unigene and Gene Ontology databases to facilitate gene annotation. Filtering functionality can help prevent the exclusion of poorly annotated, but differentially expressed, genes that reside in candidate regions that show linkage or association. Here we demonstrate the program's functionality in our study on coeliac disease (OMIM 212750), a multifactorial gluten-sensitive enteropathy. We performed a combined data analysis of a genome-wide linkage screen in 82 Dutch families with affected siblings and the microarray expression profiles of 18,110 cDNAs in 22 intestinal biopsies.

Background and Aims: The causative molecular pathways underlying the pathogenesis of coeliac disease are poorly understood. To unravel novel aspects of disease pathogenesis, we used microarrays to determine changes in gene expression of duodenal biopsies., Methods: cDNA microarrays representing 19 200 genes were used to compare gene expression profiles of duodenal biopsies from 15 coeliac disease patients with villous atrophy (Marsh III) and seven control individuals with normal biopsies (Marsh 0). In addition, the specific effect of gluten was studied by comparing the expression profiles of Marsh III lesions of seven patients exposed to gluten with four patients on a gluten free diet., Results: Comparing Marsh III with Marsh 0 lesions identified 109 genes that differed significantly (p<0.001) in expression levels between patients and controls. A large number of these genes have functions in proliferation and differentiation pathways and might be important for correct development of crypt-villous units. Alterations in these pathways may lead to the characteristic hyperplasia and villous atrophy seen in coeliac disease. The analyses also revealed 120 differentially expressed genes (p<0.005) when comparing patients on a gluten free diet with those exposed to gluten. These genes further strengthen our observation of increased cell proliferation in the presence of gluten., Conclusions: Our study provides new candidate genes in the pathogenesis of coeliac disease. Based on our results, we hypothesise that villous atrophy in coeliac disease patients is due to failure in cell differentiation. These genes are involved in pathways not previously implicated in coeliac disease pathogenesis and they may provide new targets for therapy.

Background: Abnormal changes in platelets used as peripheral markers of central serotonergic functions are said to be associated with suicidality and depression, but this association has not been supported by consistent findings., Aim: This review based on selected, often quoted publications in this area focuses firstly, on obviously contradictory findings and, secondly, on potential methodological flaws explaining these discrepancies., Results: The platelet 5-HT transporter has been found to have an inconsistent association with suicidality; furthermore, the specificity of imipramine for the 5-HT transporter is most likely low, since the number of platelet impramine binding sites has not been reliably associated with platelet serotonin uptake (Vmax). Significant changes of platelet serotonin content in suicidal individuals, as described in various studies, are most likely due to washout periods that are too short to eliminate the effects of a previous drug intake, or, in violent suicide attempters, due to blood loss and dilution. Similar methodological shortcomings might account for an often-reported elevated number of platelet 5-HT(2) receptor binding sites in suicidal individuals. In many studies, the results have not been sufficiently controlled for drug effects on platelet 5-HT(2) receptors, and associations of platelet 5-HT(2) binding with selective classifications of suicidal behavior are often generalized as further evidence for an association of platelet 5-HT(2) receptors with 'suicidality'. Finally, changes in platelet MAO-B-activity in suicidal patients have not been reproducibly found, and the impact of smoking on MAO-B activity has not been controlled in any studies., Conclusions: Methodological flaws such as small sample sizes, insufficient matching criteria for controls, use of inadequate ligands in binding experiments, nonconsideration of comorbidity etc. and considerable methodological differences between studies limit their validity and comparability. It does not seem possible, at present, to integrate published findings and concepts into a plausible biological model of suicidality.

