Radiation Damage of Myoglobin Crystals in Weak Stationary Electric and Magnetic Fields.

Radiation damage is one of the bottlenecks in the field of structural biology. Cryo-cooling of protein crystals provided a breakthrough in the 1980s and resulted in significant reductions in radiation damage. Other factors positively influencing the progression of damage include the application of radical scavengers and reductions in the experimental beam size. Here we study the impact on radiation damage of applying static magnetic and electric fields during protein diffraction experiments, ultimately probing the Lorenz force effect on primary photoelectrons and secondary Auger electrons, which both contribute to the damage process. The design of a special mounting pin using graphene for applying electric fields on a crystalline sample is described. Analyses of myoglobin protein crystals exposed to the fields of ~40 mT and -300 V show a slower global radiation damage rate and also changes in the progression of specific damage process on the molecular level, in particular at doses extending beyond the Garman limit of 30 MGy.

ADRA1A gene is associated with BMI in chronic schizophrenia patients exposed to antipsychotics.

Noradrenaline and adrenaline are neurotransmitters of the sympathetic nervous system that interact with various adrenergic receptor (ADR) subtypes, and this regulates the basal metabolic rate, thermogenesis and efficiency of energy utilization. We examined a possible role of the gene coding for ADRA1A receptor in weight gain in schizophrenia subjects exposed to antipsychotics. A total of 401 schizophrenia in-patients treated with antipsychotics for >2 years were recruited and a final 394 DNA samples were genotyped. Their body mass indexes (BMIs) were recorded for 12 months and parameterized to be correlated in regression. Among the 58 single-nucleotide polymorphisms (SNPs) genotyped, 44 valid SNPs, which had minor allele frequency > or =0.03, were analyzed in statistics. Linear regression model with age, gender, diabetes, use of typical antipsychotics and use of atypical antipsychotics as covariates, with or without gender interaction, showed evidence of associations between the ADRA1A gene and BMI. Most of the SNPs associated with BMI are located in the promoter and intron regions, and being female appeared to enhance the gene effect. Our study suggests that the ADRA1A gene is involved in weight gain among schizophrenia patients treated with antipsychotics. Further molecular dissection of the ADRA1A gene warrants better understanding on weight gain mechanisms in schizophrenia.

Performance of a neuro-fuzzy model in predicting weight changes of chronic schizophrenic patients exposed to antipsychotics.

Artificial intelligence has become a possible solution to resolve the problem of loss of information when complexity of a disease increases. Obesity phenotypes are observable clinical features of drug-naive schizophrenic patients. In addition, atypical antipsychotic medications may cause these unwanted effects. Here we examined the performance of neuro-fuzzy modeling (NFM) in predicting weight changes in chronic schizophrenic patients exposed to antipsychotics. Two hundred and twenty inpatients meeting DSMIV diagnosis of schizophrenia, treated with antipsychotics, either typical or atypical, for more than 2 years, were recruited. All subjects were assessed in the same study period between mid-November 2003 and mid-April 2004. The baseline and first visit's physical data including weight, height and circumference were used in this study. Clinical information (Clinical Global Impression and Life Style Survey) and genotype data of five single nucleotide polymorphisms were also included as predictors. The subjects were randomly assigned into the first group (105 subjects) and second group (115 subjects), and NFM was performed by using the FuzzyTECH 5.54 software package, with a network-type structure constructed in the rule block. A complete learned model trained from merged data of the first and second groups demonstrates that, at a prediction error of 5, 93% subjects with weight gain were identified. Our study suggests that NFM is a feasible prediction tool for obesity in schizophrenic patients exposed to antipsychotics, with further improvements required.

Prediction of optimal lithium doses for Taiwanese psychiatric patients.

BACKGROUND: Standard dosing regimens with lithium usually require significant time before the optimal serum concentration is achieved. METHOD: The present study compares a new method (Method 1) with two existing ones (Methods 2 and 3) for dosing of individual patients with lithium, to more rapidly achieve optimum concentrations. Thirty patients, who were on lithium therapy and who met the inclusion criteria, were evaluated. RESULTS: The correlation coefficient between true and expected concentrations were 0.518 (P < 0.01), 0.555 (P < 0.01) and 0.424 (P = 0.019) respectively. The mean +/- standard deviation of the difference from the expected concentrations of three methods were 0.02 +/- 0.20 mEq/L, 0.31 +/- 0.22 and -0.27 +/- 0.25 respectively. CONCLUSION: Method 1 was the least biased and most accurate among three methods for predicting the optimal therapeutic lithium dose in Taiwanese psychiatric patients.

