Large-scale analysis of structural brain asymmetries during neurodevelopment: Associations with age and sex in 4265 children and adolescents.

Only a small number of studies have assessed structural differences between the two hemispheres during childhood and adolescence. However, the existing findings lack consistency or are restricted to a particular brain region, a specific brain feature, or a relatively narrow age range. Here, we investigated associations between brain asymmetry and age as well as sex in one of the largest pediatric samples to date (n = 4265), aged 1-18 years, scanned at 69 sites participating in the ENIGMA (Enhancing NeuroImaging Genetics through Meta-Analysis) consortium. Our study revealed that significant brain asymmetries already exist in childhood, but their magnitude and direction depend on the brain region examined and the morphometric measurement used (cortical volume or thickness, regional surface area, or subcortical volume). With respect to effects of age, some asymmetries became weaker over time while others became stronger; sometimes they even reversed direction. With respect to sex differences, the total number of regions exhibiting significant asymmetries was larger in females than in males, while the total number of measurements indicating significant asymmetries was larger in males (as we obtained more than one measurement per cortical region). The magnitude of the significant asymmetries was also greater in males. However, effect sizes for both age effects and sex differences were small. Taken together, these findings suggest that cerebral asymmetries are an inherent organizational pattern of the brain that manifests early in life. Overall, brain asymmetry appears to be relatively stable throughout childhood and adolescence, with some differential effects in males and females.

Gender differences in psychiatric comorbidity: a population-based study of 40 000 adults with attention deficit hyperactivity disorder.

OBJECTIVE: We aimed at determining whether gender modified associations between ADHD and psychiatric comorbidities in adults. METHOD: We identified adults with ADHD by linking Norwegian national registries and compared them with the remaining adult population (born 1967-1997, ADHD and bipolar during 2004-2015, other psychiatric disorders 2008-2015). Prevalence differences (PDs) and prevalence ratios (PRs) of psychiatric disorders were determined by Poisson regression. Interaction by gender was evaluated on additive (PDs) and multiplicative (PRs) scales. Proportions of psychiatric disorders attributable to ADHD were calculated. RESULTS: We identified 40 103 adults with ADHD (44% women) and 1 661 103 adults (49% women) in the remaining population. PDs associated with ADHD were significantly larger in women than in men for anxiety, depression, bipolar and personality disorders, for example depression in women: 24.4 (95% CI, 23.8-24.9) vs. in men: 13.1 (12.8-13.4). PDs were significantly larger in men for schizophrenia and substance use disorder (SUD), for example SUD in men: 23.0 (22.5-23.5) vs. in women: 13.7 (13.3-14.0). Between 5.6 and 16.5% of psychiatric disorders in the population were attributable to ADHD. CONCLUSION: The association between ADHD and psychiatric comorbidities differed significantly among men and women. Clinicians treating adults with ADHD should be aware of these frequent and gender-specific comorbidities, such that early treatment can be offered.

Prevalence and clinical correlates of insomnia in adults with attention-deficit hyperactivity disorder.

OBJECTIVE: To investigate the prevalence of insomnia in adults with Attention-deficit hyperactivity disorder (ADHD) and its association with clinical subtypes, current ADHD symptoms, and stimulant treatment. METHOD: We obtained diagnostic information, symptom rating scales and treatment history from clinically ascertained adult ADHD patients diagnosed according to DSM-IV criteria (n = 268, mean age 38.1 years) and randomly selected population controls (n = 202, mean age 36.5 years). The Bergen Insomnia Scale (BIS) was used to measure insomnia. ADHD symptom domains were self-rated using the Adult ADHD Self-Rating Scale. RESULTS: Insomnia was far more frequent among adults with ADHD (66.8%) than in the population controls (28.8%) (P < 0.001). Insomnia was more common in adults with the combined subtype than in those with the inattentive subtype (79.7% and 55.6%, respectively) (P = 0.003). For self-reported current ADHD symptoms, inattention was strongly correlated to insomnia. Patients currently using stimulant treatment for ADHD reported a lower total insomnia score compared to patients without medication (P < 0.05). CONCLUSION: Insomnia was highly prevalent among adults with ADHD. The lower insomnia score in patients on current stimulant treatment suggests that stimulant treatment is not associated with worsening of insomnia symptoms in adult ADHD patients.