The objective of this study was to determine whether the presence of gastrointestinal symptoms, as defined by item 12 of Hamilton-Rating-Scale for Depression, is related to kinetic characteristics of platelet-5HT uptake in patients with major depression. The clinical picture of depression in patients with severe form of appetite loss with difficulties of eating (item 12 = 2) and weight loss was characterized by the combination of depressed mood with somatic symptoms of anxiety, sleep disturbances, decreased activity and the presence of nausea. The high frequency of relatively low Vmax and Km of 5HT uptake in this group (n = 12), all in the lower range of controls, resulted in significantly lower mean values compared with patients without gastrointestinal symptoms (n = 16; item 12 = 0) or 57 healthy subjects (Vmax = 1.36 +/- 0.27 vs. 2.14 +/- 0.85 vs. 2.05 +/- 0.74 nMol 5HT/10(9)plat.x min; Km = 382 +/- 68 vs. 467 +/- 94 vs. 492 +/- 123 nM respectively). Although our finding needs confirmation, it seems that in the research for serotonergic mechanisms in major depression, it makes sense to look at depressed patients with or without somatic symptoms separately. Based on findings in 5HT transporter knock-out mice (J. Neurosci. 15 (2001) 6348), we assume that the low apparent Vmax of platelet-5HT uptake reflects the low expression of 5HT transporter not only in platelets, but also in the gut mucosa and enteric serotonergic neurons, which probably increases the risk of typical gastrointestinal symptoms such as appetite loss and nausea occurring in some depressed patients.

A genome scan was performed in obese type 2 diabetes mellitus pedigrees to identify susceptibility loci involved in obesity-driven type 2 diabetes mellitus. We studied the 20% most obese diabetes pedigrees from a confined Dutch population from around the town of Breda. Previously we, and others, have already shown that a susceptibility locus influencing obesity in diabetes may reside on chromosome 18p11. We now report evidence to also suggest linkage for type 2 diabetes in these obese pedigrees on chromosome regions 11p (genome-wide P-value

Correlation vector methods were tested for their usefulness in ligand-based virtual screening. Three molecular descriptors--two based on potential pharmacophore points and one on partial atom charges--and three similarity measures--the Manhattan distance, the Euclidian distance and the Tanimoto coefficient--were compared. The alignment-free descriptors seem to be particularly applicable when a course-grain filtering of data sets is required in combination with a high execution speed. Significant enrichment of actives was obtained by retrospective analysis. The cumulative percentages for all three descriptors allow for the retrieval of up to 78% of the active molecules in the first five percent of the reference database. Different descriptors retrieved only weakly overlapping sets of active molecules among the top-ranking compounds. If a single similarity index is to be used, the Manhattan distance seems to be particularly applicable. Generally, none of the three different descriptors tested in this study clearly outperformed the others. The suitability of a descriptor critically depends on the ligand-receptor interaction under investigation. For ligand-based similarity searching it is recommended to exploit several descriptors in parallel.

Based on the assumption that there is a biological basis behind the individual differences in the speed with which an antidepressant produces its therapeutic effects, we compared initial serotonin (5HT) uptake characteristics in platelets of rapid (2 weeks), slow (4 weeks) and non-responders in a group of 47 depressed patients who were treated with amitriptyline for at least 4 weeks. A response was defined as a reduction in the Hamilton Depression Rating Scale score of > or =50% from baseline. In 16 rapid responders, a significantly higher mean 5HT uptake efficiency (Vmax/Km) corresponded with a significantly higher 5HT uptake activity at a low, physiological substrate concentration in comparison with the 15 non-responders or the 16 slow responders (33.1+/-7.8 versus 25.5+/-7.7 versus 24.1+/-5.8 pMol [3H]-5HT/10(9) plat. x 5 min, respectively). The findings indicate that pre-treatment 5HT uptake activity contributes to the individual variability in response time to amitriptyline treatment.

Objective: To analyze the spectrum of relationships between clinical effects of amitriptyline (At) treatment after 2 and 4 weeks (wks) and plasma levels of At, nortriptyline (Nt), At+Nt, demethylation rate of At, treatment modalities, age, and gender., Methods: Patients with major depression (ICD 10: F31-F33) and a HAMD-21 total score of 15-41 received At on a dosage schedule chosen by the doctor for at least 4 wks. Plasma drug levels were assessed at baseline and at wks 2 and 4., Results: Of the 58 patients enrolled in the study, 47 (15males, 32 females) were eligible for statistical analysis. An early response by wk 2 (decrease in HAMD-21 score of at least 50 % from baseline) was observed in 34.0 % of patients, and after 4 wks, the response rate was 63.8 % (males 86.6 %, females 53.1 %). There was a low, negative, and significant correlation between percent reduction in HAMD and steady state At concentration only at wk 2 (n = 47 r Sp. = -0.306 p < 0.05). However, the correlation was dependent on the degree of At demethylation and treatment modalities. A ratio of Nt/At >1 was observed in 23 patients; of these, 11 (47.8 %) were non-responders by wk 4. A low rate of demethylation (Nt/At 1 group, the non-responders were distributed in the whole range of observed At plasma concentrations (20-150 ng/ml). Only in patients with Nt/At