Crystal structures of a novel ferric reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus and its complex with NADP+.

BACKGROUND: Studies performed within the last decade have indicated that microbial reduction of Fe(III) to Fe(II) is a biologically significant process. The ferric reductase (FeR) from Archaeoglobus fulgidus is the first reported archaeal ferric reductase and it catalyzes the flavin-mediated reduction of ferric iron complexes using NAD(P)H as the electron donor. Based on its catalytic activity, the A. fulgidus FeR resembles the bacterial and eukaryotic assimilatory type of ferric reductases. However, the high cellular abundance of the A. fulgidus FeR (approximately 0.75% of the total soluble protein) suggests a catabolic role for this enzyme as the terminal electron acceptor in a ferric iron-based respiratory pathway [1]. RESULTS: The crystal structure of recombinant A. fulgidus FeR containing a bound FMN has been solved at 1.5 A resolution by multiple isomorphous replacement/ anomalous diffraction (MIRAS) phasing methods, and the NADP+- bound complex of FeR was subsequently determined at 1.65 A resolution. FeR consists of a dimer of two identical subunits, although only one subunit has been observed to bind the redox cofactors. Each subunit is organized around a six-stranded antiparallel beta barrel that is homologous to the FMN binding protein from Desulfovibrio vulgaris. This fold has been shown to be related to a circularly permuted version of the flavin binding domain of the ferredoxin reductase superfamily. The A. fulgidus ferric reductase is further distinguished from the ferredoxin reductase superfamily by the absence of a Rossmann fold domain that is used to bind the NAD(P)H. Instead, FeR uses its single domain to provide both the flavin and the NAD(P)H binding sites. Potential binding sites for ferric iron complexes are identified near the cofactor binding sites. CONCLUSIONS: The work described here details the structures of the enzyme-FMN, enzyme-FMN-NADP+, and possibly the enzyme-FMN-iron intermediates that are present during the reaction mechanism. This structural information helps identify roles for specific residues during the reduction of ferric iron complexes by the A. fulgidus FeR.

Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry.

The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 molecular and fragment ion mass values of high accuracy. Applying these spectra to the detection and localization of errors and modifications in the DNA-derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli. Direct fragmentation of the multiply-charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences. For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses. Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue. For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide. The first 107 residues of ThiC (74 kDa) were shown to be correct, with C-terminal heterogeneity indicated. For ThiG (predicted Mr = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop codon for the first protein. MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences.

Twelve-nucleotide repeat polymorphism of D4 dopamine receptor gene in Chinese familial schizophrenic patients.

BACKGROUND: Disturbances in dopaminergic transmission have been implicated in the etiology of schizophrenia. Catalano et al reported an association between delusional disorder and the number of a 12-nucleotide (bp) repeat sequence in the first exon of dopamine D4 receptor gene (DRD4), which indicated a possible role of this polymorphism in the pathogenesis of psychotic disorders. METHODS: DNA of 42 Chinese controls, 50 sporadic schizophrenic patients, and 30 familial schizophrenic probands were collected. Genotype of the 12-bp repeat polymorphism of DRD4 was determined with polymerase chain reaction and agarose gel electrophoresis. Genotypic and allelic frequencies were compared among the three groups using the chi-square test. RESULTS: Forty-three (86%) sporadic schizophrenic patients, 25 (83.3%) familial schizophrenic probands, and 35 (83.3%) controls were A1 (two 12-bp repeat) homozygotes. One (2.0%) sporadic schizophrenic and 1 familial schizophrenic patient were A2 (one 12-bp repeat) homozygotes. There was no significant difference in allelic and genotypic distributions among the three groups. CONCLUSIONS: The present data do not support an association between schizophrenia and any specific allele of the 12-bp repeat polymorphism of DRD4. Significance of familial/sporadic division of schizophrenia cannot be supported regarding this repeat polymorphism.

Association study of serotonin-6 receptor variant (C267T) with schizophrenia and aggressive behavior.