The genetics of attention deficit/hyperactivity disorder in adults, a review.

The adult form of attention deficit/hyperactivity disorder (aADHD) has a prevalence of up to 5% and is the most severe long-term outcome of this common neurodevelopmental disorder. Family studies in clinical samples suggest an increased familial liability for aADHD compared with childhood ADHD (cADHD), whereas twin studies based on self-rated symptoms in adult population samples show moderate heritability estimates of 30-40%. However, using multiple sources of information, the heritability of clinically diagnosed aADHD and cADHD is very similar. Results of candidate gene as well as genome-wide molecular genetic studies in aADHD samples implicate some of the same genes involved in ADHD in children, although in some cases different alleles and different genes may be responsible for adult versus childhood ADHD. Linkage studies have been successful in identifying loci for aADHD and led to the identification of LPHN3 and CDH13 as novel genes associated with ADHD across the lifespan. In addition, studies of rare genetic variants have identified probable causative mutations for aADHD. Use of endophenotypes based on neuropsychology and neuroimaging, as well as next-generation genome analysis and improved statistical and bioinformatic analysis methods hold the promise of identifying additional genetic variants involved in disease etiology. Large, international collaborations have paved the way for well-powered studies. Progress in identifying aADHD risk genes may provide us with tools for the prediction of disease progression in the clinic and better treatment, and ultimately may help to prevent persistence of ADHD into adulthood.

A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication.

Attention-Deficit/Hyperactivity Disorder (ADHD) has a very high heritability (0.8), suggesting that about 80% of phenotypic variance is due to genetic factors. We used the integration of statistical and functional approaches to discover a novel gene that contributes to ADHD. For our statistical approach, we started with a linkage study based on large multigenerational families in a population isolate, followed by fine mapping of targeted regions using a family-based design. Family- and population-based association studies in five samples from disparate regions of the world were used for replication. Brain imaging studies were performed to evaluate gene function. The linkage study discovered a genome region harbored in the Latrophilin 3 gene (LPHN3). In the world-wide samples (total n=6360, with 2627 ADHD cases and 2531 controls) statistical association of LPHN3 and ADHD was confirmed. Functional studies revealed that LPHN3 variants are expressed in key brain regions related to attention and activity, affect metabolism in neural circuits implicated in ADHD, and are associated with response to stimulant medication. Linkage and replicated association of ADHD with a novel non-candidate gene (LPHN3) provide new insights into the genetics, neurobiology, and treatment of ADHD.

Meta-analysis of brain-derived neurotrophic factor p.Val66Met in adult ADHD in four European populations.

Attention-deficit hyperactivity disorder (ADHD) is a multifactorial, neurodevelopmental disorder that often persists into adolescence and adulthood and is characterized by inattention, hyperactivity and impulsiveness. Before the advent of the first genome-wide association studies in ADHD, genetic research had mainly focused on candidate genes related to the dopaminergic and serotoninergic systems, although several other genes had also been assessed. Pharmacological data, analysis of animal models and association studies suggest that Brain-Derived Neurotrophic Factor (BDNF) is also a strong candidate gene for ADHD. Several polymorphisms in BDNF have been reported and studied in psychiatric disorders but the most frequent is the p.Val66Met (rs6265G > A) single nucleotide polymorphism (SNP), with functional effects on the intracellular trafficking and secretion of the protein. To deal with the inconsistency raised among different case-control and family-based association studies regarding the p.Val66Met contribution to ADHD, we performed a meta-analysis of published as well as unpublished data from four different centers that are part of the International Multicentre Persistent ADHD CollaboraTion (IMpACT). A total of 1,445 adulthood ADHD patients and 2,247 sex-matched controls were available for the study. No association between the p.Val66Met polymorphism and ADHD was found in any of the four populations or in the pooled sample. The meta-analysis also showed that the overall gene effect for ADHD was not statistically significant when gender or comorbidity with mood disorders were considered. Despite the potential role of BDNF in ADHD, our data do not support the involvement of p.Val66Met in the pathogenesis of this neuropsychiatric disorder.

