H(2)O(2) increases de novo synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin via GTP cyclohydrolase I and its feedback regulatory protein in vitiligo.

Patients with vitiligo accumulate up to 10(-3) mol/L concentrations of H(2)O(2) in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). De novo synthesis of 6BH(4) is dependent on the rate-limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH(4) and the essential amino acid L-phenylalanine. In the study presented here we wanted to investigate whether H(2)O(2) affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration-dependent regulation of rhGTPCHI where 100 micromol/L H(2)O(2) was the optimum concentration for the activation of the enzyme and >300 micromol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via L-phenylalanine and 6BH(4). In vitiligo a constant upregulation of 6BH(4) de novo synthesis results from epidermal build up of L-phenylalanine that is not controlled by H(2)O(2). Taking the results together, 6BH(4) de novo synthesis is controlled by H(2)O(2) in a concentration-dependent manner, but H(2)O(2)-mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex.

Effects of volume and concentration of lidocaine on epidural anaesthesia in pregnant females.

BACKGROUND AND OBJECTIVE: The effects of altering the concentration of a local anaesthetic on the development of epidural anaesthesia in pregnant females are unclear. We compared the anaesthetic effects of a constant dose of two different concentrations of epidural lidocaine for Caesarean section. METHODS: After Institutional Review Board approval and informed consent, patients undergoing elective Caesarean section were randomized to receive either lidocaine 1% 30 mL (+epinephrine 5 microg mL(-1)) or lidocaine 2% 15 mL (+epinephrine 5 microg mL(-1)) (n = 20 each) for epidural anaesthesia at the L1-L2 interspace. The spread of the sensory block to pinprick and the degree of motor block (modified Bromage scale) were measured at 5, 10, 15, 20 and 30 min after injection. RESULTS: No significant differences in the progression of analgesia and motor block were observed at any time between 1 and 2% lidocaine. The maximum cephalad spread was observed 30 min after injection; the median was at T4 (range T3-T5) and at T4 (range T3-T6) for lidocaine 1 and 2%, respectively. CONCLUSIONS: The same doses but different volumes of lidocaine 1 and 2% produced comparable anaesthetic effects in pregnant females. The effects of epidural anaesthesia depend primarily on the total dose of the local anaesthetic.

Neuroleptic malignant syndrome with prolonged catatonia in a dopa-responsive dystonia patient.

The authors describe a patient with dopa-responsive dystonia who developed neuroleptic malignant syndrome with prolonged catatonia following treatment with neuroleptic agents. Use of these agents probably expanded the patient's neuronal dysfunction beyond the nigrostriatal system to involve multiple dopaminergic systems. Electroconvulsive treatment alleviated the prolonged catatonia.

Identification and heterologous expression of the cytochrome P450 oxidoreductase from the white-rot basidiomycete Coriolus versicolor.

A cDNA encoding cytochrome P450 oxidoreductase (CPR) from the lignin-degrading basidiomycete Coriolus versicolor was identified using RT-PCR. The full-length cDNA consisted of 2,484 nucleotides with a poly(A) tail, and contained an open reading frame. The G+C content of the cDNA isolated was 60%. A deduced protein contained 730 amino acid residues with a calculated molecular weight of 80.7 kDa. The conserved amino acid residues involved in functional domains such as FAD-, FMN-, and NADPH-binding domains, were all found in the deduced protein. A phylogenetic analysis demonstrated that C. versicolor CPR is significantly similar to CPR of the basidiomycete Phanerochaete chrysosporium and that they share the same major branch in the fungal cluster. A recombinant CPR protein was expressed using a pET/ Escherichia coli system. The recombinant CPR protein migrated at 81 kDa on SDS polyacrylamide gel electrophoresis. It exhibited an NADPH-dependent cytochrome c reducing activity.

Metabolic response against sulfur-containing heterocyclic compounds by the lignin-degrading basidiomycete Coriolus versicolor.