The relative abundance of serotonin type 6 receptors (5-HT6) in some limbic regions and the high affinity of some antipsychotics to 5-HT6 receptors suggest that they might be involved in the pathogenesis of schizophrenic disorders. In a population-based association study, we tested the hypothesis that the allelic variant, C267T, of the human 5-HT6 gene confers susceptibility to schizophrenic disorders and associated aggressive behavior. We genotyped 5-HT6 receptors in 186 patients with schizophrenic disorders and 163 controls. The results demonstrated no significant difference in genotype or allele frequencies between patients with or without aggressive behaviors. However, genotype distribution was significantly different between schizophrenic patients and control subjects. This suggests that the 5-HT6 gene may play a role in the pathogenesis of schizophrenic disorders.

A double-blind comparative study of clozapine versus chlorpromazine on Chinese patients with treatment-refractory schizophrenia.

Clozapine has been shown to have superior effectiveness compared with classic neuroleptics in treating refractory schizophrenia in Caucasians, but its efficacy and safety in Chinese have not been adequately studied. Forty Chinese schizophrenic patients were recruited in a 12-week, double-blind, comparative trial. Twenty-one patients were randomly assigned to clozapine treatment and 19 to chlorpromazine treatment. The average dose was 543 +/- 157 and 1163 +/- 228 mg/day for clozapine and chlorpromazine, respectively. The results showed that six clozapine-treated patients (28.6%) had more than 20% improvement in Brief Psychiatric Rating Scale score and were classified as responders, whereas none of the chlorpromazine-treated patients was classified as a responder. The degree of improvement in positive symptoms, negative symptoms and Brief Psychiatric Rating Scale scores in the clozapine group was inversely correlated with the severity of negative symptoms at entry into the trial. Two clozapine-treated patients were withdrawn from the study, one because of leukopenia and nausea, and the other because of vomiting and hypotension. Chlorpromazine treatment was prematurely discontinued in two patients, because of jaundice and over sedation in one, and because of severe weight loss in the other (9 kg). The rate of moderate-to-severe sialorrhea was high in clozapine-treated patients (28.6%). Two clozapine-treated patients and two chlorpromazine-treated patients showed significant improvement in previously existing tardive dyskinesia and one chlorpromazine-treated patient exhibited aggravation of tardive dyskinesia. The results of this study indicate that clozapine treatment might have advantages over chlorpromazine for Chinese schizophrenic patients who are refractory to typical neuroleptic treatment.

Association study of the p53-gene Pro72Arg polymorphism in schizophrenia.

The p53 tumor-suppressor gene, encoding a phosphoprotein, is a key element in maintaining genomic stability and cell apoptosis. It is also implicated in nervous-system development. In order to examine the role of the p53 gene for the pathogenesis of schizophrenic disorders, patients (n=155) and control subjects (n=168) were genotyped for the p53-Pro72Arg polymorphism. The results demonstrated no association with schizophrenia and/or age of onset for this polymorphism.

Severity of psychosis syndrome and change of metabolic abnormality in chronic schizophrenia patients: severe negative syndrome may be related to a distinct lipid pathophysiology.

BACKGROUND: Metabolic abnormality is common among schizophrenia patients. Some metabolic traits were found associated with subgroups of schizophrenia patients. OBJECTIVES: We examined a possible relationship between metabolic abnormality and psychosis profile in schizophrenia patients. METHOD: Three hundred and seventy-two chronic schizophrenia patients treated with antipsychotics for more than 2 years were assessed with the Positive and Negative Syndrome Scale. A set of metabolic traits was measured at scheduled checkpoints between October 2004 and September 2006. RESULTS: Multiple regressions adjusted for sex showed negative correlations between body mass index (BMI) and total score and all subscales; triglycerides (TG) was negatively correlated with total score and negative syndrome, while HDLC was positively correlated with negative syndrome. When sex interaction was concerned, total score was negatively correlated with BMI but not with others; negative syndrome was negatively correlated with BMI and positively with HDLC. No metabolic traits were correlated with positive syndrome or general psychopathology. CONCLUSIONS: Loss of body weight is a serious health problem in schizophrenia patients with severe psychosis syndrome, especially the negative syndrome. Schizophrenia patients with severe negative syndrome may have a distinct lipid pathophysiology in comparison with those who were less severe in the domain.