Association between catechol O-methyltransferase (COMT) haplotypes and severity of hyperactivity symptoms in adults.

It has been suggested that symptoms of attention-deficit/hyperactivity disorder (ADHD) is related to low dopamine levels in the prefrontal cortex. The enzyme catechol O-methyltransferase (COMT), which degrades dopamine and other catecholamines, is important for monoamine signaling in this brain-region, but genetic studies of the functional Val158Met (rs4680) polymorphism in ADHD have been inconsistent. However, recently it was shown that also common synonymous COMT variants modulate total COMT enzymatic activity by affecting the expression of the gene [Nackley et al. (2006); Science 314(5807):1930-1933]. We therefore hypothesized that analysis of haplotypes could reveal more about the association between COMT and ADHD symptoms than the Val158Met polymorphism alone. SNPs rs6269, rs4633, rs4818, and rs4680, tagging the common putative functional COMT haplotypes, were genotyped in 435 adult subjects with a clinical diagnosis of ADHD and 383 controls and analyzed for association with ADHD and the hyperactivity/impulsivity and inattention dimensions from the Adult ADHD Self-Report Scale (ASRS). All markers showed a trend for association with the hyperactivity/impulsivity scale, peaking at marker rs6269 (P = 0.007). Haplotype analysis revealed that the rs6269 risk allele tags the suggested high COMT-activity haplotype, which is associated with the highest hyperactivity/impulsivity score in our sample (P = 0.01). Our results also suggest that there is a stepwise decreased hyperactivity/impulsivity score associated with the proposed mid and low activity haplotypes described previously. In conclusion, we suggest that COMT haplotype variation is associated primarily with the hyperactivity/impulsivity dimension of ADHD and point to the importance of testing this hypothesis in future studies.

Oxygen dependence of tyrosine hydroxylase.

The effects of dioxygen on tyrosine hydroxylase (TH) activity was studied, measuring the formation of DOPA from tyrosine, (3)H(2)O from 3,5-(3)H-tyrosine, or by direct oxygraphic determination of oxygen consumption. A high enzyme activity was observed during the initial 1-2 min of the reactions, followed by a decline in activity, possibly related to a turnover dependent substoichiometrical oxidation of enzyme bound Fe(II) to the inactive Fe(III) state. During the initial reaction phase, apparent K (m)-values of 29-45 microM for dioxygen were determined for all human TH isoforms, i.e. 2-40 times higher than previously reported for TH isolated from animal tissues. After 8 min incubation, the K (m) (O(2))-values had declined to an average of 20 +/- 4 microM. Thus, TH activity may be severely limited by oxygen availability even at moderate hypoxic conditions, and the enzyme is rapidly and turnover dependent inactivated at the experimental conditions commonly employed to measure in vitro activities.

Genetic analyses of dopamine related genes in adult ADHD patients suggest an association with the DRD5-microsatellite repeat, but not with DRD4 or SLC6A3 VNTRs.

