Cannabidiol enhances anandamide signaling and alleviates psychotic symptoms of schizophrenia.

Cannabidiol is a component of marijuana that does not activate cannabinoid receptors, but moderately inhibits the degradation of the endocannabinoid anandamide. We previously reported that an elevation of anandamide levels in cerebrospinal fluid inversely correlated to psychotic symptoms. Furthermore, enhanced anandamide signaling let to a lower transition rate from initial prodromal states into frank psychosis as well as postponed transition. In our translational approach, we performed a double-blind, randomized clinical trial of cannabidiol vs amisulpride, a potent antipsychotic, in acute schizophrenia to evaluate the clinical relevance of our initial findings. Either treatment was safe and led to significant clinical improvement, but cannabidiol displayed a markedly superior side-effect profile. Moreover, cannabidiol treatment was accompanied by a significant increase in serum anandamide levels, which was significantly associated with clinical improvement. The results suggest that inhibition of anandamide deactivation may contribute to the antipsychotic effects of cannabidiol potentially representing a completely new mechanism in the treatment of schizophrenia.

Generation and characterization of electron bunches with ramped current profiles in a dual-frequency superconducting linear accelerator.

We report on the successful experimental generation of electron bunches with ramped current profiles. The technique relies on impressing nonlinear correlations in the longitudinal phase space using a superconducing radio frequency linear accelerator operating at two frequencies and a current-enhancing dispersive section. The produced ~700-MeV bunches have peak currents of the order of a kilo-Ampère. Data taken for various accelerator settings demonstrate the versatility of the method and, in particular, its ability to produce current profiles that have a quasilinear dependency on the longitudinal (temporal) coordinate. The measured bunch parameters are shown, via numerical simulations, to produce gigavolt-per-meter peak accelerating electric fields with transformer ratios larger than 2 in dielectric-lined waveguides.

Retinal microstructure in patients with EFEMP1 retinal dystrophy evaluated by Fourier domain OCT.

OBJECTIVES: To investigate retinal microstructure of patients affected with malattia leventinese (MLVT) and mutation in the EFEMP1 gene using high-resolution optical coherence tomography (OCT). METHODS: Patients diagnosed with MLVT received a comprehensive eye exam, full-field and multifocal electroretinogram testing and imaging with a high-resolution Fourier domain OCT (Fd-OCT, UC Davis Medical Center, Davis, USA; axial resolution: 4.5 microm, acquisition speed: 9 frames s(-1), 1000 A scans s(-1)) combined with a flexible scanning head (Bioptigen Inc. Durham, NC, USA). RESULTS: Two related patients aged 30 and 60 years, with MLVT and identified c.R345W mutation in the EFEMP1 gene, were tested. Mother and daughter showed a variable phenotype with reduced vision function in the younger patient, whereas the mother had a 'form frustre'. Fd-OCT revealed extensive or focal sub-retinal pigment epithelium (RPE) deposits, separation of RPE and Bruch's membrane, and disruption of the photoreceptor outer and inner segment layers. No outer retinal changes were visible outside areas with sub-RPE deposits. CONCLUSION: Retinal structure in EFEMP1 retinal dystrophy is reflected by morphological changes within the RPE/Bruch's membrane complex with accumulation of sub-RPE material associated with disrupted photoreceptor integrity. The pattern of microstructural retinal abnormalities is similar but with a different extent in patients with variable phenotypes.

Independent protein-profiling studies show a decrease in apolipoprotein A1 levels in schizophrenia CSF, brain and peripheral tissues.