Paroxetine in the treatment of Chinese patients with depressive episode: a double-blind randomized comparison with imipramine.

BACKGROUND: Paroxetine is a potent inhibitor of serotonin re-uptake. Although it has been widely used as an antidepressant in western countries, its efficacy and side effects in the Chinese are unknown. METHODS: Patients with major depressive episode were recruited from the outpatient clinic and the acute wards of the Department of Psychiatry, Veterans General Hospital-Taipei in 1994. Severity of depression was evaluated with Hamilton Rating Scale for Depression (HAM-D), Clinical Global Impression (CGI) and the adverse effects were evaluated with Treatment Emergent Symptom Scale (TESS). Forty Chinese patients with HAM-D scores > or = 18 at the beginning of the first dose were randomized to receive paroxetine (20 to 30 mg/day) or imipramine (100 to 125 mg/day). The dosage was designed as a fixed-adjustable regimen for an active treatment period of six weeks. The patients and the clinical investigators were both blind to the medication until the end of the trial. RESULTS: Five patients violating the trial protocol were excluded from this study, leaving 35 patients for efficacy and adverse effect analyses. Sixty-seven percent (12/18) of paroxetine-treated patients and 65% (11/17) of imipramine-treated patients showed a 50% or more reduction in HAM-D scores. The rate of patients whose mood recovered to normal or near-normal (borderline) was higher in the paroxetine group (66.7%) than in the imipramine group (35.3%), but the difference was not statistically significant (p = 0.13). At the end of this trial, the mean reduction of HAM-D scores was similar between groups (20.2 +/- 9.1 vs 15.3 +/- 8.4, p > 0.1). Three patients in the paroxetine group and two patients in the imipramine group withdrew prematurely due to adverse effects of impatience. Adverse effects of anticholinergic effects were reported more frequently in the imipramine group than in the paroxetine group (p = 0.01). CONCLUSIONS: In comparison with imipramine, paroxetine affords fewer anticholinergic adverse effects without the sacrifice of efficacy in the treatment of depressed Chinese patients.

Characterization of the Bacillus subtilis thiC operon involved in thiamine biosynthesis.

The characterization of a three-gene operon (the thiC operon) at 331 min, which is involved in thiamine biosynthesis in Bacillus subtilis, is described. The first gene in the operon is homologous to transcription activators in the lysR family. The second and third genes (thiK and thiC) have been subcloned and overexpressed in Escherichia coli. ThiK (30 kDa) catalyzes the phosphorylation of 4-methyl-5-(beta-hydroxyethyl)thiazole. ThiC (27 kDa) catalyzes the substitution of the pyrophosphate of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate to yield thiamine phosphate. Transcription of the thiC operon is not regulated by thiamine or 2-methyl-4-amino-5-hydroxymethylpyrimidine and is only slightly repressed by 4-methyl-5-(beta-hydroxyethyl)thiazole.

Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution.

The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution. Thiamin phosphate synthase is an alpha/beta protein with a triosephosphate isomerase fold. The active site is in a pocket formed primarily by the loop regions, residues 59-67 (A loop, joining alpha3 and beta2), residues 109-114 (B loop, joining alpha5 and beta4), and residues 151-168 (C loop, joining alpha7 and beta6). The high-resolution structure of thiamin phosphate synthase complexed with its reaction products described here provides a detailed picture of the catalytically important interactions between the enzyme and the substrates. The structure and other mechanistic studies are consistent with a reaction mechanism involving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate at the active site to give the pyrimidine carbocation. Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction. The ionization step is catalyzed by orienting the C-O bond perpendicular to the plane of the pyrimidine, by hydrogen bonding between the C4' amino group and one of the terminal oxygen atoms of the pyrophosphate, and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme.

Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase.

Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme-substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures.

Thiamin biosynthesis in Escherichia coli. Identification of ThiS thiocarboxylate as the immediate sulfur donor in the thiazole formation.

ThiFSGH and ThiI are required for the biosynthesis of the thiazole moiety of thiamin in Escherichia coli. The overproduction, purification, and characterization of ThiFS and the identification of two of the early steps in the biosynthesis of the thiazole moiety of thiamin are described here. ThiS isolated from E. coli thiI+ is post-translationally modified by converting the carboxylic acid group of the carboxyl-terminal glycine into a thiocarboxylate. The thiI gene plays an essential role in the formation of the thiocarboxylate because ThiS isolated from a thiI- strain does not contain this modification. ThiF catalyzes the adenylation by ATP of the carboxyl-terminal glycine of ThiS. This reaction is likely to be involved in the activation of ThiS for sulfur transfer from cysteine or from a cysteine-derived sulfur donor.