In biological suicide research, low cerebrospinal fluid-5-hydroxyindolacetic acid (CSF-5HIAA) concentrations have been associated with suicidality, aggression and impulsivity. However, it frequently appears that the interpretation of existing study results is flawed. The analysis of various published findings suggests that contaminating factors like impulsivity or depressive symptoms in suicide attempters are often not taken into consideration at the time of suicide. The seemingly 'robust' association of low CSF-5HIAA concentration with 'suicidality' and 'aggression' is in fact rather weak. Reported associations of subgroups of suicidal behavior (e.g. violent suicide attempts) with low CSF-5HIAA concentrations are likely to represent somewhat premature translations of findings from studies that have flaws in methodology. Furthermore, the perception of 'suicidal behavior' as autoaggressive behavior or inwardly directed aggression in the view of the authors may not be useful in biological suicide research. The construct of aggressivity is insufficiently defined, resulting in difficulties to interpret empirical data. Some evidence exists, however, that reduced CSF-5HIAA concentrations might be related to certain depressive symptoms and changes in impulsivity. More carefully designed studies are required to overcome the existing methodological shortcomings.

Objective: To determine: (i) whether general practitioners have difficulty with drug dosing; (ii) what information sources they currently use to help them with drug dosing; (iii) their views on the potential value of decision support software for drug dosing., Design: Questionnaire survey., Setting: Nottingham, U.K., Participants: 263 general practitioners (GPs)., Results: The response rate was 78% (263/336). Most GPs reported difficulties with drug dosing for children, the elderly and patients with renal impairment. Compared with 'patients in general', GPs had particular difficulties in drug dosing for these specific groups (P < 0.001). Paper-based formularies were the most common source of information for help with drug doses. Nevertheless, most GPs had positive views on the potential usefulness of computerized decision support., Conclusion: GPs commonly have problems in drug dosing for certain groups of patients. The development and use of computerized decision support might help GPs in these situations.

The dependence of platelet-5HT content on apparent kinetic parameters of 5HT uptake was analyzed in 56 healthy subjects and 47 depressed patients, who had not been taking psychotropic medication for several months. There were no significant relationships between apparent Vmax or Km and platelet-5HT content in both groups. However, the ratio of Vmax to Km, as a measure of apparent 5HT uptake efficiency, significantly correlated with the platelet-5HT concentration in healthy subjects (r=0.627 p<0.001). Female controls showed a higher correlation coefficient (r=0.723) than male controls (r=0.457). A marked deviation from the linear relationship between 5HT content and the ratio Vmax/K was observed in female depressed patients (r=0.250 n.s.). In male depressed patients the correlation coefficient (r=0.485 p<0.05) was similar to male healthy subjects, but the regression equations differed significantly in slope and intercept. Dividing controls and patients in subgroups with low, median and high net uptake rates, it was found that the frequencies of these uptake rate classes were 24.6%, 33.3%, 42.1% in controls and 38.3%, 44.7%, 17.0% in patients respectively. Patients and controls with low net uptake rate showed very similar uptake kinetics and uptake efficiencies, but the lack of a significant correlation between 5HT content and the ratio Vmax/Km differentiated patients from controls. The status of the serotonergic system, expressed as relationship between 5HT content and uptake efficiency, was very similar in patients and controls in the range of medium net uptake rate. A trend toward higher values of uptake efficiency was apparent in patients with high net uptake rate but the platelet-5HT content was similar to corresponding controls. Mean scores on the HAMD scale (total score and psychic anxiety item) were significantly higher in the low net uptake rate group of patients than in those with a high net uptake rate.