The fungal conversions of sulfur-containing heterocyclic compounds were investigated using the lignin-degrading basidiomycete Coriolus versicolor. The fungus metabolized a series of sulfur compounds--25 structurally related thiophene derivatives--via several different pathways. Under primary metabolic conditions, C. versicolor utilized thiophenes, such as 2-hydroxymethyl-, 2-formyl-, and 2-carboxyl-thiophenes, as a nutrient sulfur source for growth; thus, the fungus degraded these compounds more effectively in a non-sulfur-containing medium than in conventional medium. The product analysis revealed that several redox reactions, decarboxylation reactions, and C-S cleavage reactions were involved in the fungal conversion of non-aromatic thiophenes. On the other hand, benzothiophene (BT) and dibenzothiophene (DBT) skeletons were converted to water-soluble products. All the products and metabolic intermediates were more hydrophilic than the starting substrates. These metabolic actions seemed to be a chemical stress response against exogenously added xenobiotics. These metabolic reactions were optimized under ligninolytic conditions, also suggesting the occurrence of a fungal xenobiotic response. Furthermore, the fungus converted a series of BTs and DBTs via several different pathways, which seemed to be controlled by the chemical structure of the substrates. DBT, 4-methylDBT, 4, 6-dimethylDBT, 2-methylBT, and 7-methylBT were immediately oxidized to their S-oxides. BTs and DBTs with the hydroxymethyl substituent were converted to their xylosides without S-oxidation. Those with carboxyl and formyl substituents were reduced to form a hydroxymethyl group, then xylosidated. These observations strongly suggested the involvement of a fungal substrate-recognition and metabolic response mechanism in the metabolism of sulfur-containing heterocyclic compounds by C. versicolor.

Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AAV) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AAV vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ) into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AAV-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AAV vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.

Identification and characterization of novel cytochrome P450 genes from the white-rot basidiomycete, Coriolus versicolor.

Using a reverse-transcription polymerase chain reaction (RT-PCR) technique, cytochrome P450 genes were cloned from the lignin-degrading basidiomycete, Coriolus versicolor. One possible P450 gene was identified, which consisted of 1,672 nucleotides and a poly(A) tail and encoded a deduced protein containing 449 amino acids. The deduced amino acid sequence revealed the presence of the P450 heme-binding motif, strongly suggesting that this protein belongs to the P450 superfamily, then designated CYP512A1. The deduced protein showed sequential similarity to other known P450s from several micro-organisms, such as Aspergillus terreus, Gibberella fujikuroi, and Neurospora crassa, with 30-35% identity. Since the identity of the amino id sequence was less than 40% with any other P450s, this protein was suggested to be the first member of a new family of cytochrome P450. In addition, a differential display RT-PCR analysis showed the expression of the other P450 genes, which were up-regulated by the addition of dibenzothiophene and 4-methyldibenzothiophene-5-oxide. Using the 5' rapid amplification of cDNA ends method, a 520-nucleotide sequence, including the P450 motif-coding region, was determined for one clone. The deduced protein showed high similarity to CYP512A1 but less than 40% identity with P450s from other organisms. A chemical stress-responsive expression of P450 is suggested for the first time in basidiomycetes.

Values of the bispectral index do not parallel the hemodynamic response to the rapid increase in isoflurane concentration.

PURPOSE: To investigate the changes in hemodynamic variables and bispectral index (BIS) in response to a rapid increase in isoflurane or sevoflurane concentration. METHOD: Thirty adult patients were anesthetized with either isoflurane (isoflurane group) or sevoflurane (sevoflurane group). Two minutes after induction of anesthesia with thiamylal, the inspired concentrations of isoflurane and sevoflurane were rapidly increased from 0.5 minimum alveolar anaesthetic concentration (MAC) to 3 MAC and maintained for five minutes. Heart rate (HR), mean arterial pressure (MAP), and BIS were measured every minute. RESULTS: An increase in the anesthetic concentration caused increases in HR and MAP in the isoflurane group and a decrease in MAP in the sevoflurane group. Consequently, HR and MAP in the isoflurane group were significantly higher than those in the sevoflurane group. After inhalation of high concentrations, BIS significantly and progressively decreased in both groups. CONCLUSION: BIS values decrease after a step increase in volatile agent concentration, whether or not a hyperdynamic action occurs.

A new splicing variant for human tyrosine hydroxylase in the adrenal medulla.