Although some insights into the etiology of schizophrenia have been gained, an understanding of the illness at the molecular level remains elusive. Recent advances in proteomic profiling offer great promise for the discovery of markers underlying pathophysiology of diseases. In the present study, we employed two high-throughput proteomic techniques together with traditional methods to investigate cerebrospinal fluid (CSF), brain and peripheral tissues (liver, red blood cells and serum) of schizophrenia patients in an attempt to identify peripheral/surrogate disease markers. The cohorts used to investigate each tissue were largely independent, although some CSF and serum samples were collected from the same patient. To address the major confounding factor of antipsychotic drug treatment, we also included a large cohort of first-onset drug-naive patients. Apolipoprotein A1 (apoA1) showed a significant decrease in expression in schizophrenia patients compared to controls in all five tissues examined. Specifically, using SELDI-TOF mass spectrometry, apoA1 was found decreased in CSF from schizophrenia patients (-35%, P=0.00001) and, using 2D-DIGE, apoA1 was also found downregulated in liver (-30%, P=0.02) and RBCs (-60%, P=0.003). Furthermore, we found a significant reduction of apoA1 in sera of first-onset drug-naive schizophrenia patients using enzyme-linked immunosorbent assay (-18%, P=0.00008) and in two investigations of post-mortem brain tissue using western blot analysis (-35%, P=0.05; -51%, P=0.05). These results show that apoA1 is consistently downregulated in the central nervous system as well as peripheral tissues of schizophrenia patients and may be linked to the underlying disease mechanism.

[Efficacy and tolerability of quetiapine in hospitalised schizophrenic patients during the acute treatment phase: a PMS study]. / Wirksamkeit und Verträglichkeit von Quetiapin in der stationären Akutbehandlung schizophrener Psychosen: Eine Anwendungsbeobachtung.

We performed a post marketing surveillance study (PMS study) to reveal the efficacy and tolerability of orally administered quetiapine in the treatment of acute psychosis over a period of up to three weeks. 398 respective inpatients were assessed in 88 psychiatric departments in Germany by use of the Clinical Global Impression Scale (CGI) and the Brief Psychiatric Rating Scale (BPRS) as well as the clinical impression of psychiatric raters. Safety and tolerability were assessed by vital parameters such as blood pressure, hearth rate and weight as well as the clinical impression of the psychiatric raters. In addition, dosing, concomitant and/or previous pharmacotherapies as well as certain aspects of psychiatric and medical history were documented. A significant reduction of psychopathology was found during three weeks of drug treatment. Daily dosages of quetiapine between 400 and 1200 mg were well tolerated with a limited number of adverse and no serious adverse events. Noteworthy, more than 35 % of all patients received and tolerated excellently more than 800 mg of quetiapine per day under naturalistic treatment conditions, well above the approved maximum daily dosage. This study reflects the clinical efficacy and good tolerability of quetiapine under real world treatment conditions and is in line with the results of the controlled clinical trials of phase II and III.

Photodynamic therapy for choroidal neovascularization in patients with multifocal choroiditis and panuveitis.

PURPOSE: To evaluate the visual benefit of photodynamic therapy (PDT) with verteporfin in patients with choroidal neovascularization (CNV) secondary to multifocal choroiditis and panuveitis over a longer follow-up period. METHODS: A total of 14 eyes of 12 patients (mean age 34 years) with a classic subfoveal CNV (13/14) or juxtafoveal CNV (1/14) were treated with PDT. Visual outcome was assessed by best-corrected visual acuity (VA). Morphologic characteristics of CNVs such as localization, size, and activity were monitored by fluorescein angiography. RESULTS: Patients were followed for 3 to 45 months (mean 23 months). During this period, one to six PDTs (mean 2.4) were performed. At the time of the first PDT no acute inflammation was seen in the affected eyes. Improved or stabilized visual function (VA loss < or = 2 lines in the Early Treatment Diabetic Retinopathy Study chart) was observed in 71.4% of the eyes. A total of 78% of the eyes showed an inactive scar in the area of CNV after PDT. Treatment failure after PDT occurred due to uninfluenced CNV growth. No further complications were observed. CONCLUSIONS: PDT in subfoveal or juxtafoveal classic CNV secondary to multifocal choroiditis and panuveitis stabilized or improved VA in the majority of patients over a longer follow-up period. No risk factor for failed VA rehabilitation could be defined.