Kava-kava, a psychoactive beverage, induces relaxation, improves social interaction, promotes sleep and plays an important role in the sociocultural life in the islands of the South Pacific. On the other hand, standardized extracts of kava-kava roots are used for the therapy of anxiety, tension and restlessness. Kava pyrones, the major constituents of kava kava, are generally considered to be responsible for the pharmacological activity in humans and animals. To obtain more information on the mechanisms by which kava-kava exerts psychotropic properties we investigated the in vitro effects of kava-kava extract and pure synthetic kava pyrones on human platelet MAO-B, in comparison to amitriptyline, imipramine and brofaromine. Kava-kava extract was found to be a reversible inhibitor of MAO-B in intact platelets (IC50 24 microM) and disrupted platelet homogenates (IC50 1.2 microM). Structural differences of kava pyrones resulted in a different potency of MAO-B inhibition. The order of potency was desmethoxyyangonin > (+/-)-methysticin > yangonin > (+/-)-dihydromethysticin > (+/-)- dihydrokavain > (+/-)-kavain. The two most potent kava pyrones, desmethoxyyangonin and (+/-)-methysticin displayed a competetive inhibition pattern with mean Ki 0.28 microM and 1.14 microM respectively. The inhibition of MAO-B by kava pyrone-enriched extracts might be an important mechanism for their psychotropic activity.

Vascular complications following orthopedic procedures at the lower extremity are mainly thrombotic or thromboembolic events. Damages to vascular structures during operations close to the knee joint, like injuries of the popliteal artery, are extremely rare and receive a reserved consideration in the evaluation of postoperative complications. The extent of the vessel injury affects the grade of clinical symptoms-from acute arterial bleeding as far as slowly developing disturbances of haemodynamic, with effects to the different functions and pattern of the concerned tissues. Arterial bleeding into muscle compartments do not necessarily appear as an acute emergency, therefore it may occasionally be difficult to place the clinical symptoms into the right context of pathophysiological processes. In the present case a deep vein thrombosis of the affected extremity was initiated by a haemorrhage into the fossa popliteal and the compartments of the lower leg after injury of the popliteal artery. Subsequently a compartment syndrome, a false aneurysm of the popliteal artery and an arteriovenous fistula from popliteal artery into a lower leg vein developed.

Radioiododestannylation has been employed to prepare a series of radioiodinated vinyl alcohols, two of which were evaluated for their tissue distribution in rats. The radioiodination method utilized tri-alkylstannylvinyl intermediates that can be prepared in good yields from 1,2-bis(tributylstannyl) ethylene and the appropriate ketones or by hydrostannation of the corresponding ethynyl alcohols. The radiolabeling proceeded at the no-carrier-added level to give, after HPLC separation, the desired products in 85-95% isolated yields. Tissue distribution studies in the rat indicated that the agents were rapidly extracted by the brain and then quickly cleared. No retention of activity was observed in the nonmetabolizing or nonexcretory organs. Chemically, the use of the E-tributylstannylvinyl lithium followed by radioiodination controlled stereochemistry and eliminated the separation of E- and Z-iodovinyl isomers. Biologically, the compounds behaved as neutral, diffusible and nonmetabolized radiotracers that may be used for the evaluation of cerebral blood flow, potentially with the short-lived radionuclide I-122 or other short-lived radiohalogens.