It has been reported that several mRNA isoforms of tyrosine 3-monooxygenase (tyrosine hydroxylase; TH) occur only in primates. New TH isoforms produced by skipping of exon 3 in the adrenal medulla of patients with progressive supranuclear palsy (PSP) have recently been reported, J. Neurochem. 67 (1996) 19. Here, we looked for the presence of new TH isoforms in control brains and adrenal medulla and in brains from patients with PSP. We found a novel type of TH mRNA in the adrenal medulla from one of the control subjects. The mRNA lacked exon 4, resulting in a premature stop codon at amino acid 147. This result suggests the importance of alternative splicing in the regulation of TH activity.

Catecholamines and serotonin are differently regulated by tetrahydrobiopterin. A study from 6-pyruvoyltetrahydropterin synthase knockout mice.

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.

Analgesic effect of epidural neostigmine after abdominal hysterectomy.

STUDY OBJECTIVE: To evaluate the effects of epidurally administered neostigmine on pain after abdominal hysterectomy. DESIGN: Prospective, randomized, double-blind study. SETTING: Teaching hospital. PATIENTS: 45 ASA physical status I adult patients scheduled for abdominal hysterectomy. INTERVENTIONS: All patients received identical general and epidural anesthesia. At the end of the surgery, they received epidural bupivacaine (10 mg) with either saline (control group, n = 15), 5 micro g/kg (5-micro g group, n = 15), or 10 micro g/kg neostigmine (10-micro g group, n = 15). Postoperatively, 50 mg diclofenac suppository was given for pain relief on patient demand. MEASUREMENTS AND MAIN RESULTS: The time to first diclofenac administration and the number of times diclofenac was required during the first 24 postoperative hours were recorded. Pain was assessed using a 10-cm visual analog pain scale (VAS) at rest at the first diclofenac request, and at 15 and 24 hours after surgery. The time to first diclofenac administration was significantly longer (p < 0.05) in the 10-micro g group (223 +/- 15 min) than in the control (78 +/- 17 min) or 5-micro g groups (88 +/- 18 min). However, epidural neostigmine at both doses did not reduce the number of postoperative diclofenac administrations. There were no differences in VAS among the three groups. CONCLUSIONS: Epidural neostigmine of 10 micro g/kg in bupivacaine provides a longer duration of analgesia than does bupivacaine alone or with 5 micro g/kg of neostigmine after abdominal hysterectomy.

Emotional stress-induced 'ampulla cardiomyopathy': discrepancy between the metabolic and sympathetic innervation imaging performed during the recovery course.

Four patients had the clinical features of 'ampulla cardiomyopathy', consisting of acute-onset transient left ventricular apical akinesis with basal normokinesis, normal coronary angiogram, ST-segment elevation and subsequent giant T wave inversion, which mimicked acute coronary syndrome, the onset of which occurred shortly after extreme mental stress. Myocardial necrosis was minimal, although 2 patients showed elevated serum catecholamine levels in the acute phase. Each patient underwent serial cardiac radionuclide single-photon emission computed tomography of myocardial functional sympathetic innervation, fatty acid metabolism and perfusion using I-123-metaiodobenzyl-guanidine (MIBG), I-123-beta-metyl-iodophenyl pentadecanoic acid (BMIPP) and thallium-201 (201Tl), respectively. In the acute phase, MIBG and BMIPP imaging showed an uptake defect in the apical region, whereas 201Tl uptake was mildly decreased. When assessed semi-quantitatively, the MIBG images had higher defect scores from the acute phase throughout the year of observation compared with BMIPP, and 201Tl. These observations suggest that the primary cause of ampulla cardiomyopathy is related to a disturbance of the cardiac sympathetic innervation.

Perioperative intravenous flurbiprofen reduces postoperative pain after abdominal hysterectomy.