Impact of intravenous oxygen therapy on the expression of reticulocyte-type 15-lipoxygenase in human volunteers.

Intravenous oxygen infusion is used in complementary medicine to fight inflammatory disorders and repeated application of this therapeutic method leads to an increase in eosinophilic granulocytes in the peripheral blood. Since this subset of human peripheral leukocytes are known to express large amounts of the reticulocyte-type 15-lipoxygenase (15-lipoxygenase 1), which was suggested to exhibit anti-inflammatory activities, we profiled expression of this enzyme in the peripheral blood during the time course of typical oxygen infusion therapy. For this purpose seven volunteers were treated with intravenous infusion of oxygen gas for 4 weeks and the time-course of 15-lipoxygenase expression as well as the eosinophil count were monitored during and after the treatment interval. We found that 15-lipoxygenase 1 expression and the eosinophil count were significantly increased during the treatment period but returned to normal after the therapy was stopped. There was a striking correlation between the relative number of 15-lipoxygenase transcripts and the eosinophil counts suggesting eosinophils as major source of 15-lipoxygenase 1 expression. Since 15-lipoxygenase has been implicated in the resolving phase of acute inflammatory diseases the anti-inflammatory effects of intravenous oxygen infusion may be explained at least in part by our experimental findings.

Multiple ionization of atom clusters by intense soft X-rays from a free-electron laser.

Intense radiation from lasers has opened up many new areas of research in physics and chemistry, and has revolutionized optical technology. So far, most work in the field of nonlinear processes has been restricted to infrared, visible and ultraviolet light, although progress in the development of X-ray lasers has been made recently. With the advent of a free-electron laser in the soft-X-ray regime below 100 nm wavelength, a new light source is now available for experiments with intense, short-wavelength radiation that could be used to obtain deeper insights into the structure of matter. Other free-electron sources with even shorter wavelengths are planned for the future. Here we present initial results from a study of the interaction of soft X-ray radiation, generated by a free-electron laser, with Xe atoms and clusters. We find that, whereas Xe atoms become only singly ionized by the absorption of single photons, absorption in clusters is strongly enhanced. On average, each atom in large clusters absorbs up to 400 eV, corresponding to 30 photons. We suggest that the clusters are heated up and electrons are emitted after acquiring sufficient energy. The clusters finally disintegrate completely by Coulomb explosion.

Transient experimental anemia in cholesterol-fed rabbits induces systemic overexpression of the reticulocyte-type 15-lipoxygenase and protects from aortic lipid deposition.

Oxidative modification of low-density lipoprotein has been implicated in atherogenesis and the lipid peroxidizing enzyme 12/15-lipoxygenase (12/15-LOX) was suggested to be involved. For this study, we induced a strong and long-lasting systemic overexpression of the 15-LOX, in female New Zealand White rabbits by transient experimental anemia. After the hematopoietic parameters had returned to normal, these animals and age-matched controls were fed a lipid-rich Western-type diet for 10 weeks. Analyzing the lipid deposition in the aortic wall, we found that the 15-LOX overexpressing rabbits deposited significantly (P<0.01) less cholesteryl linoleate in the thoracic aorta than the corresponding controls. Similar results were obtained when free cholesterol and cholesteryl oleate were quantified. However, in the aortic arch where lipid deposition was much more severe a similar trend was observed, but the effects were not significant any more. Comparative determination (lipoxygenase overexpressing vs. control animals) of various plasma parameters as well as histological inspections of major organs did not reveal any indications for major organ malfunction. These data suggest that transient experimental anemia, which is accompanied by a long-lasting overexpression of the reticulocyte-type 15-LOX protects cholesterol-fed rabbits from lipid deposition in the aortic wall.