Background: Certain antigens of the HIV-1, e.g., gp120-envelop proteins, can be expressed on the membrane of HIV-infected cells. Little is known about the membrane expression of other HIV-antigens and their interaction with specific antibodies., Objective: To develop murine monoclonal antibodies (mAbs) to the p24-core protein of HIV-1 and to characterise their binding sites and biological activities on HIV-infected T cells., Methods: Monoclonal antibodies were developed from mice hyperimmunised with a recombinant p24-core protein from HIV-1. Two mAbs were epitope-mapped on overlapping peptides and characterised for their reactivity with non-fixed HIV-infected T cells by immunofluorescence staining and flow cytometric analysis. Their biological activities were studied for antibody-dependent cellular cytotoxicity (ADCC) and suppression of viral spread in vitro., Results: The epitopes of two selected mAbs were located on the amino terminal region of p24 in the regions 147-152 aa and 178-187 aa, respectively. The antibodies were able to react with living HIV-1 infected cells. The expression of the antigens was time-dependent after the infection of certain cell lines by HIV-1. The mAbs mediated a strong HIV-1-specific ADCC and were able to delay the spread of HIV-1 for about 6 days in cell cultures., Conclusions: Certain epitopes of the p24-core protein of HIV-1 can be expressed on living, HIV-infected T cells and are recognised by specific antibodies. Such antibodies can destroy infected cells by ADCC or delay the virus spread, and therefore, should be considered in immunisation strategies against HIV.

Determination of percentages of CD4+ and CD8+ T cells from patients with human immunodeficiency virus infection is usually done by flow cytometric analysis. We compared a cell marker ELISA with flow cytometry for quantitation of CD4 and CD8 molecules on T lymphocytes, and correlated the values both with the number of CD4+ and CD8+ T lymphocytes and with clinical data. Results by cell marker ELISA (y) correlated well with those by flow cytometric analysis (x); r = 0.69, P < 0.001 (y = 0.01x + 3.9) for CD4; r = 0.81, P < 0.001 (y = 0.03x + 5.4) for CD8; n = 343. The ELISA detected changes in numbers of CD8 molecules on the cells earlier than flow cytometry recognized changes in CD8+ T-cell counts. The advantages of the ELISA are the small sample volume required (0.5 mL of blood), its internal standardization by a CD4+/CD8+ cell line, and its simple and fast performance. The cell marker ELISA appears to be an efficient alternative to flow cytometry.

Murine monoclonal antibodies (Mabs) to the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were tested for their ability to inhibit the replication and spread of the virus in permanent cell cultures (Molt4/8, K37, H9) and in the culture of II-2 stimulated T cells of healthy donors. After addition of ascitic fluid containing monoclonal anti-p24 antibodies or purified anti-p24 antibodies or the respective control to co-cultures of infected and non-infected cells, HIV-1 replication was evaluated by determining the percentage of infected cells and the activity of reverse transcriptase (RT) in cell-free supernatant. In addition, the supernatant's infectivity was determined. FACS analysis demonstrated p24 antigen in about 40% of unfixed HIV-1 infected cells at the cell membrane. Monoclonal anti-p24 antibodies of different epitope specificity added to the cells but not to the virus delayed the spread of HIV-1 infection in permanent cell culture. Furthermore, anti-p24 Mabs inhibited the release of RT-active virus particles by HIV-1 infected cell lines or II-2 stimulated T-lymphocytes, respectively, up to 60%. The mode of action of anti-p24 antibodies after HIV-1 infection is discussed on the basis of the data obtained.

Out of 100 psychotic patients who underwent neurobiochemical examination, in 24 cases activity of phenylalanine hydroxylase in the thrombocytes was found to be absent. 38% of these cases were susceptible to stimulation in vitro by means of folic acid derivative, without actually reaching a normal level of activity. The patients are presented first as a homgeneous group, then in the two major diagnostic divisions, and divided according to the biochemical data. An account is given of early experiences in the treatment of the more intractable cases.

Some cytochemical methods for determination of monocytes, neutrophilic and eosinophilic granulocytes were modified on the basis of manifestation of the respective marker enzymes (alpha-naphthyl-acetatesterase, naphthol-AS-D-chloracetatesterase and cyano-resistant peroxidase). The modification presented formation of economic methods by using some common reagents for identification of single esterases and peroxidases.

Beta-Phenylethylamine can be detected in the cisternal cerebrospinal fluid of healthy controls and acute endogenous psychoses. One group of the patients shows the same concentrations as the psychiatrically healthy controls. In the other groups beta-phenylethylamine was found to be elevated in some cases up to a very high level. The results suggest a link between a disturbance of the phenylethylamine pathway and certain endogenous psychoses.