PURPOSE: To assess whether perioperative intravenous administration of flurbiprofen, a non-steroidal anti-inflammatory drug, reduced postoperative pain after abdominal hysterectomy. METHODS: Forty-five patients undergoing abdominal hysterectomy were randomly assigned to one of three groups of equal size. A control group (CONT) received a placebo 30 min before and at the end of surgery. The other two groups, PRE and POST, received 1 mg x kg(-1) flurbiprofen iv 30 min before and at the end of surgery, respectively. All patients received identical general and epidural anesthesia. Postoperatively, 50 mg diclofenac pr was given for pain relief on patient demand. One of the authors assessed pain using a 10 cm visual analog scale at rest and during coughing at the first request for diclofenac, and at 15, 24, 48, and 72 hr after surgery. The number of times diclofenac was required during the first 24 hr after surgery was also recorded. RESULTS: The number of diclofenac requests in the PRE (1.8 +/- 0.4) and POST groups (2.0 +/- 0.4) were less than in the CONT group (3.0 +/- 0.4). The PRE group showed lower visual analog scale at rest at 15 and 24 hr and on coughing at 24, 48, and 72 hr after surgery than the CONT and POST groups. CONCLUSION: Intravenous 1 mg x kg(-1) flurbiprofen administered during anesthesia reduces postoperative rescue analgesic requirement after abdominal hysterectomy. Moreover, flurbiprofen is more effective when given before than after surgery.

A quantitative-trait analysis of human plasma-dopamine beta-hydroxylase activity: evidence for a major functional polymorphism at the DBH locus.

Dopamine-beta-hydroxylase (D beta H) catalyzes the conversion of dopamine to norepinephrine and is released from sympathetic neurons into the circulation. Plasma-D beta H activity varies widely between individuals, and a subgroup of the population has very low activity levels. Mounting evidence suggests that the DBH structural gene is itself the major quantitative-trait locus (QTL) for plasma-D beta H activity, and a single unidentified polymorphism may account for a majority of the variation in activity levels. Through use of both sequencing-based mutational analysis of extreme phenotypes and genotype/phenotype correlations in samples from African American, European American (EA), and Japanese populations, we have identified a novel polymorphism (--1021C-->T), in the 5' flanking region of the DBH gene, that accounts for 35%--52% of the variation in plasma-D beta H activity in these populations. In EAs, homozygosity at the T allele predicted the very low D beta H-activity trait, and activity values in heterozygotes formed an intermediate distribution, indicating codominant inheritance. Our findings demonstrate that --1021C-->T is a major genetic marker for plasma-D beta H activity and provide new tools for investigation of the role of both D beta H and the DBH gene in human disease.

Glial cell line-derived neurotrophic factor in the substantia nigra from control and parkinsonian brains.

Glial cell line-derived neurotrophic factor (GDNF) was measured for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients by highly sensitive sandwich enzyme-linked immunosorbent assay. In both groups, the levels of GDNF in the various brain regions were lower (pg/mg protein) than those of brain-derived growth factor (ng/mg order), and were significantly higher in the nigro-striatal dopaminergic regions (substantia nigra, caudate nucleus, putamen) than in the cerebellum and frontal cortex (P < 0.05). However, the content of GDNF in the dopaminergic regions showed no significant difference between parkinsonian and control patients.

X-linked dilated cardiomyopathy with a large hot-spot deletion in the dystrophin gene.

A 36-year-old Japanese man was hospitalized with coughing and exertional dyspnea (NYHA class I). He was diagnosed as having congestive heart failure, and was treated with diuretics and a beta-adrenergic blocking agent. He responded well to the treatment and his symptoms completely disappeared within a few days. Based on his clinical, laboratory, and molecular genetic findings, he was diagnosed as having X-linked dilated cardiomyopathy (XLDCM). He was found to have a large deletion in the dystrophin gene, involving exons 45-55. This is the first report on a Japanese XLDCM patient with a mutation in the central hot-spot region of this gene.

Quantification of tyrosine hydroxylase mRNA.

The main biochemical characteristic of Parkinson's disease (PD) is reduction of the neurotransmitter dopamine and the dopamine-synthesizing enzyme system, including tyrosine hydroxylase (TH, tyrosine 3-monooxygenase, EC 1.14.16.2) and tetrahydrobiopterin (BH(4) co-factor, in nigrostriatal neurons (1). The deficiency in dopamine-synthesizing enzymes is accompanied by cell loss, which is thought to be caused by unknown exogenous environmental factors as well as endogenous genetic factors.