Serotonin 6 receptor polymorphism in schizophrenia: frequency, age at onset and cognitive function.

The relative abundance of serotonin 6 receptor (5HT6) in some limbic regions and the high affinity of some antipsychotics for 5HT6 suggest that the 5HT6 gene might play a role in the pathogenesis of schizophrenic disorders. A recent study reported an association between a C267T polymorphism of the 5HT6 gene and schizophrenia. In order to test whether the 5HT6 gene plays a role in the pathogenesis of schizophrenic disorders, patients (n = 148) and control subjects (n = 160) were genotyped for 5HT6. We also investigated the relationship between genotypes and patients' age at onset and cognitive function in schizophrenic patients. Cognitive function in the patients was evaluated by the Mini-Mental State Examination (MMSE). The results demonstrated no significant differences in genotype or allele frequencies between controls and patients. In the patient group, age at onset and MMSE score did not differ significantly among the three 5HT6 genotpyes. The results of this study suggest that the 5HT6 C267T polymorphism plays no major role in susceptibility to the development of schizophrenia and is not related to cognitive impairment or age at onset in schizophrenic patients. Further studies of the relation between 5HT6 polymorphism and the symptoms and the therapeutic response in schizophrenic patients may help to elucidate the role of 5HT6 in the pathogenesis of schizophrenia.

No evidence for association of alpha 1a adrenoceptor gene polymorphism and clozapine-induced urinary incontinence.

Clozapine is an effective atypical antipsychotic that has high affinity for many neurotransmitter receptors. Among the adverse effects of clozapine, urinary incontinence is commonly found and is suggested to be caused by alpha-adrenergic blockade. We tested the hypothesis that clozapine-induced urinary incontinence is related to a genetic variant of the alpha(1a)-adrenoceptor. We also tested whether the alpha(1a)-receptor gene confers susceptibility to schizophrenic disorders. Our result indicated that the alpha(1a)-adrenoceptor gene polymorphism investigated plays no major role in the pathogenesis of schizophrenia or in clozapine-induced urinary incontinence. Considering the superior effects of clozapine and its potent adrenergic antagonistic effects, it is of interest to investigate the association between this polymorphism and the treatment response.

Apolipoprotein E genotype and schizophrenia.

Schizophrenic disorders are complex genetic disorders and may involve multiple genes of small effect. The presence of apolipoprotein E (apoE) is associated with several neuropsychiatric disorders. Previous studies on apoE genotype distribution in schizophrenia have reported conflicting findings. We studied the genotype frequencies in a large group of schizophrenic patients. The genotype distribution was significantly different between the schizophrenic patients and the control subjects. Persons who were sigma3 carriers have an increased risk of schizophrenia. This result suggests that apoE isoforms may play a functional role in the pathogenesis of schizophrenic disorders. Some possible mechanisms regarding the effect of apoE on the development of schizophrenia are discussed.

Thiamin biosynthesis in prokaryotes.

Twelve genes involved in thiamin biosynthesis in prokaryotes have been identified and overexpressed. Of these, six are required for the thiazole biosynthesis (thiFSGH, thil, and dxs), one is involved in the pyrimidine biosynthesis (thiC), one is required for the linking of the thiazole and the pyrimidine (thiE), and four are kinase genes (thiD, thiM, thiL, and pdxK). The specific reactions catalyzed by ThiEF, Dxs, ThiDM, ThiL, and PdxK have been reconstituted in vitro and ThiS thiocarboxylate has been identified as the sulfur source. The X-ray structures of thiamin phosphate synthase and 5-hydroxyethyl-4-methylthiazole kinase have been completed. The genes coding for the thiamin transport system (thiBPQ) have also been identified. Remaining problems include the cloning and characterization of thiK (thiamin kinase) and the gene(s) involved in the regulation of thiamin biosynthesis. The specific reactions catalyzed by ThiC (pyrimidine formation), and ThiGH and ThiI (thiazole formation) have not yet been identified.