Structural basis for lipoxygenase specificity. Conversion of the human leukocyte 5-lipoxygenase to a 15-lipoxygenating enzyme species by site-directed mutagenesis.

Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the active-site volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "space-based" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S- via 8S- toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85-95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9 ,11, 14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5- and 15-lipoxygenases by site-directed mutagenesis and appear to support the space-based hypothesis of positional specificity.

Alterations in leukotriene synthase activity of the human 5-lipoxygenase by site-directed mutagenesis affecting its positional specificity.

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of lipoxygenases. Although the mechanistic basis of lipoxygenase specificity is not completely understood, sequence determinants for the positional specificity have been identified for various isoenzymes. In this study we altered the positional specificity of the human 5-lipoxygenase by multiple site-directed mutagenesis and assayed the leukotriene A(4) synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicos atetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A(4) as indicated by the dominant formation of leukotriene A(4) hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C(10), it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A(4) synthase activity. Surprisingly, we found that this quadruple mutant exhibited a similar leukotriene synthase activity as the wild-type enzyme in addition to its double oxygenation activity. The leukotriene synthase activity of the 8-lipoxygenating double mutant F359W + A424I was almost twice as high, and similar amounts of leukotriene A(4) hydrolysis products and double oxygenation derivatives were detected with this enzyme species. These data indicate that site-directed mutagenesis of the human 5-lipoxygenase that leads to alterations in the positional specificity favoring arachidonic acid 15-lipoxygenation does not suppress the leukotriene synthase activity of the enzyme. The residual 8-lipoxygease activity of the mutant enzyme and its augmented rate of 5-HpETE conversion may be discussed as major reasons for this unexpected result.

Early-onset severe rod-cone dystrophy in young children with RPE65 mutations.

PURPOSE: To describe the ocular phenotype of patients with RPE65 mutations in infancy and young childhood. METHODS: Four children from three families with severe early-onset visual impairment related to electrophysiologically detectable retinal dystrophy were screened for mutations in the RPE65 gene. Visual function from infancy to the age of 10 years was assessed with age-adapted methods. Clinical examinations and electroretinograms (ERGs) were also performed on the six parents. RESULTS: In all three families, patients were compound heterozygous for mutations of the RPE65 gene (ins144T/IVS1+5G-->A, R91W/Y368H, 1114delA+T457N/IVS1+5G-->A). Visual acuity was measurable in all patients at the age of 6 to 10 years, despite severe visual impairment noted during infancy and congenital nystagmus in three of the four patients. Photophobia was not a feature. Funduscopic changes were discrete, the most prominent finding being increased granularity in the macula and the periphery. Peripheral vision was well preserved, measured by Goldmann perimetry. Rod ERGs were not recordable, whereas cone ERGs were detectable in early childhood. All features taken together suggest a specific form of Leber congenital amaurosis (LCA) distinguishable on clinical grounds. ERGs were normal in five of the six parents. One father had an ERG compatible with congenital stationary night blindness unrelated to his heterozygous state for the RPE65 mutation. CONCLUSIONS: RPE65 mutations on both alleles may be associated with early-onset severe rod-cone dystrophy. Visual functions of the four patients were better than is usually seen in LCA, in particular in cases associated with retGC1 mutations. RPE65 mutations should be suspected in infants who appear to be blind in dim surroundings but react to objects in bright illumination and have nonrecordable rod ERGs and residual cone ERGs.

Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.

Impact of notch filter use on waveforms of First- and Second-Order-Kernel responses from multifocal ERGs.