Attention deficit hyperactivity disorder (ADHD) is a common and highly heritable psychiatric disorder in children and adults. Recent meta-analyses have indicated an association between genes involved in dopaminergic signaling and childhood ADHD, but little is known about their possible role in adult ADHD. In this study of adults with ADHD, we evaluated the three most commonly studied ADHD candidate genetic polymorphisms; the dopamine receptor D4 (DRD4) exon 3 VNTR repeat, a microsatellite repeat 18.5 kb upstream of the DRD5 locus and the 3'UTR dopamine transporter SLC6A3 (DAT 1) VNTR. We examined 358 clinically diagnosed adult Norwegian ADHD patients (51% males) and 340 ethnically matched controls. We found a nominally significant overall association with adult ADHD for the DRD5 microsatellite marker (P = 0.04), and a trend toward increased risk associated with the 148-bp allele consistent with recent meta-analyses. The strongest overall association (P = 0.02) and increased risk for the 148-bp allele [odds ratio (OR) = 1.27 (95% CI: 1.00-1.61)] were seen in the inattentive and combined inattentive/hyperactive group as previously reported for childhood ADHD. No association was found for the DRD4 or SLC6A3 polymorphisms in this patient sample. In conclusion, our results among adults with a clinical diagnosis of ADHD support an association between ADHD and the DRD5 locus, but not the DRD4 or SLC6A3 loci. It is possible that the latter polymorphisms are associated with a transient form of ADHD with better long-term clinical outcome.

Characterization of the cyclic adenosine 3':5'-monophosphate effector system in hormone-dependent and hormone-independent rat mammary carcinomas.

We have compared the properties of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases I and II in hormone-dependent/cAMP-sensitive (DMBA tumor) and hormone-independent/cAMP-resistant (DMBA 1 tumor) rat mammary carcinomas. cAMP-resistance was not due to less total kinase in the hormone-independent tumor, grossly altered distribution between soluble and particulate forms of the kinase (80% soluble in either tumor), alteration in the relative proportion of isozymes I and II of the protein kinase (the soluble and the particulate fraction from both tumors contained about 50% of either isozyme), or a decreased sensitivity towards cAMP (both isozymes had affinities for cAMP and its derivatives that corresponded closely with those of isozymes from normal tissues). Furthermore, the sensitivity of the enzymes towards thermal denaturation was identical for samples from the two tumor types. Subtle differences did, however, exist between the regulatory moieties [regulatory subunit of cAMP-dependent protein kinase II (RII)] of isozyme II from the two tumors: autophosphorylated RII from the hormone-independent tumor migrated as a doublet corresponding to Mrs 54,000 and 52,000 on sodium dodecyl sulfate-polyacrylamide gels, against Mrs 53,000 and 52,000 for RII from the hormone-dependent tumor; RII from the two tumors showed different elution profiles upon DEAE-cellulose chromatography; a considerable proportion of the soluble RII in the hormone-independent tumor formed supramolecular aggregates as judged by size-exclusion chromatography. No such microheterogeneity was noted for isozyme I. This study thus shows that the lack of cAMP-responsiveness of one tumor is related either to a defect distal to the cAMP-dependent protein kinases or to the appearance of the new subtype of RII in the resistant tumor. If the latter explanation is correct, it means that the part of the RII molecule responsible for interaction with other proteins rather than that responsible for cAMP-binding and control of protein kinase activity modulates the growth-inhibiting response to cAMP.

Implication of NOTCH1 gene in susceptibility to anxiety and depression among sexual abuse victims.