LSD (10(-7)M--10(-11)M) and 5-methoxy-N,N-dimethyltyptamine (10(-7)--10(-9)M) inhibited the high affinity uptake of [3H]-5HT into synaptosomes. These results suggest that the inhibition of the high affinity uptake is involved in the psychotomimetic action of LSD and 5MeODMT.

The pattern of gangliosides in membranes of erythrocytes was examined in healthy donors, in acute schizophrenics without neuroleptic treatment and in alcohol-dependent patients. 7 different gangliosides could be detected after extraction, purification by column chromatography and fractionation by TLC. Healthy donors were characterized by the following pattern of gangliosides: GX = 5.8%, GT1b = 6.7%, FucGD1b = 5.2%, und GD1a = 12.6%, GD3 = 9.2%, SPG = 43.5% and GM3 = 17.0%. In schizophrenic patients the GM3- and GD3-fraction were increased. No difference was found between the control group and the alcoholics.

For the investigated indolamines, the recovery following extraction with n-butanol and acetic acid ethyl ester from alkaline solution amounted to 80-90%. Using benzene only the two 5-methoxyindolamines were transferred from the alkaline medium into the organic phase. The reaction of the o-phthaldialdehyde with substituted indolamines was optimized in regard to higher fluorimetric detection sensitivity. The lower limit of determination was 2.0 ng for 5HT and 5OHDMT, and 1.0 ng for 5MeOT and 5MeODMT.

Radioiododestannylations was employed to prepare a series of four specifically labeled thienyl alcohols: 1-(5-iodo-2-thienyl)-cyclopentan-1-ol and -cyclohexan-1-ol; 17 alpha-(5-iodo-2-thienyl)-17 beta-estradiol and -estradiol-3-O-methyl ether. The method utilized 5-(trimethylstannyl)thienyl intermediates which had been prepared in good yields from 2,5-bis(trimethylstannyl)thiophene and the appropriate cyclic ketones. The trimethylstannyl substrates reacted with no-carrier-added Na125I in the presence of 30% H2O2-acetic acid (2:1) at pH 4.5 at ambient temperature for 5-15 min to give, after HPLC separation, the desired 5-[125I]iodothien-2-yl products in greater than 90% isolated yields. Although tissue distribution studies in rats were uneventful, the methodology employed to give the labeled products possesses distinct advantages compared to alternative methods for preparing radioiodinated aryl moieties.

Rats received 20, 50 or 100 mg/animal D-penicillamine i.p. twice daily on days 15, 16, 17, 18, and 19 of gestation, i.e. a total dose of 200, 500 resp. 1000 mg/animal. At all dosages the number of fetal resorptions did not increase significantly. Weight of the 20 day old embryos as well as length of the long bones in the extremities in the 100 mg-group showed a significant decrease. Numerous skeletal alterations could be observed in the 1000 mg-group such as absence, deformations or incomplete mineralisation of bones. Light microscopic examinations revealed an inhibition of the ossification as well as a decrease of number and size of the trabecula and of the thickness of the perichondrial bone sheath. A swelling of the collagenous fibrils can be demonstrated with the electron microscope. The first apatite crystals aggregate in collagen-free areas. The fusion of these aggregates to homogenously mineralized trabecula is inhibitied. In contrast to bones from untreated embryos, mineralized areas show varying content of collagen and apatide crystals. A regular spatial relationship between apatite crystals and collagenous fibrils does not develop. These findings show that even after the so-called "critical period" malformations can be pproduced by substances which disturb synthesis and maturation of the mesenchymal intercellular substance.

A rapid and simple method is described for separation of intact synaptosomes, synaptic plasma membranes and vesicles. Two synaptosome fractions were obtained by modified differential centrifugation. The rate zonal zentrifugation in a linear sucrose gradient (very low density) is suitable to obtain fractions highly enriched in synaptic plasma membranes and vesicles. Examination of the prepared fractions was done by enzyme marker activities and electron microscopy

Considering that platelet phenylalanine hydroxylase represents a rather high percentage of the total body hydroxylating activity, the encountered deficiency may very well be considered as one of the exacerbing factors of psychosis of this type. Further biochemical and clinical characterization of the described subgroup is in progress.