The filter settings of the amplifier section of ERG recording systems have large impact on the waveforms of the recorded responses. In this study, the effects of a 50 Hz notch filter were assessed for both First-Order-Kernel- (FOK) and Second-Order-Kernel-responses (SOK 1st slice) from multifocal ERGs recorded with a VERIS III system. Amplitude and phase responses of the amplifier section were recorded for typical filter settings with the notch filter active and inactive. Multifocal ERGs (MERGs) from a group of 11 normal subjects were recorded for both amplifier settings, the waveforms resulted from those recordings were compared in the time and frequency domain. To verify the results, the recordings without the notch filter were digitally filtered with a simulated notch filter and compared to the responses recorded with an active analog notch filter. The line filter has the biggest attenuation at 50 Hz with an additional phase jump of 180 degrees. The FOK responses of MERGs are assembled by frequencies below 65 Hz with main spectral components between 19 and 47 Hz. The 1st slice of the SOK consists of frequencies up to 100 Hz with main components between 19 and 84 Hz. Thus, if FOK recordings are to be analyzed, the notch filter of the amplifier can be used in order to cope with noise problems caused by the line frequency of 50 Hz. However, one must be aware that the attenuation of higher frequency components will lead to changes of the waveforms. For SOK analysis, main spectral components are attenuated and/or shifted in phase, which leads to completely different waveforms and severe distortion of the recording results.

Shape and specificity in mammalian 15-lipoxygenase active site. The functional interplay of sequence determinants for the reaction specificity.

Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases. Based on the x-ray crystal structure of rabbit 15-lipoxygenase, Ile(593) appeared to be important in defining size and shape of the substrate-binding site in 15-lipoxygenases. We found that substitution of Ile(593) with alanine shifted the positional specificity of this enzyme toward 12-lipoxygenation. To compare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or large amino acids (phenylalanine-scan) at critical positions surrounding the putative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density within the substrate binding pocket in the rabbit 15-lipoxygenase increased the share of 12-lipoxygenase products, whereas a smaller active site favored 15-lipoxygenation. Simultaneous substitution of both large and small residues in the context of either a 15- or 12-lipoxygenase indicated that there is a functional interplay of the sequence determinants for lipoxygenation specificity. If the 15-lipoxygenase active site is enlarged excessively, however, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction.

Molecular analysis of 29 pyruvate kinase-deficient patients from central Europe with hereditary hemolytic anemia.

We investigated the DNA of 29 unrelated pyruvate kinase (PK) deficiency (PKD) patients from Central Europe with hereditary nonspherocytic hemolytic anemia for mutations in the PK-L/R gene. Among 58 potentially affected alleles, 53 mutations were identified, of which 17 were different from each other. Of these 17 mutations, 13 were single-nucleotide (nt) substitutions resulting in amino acid exchanges, G787A (Gly263-Arg), G994A (Gly332-Ser), G1006T (Ala336-Ser), G1010A (Arg337-Gln), A1081G (Asn361-Asp), G1127T (Ser376-Ile), G1174A (Ala392-Thr), G1281T (Glu427-Asp), C1454T (Ser485-Phe), C1456T (Arg486-Trp), G1493A (Arg498-His), G1529A (Arg510-Gin), and C1594T (Arg532-Trp); 1 in-frame triplet deletion, 1060delAAG (delLys354); 1 in-frame triplet insertion, 1203insAGC (insSer after Cys401); 1 splicesite mutation, 101-1G-A; and 1 frameshift deletion, 628delGT. Six mutations, 628delGT, G787A, G1010A, G1127T, G1281T, and C1454T, are described for the first time. To test the hypothesis of a single origin of the most common PK mutation in the European population, G1529A, we investigated all patients at four polymorphic sites in the PK-L/R gene: C/A at nt 1705, C/T at nt 1992, the (ATT)n microsatellite in intron J, and a polymorphism (T)10/(T)19 in intron I. Nine patients homozygous for mutation G1529A were consistent in all four markers. In the group of patients homozygous for mutation G1529A, the hematologic parameters and clinical manifestations have been studied in detail. Although having an identical mutation in the PK-L/R gene, the patients are affected differently. Their appearance ranges from a very mild compensated hemolysis to a severe anemia. Possible molecular explanations are discussed.