Sexual abuse contributes to the development of multiple forms of psychopathology, including anxiety and depression, but the extent to which genetics contributes to these disorders among sexual abuse victims remains unclear. In this translational study, we first examined gene expression in the brains of rodents exposed to different early-life conditions (long, brief or no maternal separation). Hypothesizing that genes revealing changes in expression may have relevance for psychiatric symptoms later in life, we examined possible association of those genes with symptoms of anxiety and depression in a human sample of sexual abuse victims. Changes in rodent brain gene expression were evaluated by means of correspondence and significance analyses of microarrays by comparing brains of rodents exposed to different early-life conditions. Tag single-nucleotide polymorphisms (SNPs) of resulting candidate genes were genotyped and tested for their association with symptoms of anxiety and depression (Hospital Anxiety and Depression Scale) in a sample of 361 sexual abuse victims, using multinomial logistic regression. False discovery rate was applied to account for multiple testing in the genetic association study, with q-value of 0.05 accepted as significant. We identified four genes showing differential expression among animals subjected to different early-life conditions as well as having potential relevance to neural development or disorders: Notch1, Gabrr1, Plk5 and Zfp644. In the human sample, significant associations were observed for two NOTCH1 tag SNPs: rs11145770 (OR=2.21, q=0.043) and rs3013302 (OR=2.15, q=0.043). Our overall findings provide preliminary evidence that NOTCH1 may be implicated in the susceptibility to anxiety and depression among sexual abuse victims. The study also underscores the potential importance of animal models for future studies on the health consequences of early-life stress and the mechanisms underlying increased risk for psychiatric disorders.

Exome chip analyses in adult attention deficit hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) is a highly heritable childhood-onset neuropsychiatric condition, often persisting into adulthood. The genetic architecture of ADHD, particularly in adults, is largely unknown. We performed an exome-wide scan of adult ADHD using the Illumina Human Exome Bead Chip, which interrogates over 250 000 common and rare variants. Participants were recruited by the International Multicenter persistent ADHD CollaboraTion (IMpACT). Statistical analyses were divided into 3 steps: (1) gene-level analysis of rare variants (minor allele frequency (MAF)<1%); (2) single marker association tests of common variants (MAF⩾1%), with replication of the top signals; and (3) pathway analyses. In total, 9365 individuals (1846 cases and 7519 controls) were examined. Replication of the most associated common variants was attempted in 9847 individuals (2077 cases and 7770 controls) using fixed-effects inverse variance meta-analysis. With a Bonferroni-corrected significance level of 1.82E-06, our analyses of rare coding variants revealed four study-wide significant loci: 6q22.1 locus (P=4.46E-08), where NT5DC1 and COL10A1 reside; the SEC23IP locus (P=6.47E-07); the PSD locus (P=7.58E-08) and ZCCHC4 locus (P=1.79E-06). No genome-wide significant association was observed among the common variants. The strongest signal was noted at rs9325032 in PPP2R2B (odds ratio=0.81, P=1.61E-05). Taken together, our data add to the growing evidence of general signal transduction molecules (NT5DC1, PSD, SEC23IP and ZCCHC4) having an important role in the etiology of ADHD. Although the biological implications of these findings need to be further explored, they highlight the possible role of cellular communication as a potential core component in the development of both adult and childhood forms of ADHD.

p53 wild-type and p53 nullizygous Big Blue transgenic mice have similar frequencies and patterns of observed mutation in liver, spleen and brain.

Transgenic mouse mutation detection systems offer a powerful tool for analysis of spontaneous and induced mutations in vivo. Mice doubly transgenic for a null mutation of the p53 tumor suppressor gene and a lambda shuttle vector harboring the lacI gene were utilized to examine the rate and pattern of spontaneous somatic mutation of the lacI transgene in vivo. Three somatic tissues were examined: liver, spleen and brain. At 6 weeks of age, three p53 wild type (+/+) and three p53 nullizygous (-/-), lacI (+/-) male mice were analysed. The mutation frequencies in the two genotypes were similar. The mutant frequencies for wild type (+/+) and nullizygous (-/-) p53 genotypes were, respectively, 4.2 x 10(-5) and 3.6 x 10(-5) in the liver, 4.3 x 10(-5) and 3.4 x 10(-5) in the spleen and 2.8 x 10(-5-) and 3.0 x 10(-5) in the brain. When the data from the three tissues were combined, the mutant frequency was 3.7 x 10(-5) for the (+/+) genotype and 3.3 x 10(-5) for the (-/-) genotype. By sequencing both strands in the DNA-binding region of the lacI gene, 91 mutations were found. When recurrent mutations in the same mouse were excluded, a total of 67 definitely independent mutations were found. No statistically significant differences were found in the mutational spectra between the two genotypes when the three tissues were analysed individually or combined (P = 0.58). These findings suggest a need to reconsider the general form of hypothesis that the p53 gene serves as the 'guardian of the genome'.

Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: large-scale purification and physicochemical properties.

A new procedure that permits large-scale purification of tyrosine 3-monooxygenase (tyrosine hydroxylase) (L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the cytosolic fraction of bovine adrenal medulla is described. The homogenous enzyme revealed a subunit Mr of 60,000 and a specific activity of 425 nmol.min-1.mg-1. The N-terminal amino-acid sequence (27 residues) revealed 89% homology with the human pheochromocytoma enzyme as deduced from its cDNA sequence. The pure enzyme contained 0.66 +/- 0.09 mol iron, 0.13 mol zinc and 0.62 +/- 0.04 mol phosphate per mol subunit of Mr = 60,000. A broad light absorption band with its maximum around 700 nm (epsilon 700 nm = 1.3 (mM monomer)-1.cm-1) explains its blue-green color. EPR spectra at 3.6 K revealed high-spin Fe(III) (S = 5/2) in an environment of nearly axial symmetry (g values at 7.2-6.7, 4.7-5.3 and 1.9-2.0). A close correlation was observed between the absorbance at 700 nm and the intensity of the axial type of EPR spectrum. The absorption peak at 700 nm is compatible with a ligand-to-iron charge-transfer transition as a result of catecholate coordination to the iron. Physicochemical studies suggest that the enzyme does not undergo such major substrate- or cofactor-induced conformational changes as have been reported for the related enzyme, phenylalanine hydroxylase.

Pteridin-dependent hydroxylases as autoantigens in autoimmune polyendocrine syndrome type I.

Autoimmune polyendocrine syndrome type I (APS I) is characterized by autoantibodies, often directed towards tissue-specific enzymes in the affected organs. We have earlier reported the identification of tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) as autoantigens in APS I associated with intestinal dysfunction and alopecia, respectively. These two enzymes, together with phenylalanine hydroxylase (PAH), constitute the group of biopterin-dependent hydroxylases, which all are involved in the biosynthesis of neurotransmitters. A clone encoding PAH was used for in vitro transcription/translation, followed by immunoprecipitation with sera from 94 APS I patients and 70 healthy controls. Of the APS I patients, 25% had PAH antibodies, and no reactivity was detected in the controls. No association with the main clinical components of APS I was found with PAH antibodies. Altogether, 59 sera from the 94 APS I patients reacted with at least one of TPH, TH, or PAH, whereas 35 showed no reactivity. Nineteen of the sera contained antibodies towards all enzymes, 12 to TPH only and 12 to TH only. No sera showed antibodies that reacted to only PAH. An immunocompetition assay demonstrated that the reactivity against PAH represents a cross-reactivity with TPH, whereas antibodies against TPH and TH are directed towards epitopes unique for the two enzymes.

Tyrosine hydroxylase and Parkinson's disease.

A consistent neurochemical abnormality in Parkinson's disease (PD) is degeneration of dopaminergic neurons in substantia nigra, leading to a reduction of striatal dopamine (DA) levels. As tyrosine hydroxylase (TH) catalyses the formation of L-DOPA, the rate-limiting step in the biosynthesis of DA, the disease can be considered as a TH-deficiency syndrome of the striatum. Similarly, some patients with hereditary L-DOPA-responsive dystonia, a neurological disorder with clinical similarities to PD, have mutations in the TH gene and decreased TH activity and/or stability. Thus, a logical and efficient treatment strategy for PD is based on correcting or bypassing the enzyme deficiency by treatment with L-DOPA, DA agonists, inhibitors of DA metabolism, or brain grafts with cells expressing TH. A direct pathogenetic role of TH has also been suggested, as the enzyme is a source of reactive oxygen species (ROS) in vitro and a target for radical-mediated oxidative injury. Recently, it has been demonstrated that L-DOPA is effectively oxidized by mammalian TH in vitro, possibly contributing to the cytotoxic effects of DOPA. This enzyme may therefore be involved in the pathogenesis of PD at several different levels, in addition to being a promising candidate for developing new treatments of this disease.

pH-dependent release of catecholamines from tyrosine hydroxylase and the effect of phosphorylation of Ser-40.

Bovine adrenal tyrosine hydroxylase (TH) is isolated in a partially inhibited state with the feed-back inhibitors adrenaline and noradrenaline tightly coordinated to high-spin (S = 5/2) Fe(III) at the active site. In addition to the charge-transfer interaction with iron, an additional charged group in the polypeptide chain, with an apparent pKa of about 5.3 at 4 degrees C, is involved in the binding of catecholamines. Protonation of this group increases the pseudo-first order rate constant for the dissociation of the TH-[3H]noradrenaline complex more than 100-fold at 4 degrees C. At pH 7.0 and 30 degrees C, phosphorylation of Ser-40 causes a 6-fold increase in the rate constant for this dissociation.

Identification of protein phosphatase 2A as the major tyrosine hydroxylase phosphatase in adrenal medulla and corpus striatum: evidence from the effects of okadaic acid.

(i) The major sites on bovine adrenal tyrosine hydroxylase (TH) phosphorylated by calmodulin-dependent multiprotein kinase (CaM-MPK) and cyclic AMP-dependent protein kinase were shown to be Ser-19 and Ser-40, respectively, while Ser-40 was also phosphorylated slowly by CaM-MPK. (ii) Type 2A and type 2C phosphatases accounted for approximately 90% and approximately 10% of TH phosphatase activity, respectively, in extracts of adrenal medulla and corpus striatum assayed at near physiological free Mg2+ (1 mM), while type 1 and type 2B phosphatases had negligible activity towards TH. (iii) Incubation of adrenal chromaffin cells with okadaic acid increased TH phosphorylation by 206% and activity by 77%, establishing that type 2A phosphatases play a major role in regulating TH in vivo.

Crystallization and preliminary diffraction analysis of a truncated homodimer of human phenylalanine hydroxylase.

A recombinant truncated form (delta1-102/delta428-452) of the non-heme iron-dependent metalloenzyme human phenylalanine hydroxylase (hPAH, phenylalanine 4-monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a = 66.6 A, b = 108.4 A, c = 125.7 A. The calculated packing parameter (Vm) is 3.24 A3/Da with four 2-fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 A resolution.

Isolation from the microsomal fraction of rat liver of a subfraction highly enriched in uncoated endocytic vesicles with high H+-ATPase activity and a 50 kDa phosphoprotein.

A subcellular fraction, highly enriched in uncoated vesicles (UCV) with high H+-ATPase (EC 3.6.1.34) activity, was isolated from the crude microsomal fraction of rat liver homogenates by discontinuous sucrose gradient centrifugation. The UCV fraction, recovered at the interface of sucrose density 1.08 and 1.10 g/ml, was shown morphologically to be a mixture of small, smooth-surfaced univesicular and a few multivesicular structures. A permeable anion (e.g. chloride) was required for internal acidification, indicating an electroneutral proton pump. Specific inhibitors of anion transport (pyridoxal 5'-phosphate and 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid) totally inhibit proton translocation. The proton pump activity was insensitive to oligomycin, but was completely inhibited by about 5 microM of the tridentate bathophenanthroline chelate of Fe(II). The activity was also inhibited 100% by low concentrations of the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the proton conduction inhibitor N,N'-dicyclohexylcarbodiimide and the ionophore monensin. The UCV fraction contained 2 proteins of Mr 50000 (major) and 54000 (minor) which were phosphorylated by an endogenous cyclic nucleotide- and Ca2+-independent protein kinase.