Putative multiadhesive protein from the marine sponge Geodia cydonium: cloning of the cDNA encoding a fibronectin-, an SRCR-, and a complement control protein module

Sponges (Porifera) representing the simplest metazoan phylum so far have been thought to possess no basal lamina tissue structures. One major extracellular matrix protein that is also a constitutive glycoprotein of the basal lamina is fibronectin. It was the aim of the present study to identify the native protein from the marine sponge Geodia cydonium and to isolate the corresponding cDNA. In crude extracts from this sponge protein(s) of M(r) of approximately 230 and approximately 210 kDa could be visualized by Western-blotting using an anti-fibronectin [human] antibody. By PCR cloning from a cDNA library of G. cydonium we isolated a cDNA comprising one element of fibronectin, the type-III (FN3) module. The cDNA (2.3 kb long), encoding a 701 amino acid [aa] long putative "multiadhesive protein" termed MAP_GEOCY, was found to contain (i) a fibronectin-, (ii) a scavenger receptor cysteine-rich [SRCR]-, and (iii) a short consensus repeat [SCR] module. The 89 aa long fibronectin module comprises the characteristic topology and conserved aa found in fibronectin type-III (FN3) elements. The SRCR module (101 aa) features the characteristics of group B SRCR molecules. The predominant proteins belonging to this group are the mammalian WC1-, M130-, CD6- and CD5 antigens that probably are involved in immunological reactions. The SCR module (54 aa) shows the characteristics of type III SCR modules found in complement receptors. Phylogenetic analyses performed with all three building blocks of the "multiadhesive protein" showed that the respective sponge modules form independent, possibly basal, lineages in trees that include the corresponding modules from higher metazoan animals. In summary, these data demonstrate for the first time that the phylogenetically oldest Metazoa, the sponges, contain protein modules seen in higher animals in proteins of the extracellular matrix and in molecules involved in cell-mediated immune reactions in vertebrates.     

Mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase increases the affinity for phenylalanine

The aromatic amino acid hydroxylases tyrosine and phenylalanine hydroxylase both contain non-heme iron, utilize oxygen and tetrahydrobiopterin, and are tetramers of identical subunits. The catalytic domains of these enzymes are homologous, and recent X-ray crystallographic analyses show the active sites of the two enzymes are very similar. The hydroxyl oxygens of tyrosine 371 in tyrosine hydroxylase and of tyrosine 325 of phenylalanine hydroxylase are 5 and 4.5 A, respectively, away from the active site iron in the enzymes. To determine whether this residue has a role in the catalytic mechanism as previously suggested [Erlandsen, H., et al. (1997) Nat. Struct. Biol. 4, 995-1000], tyrosine 371 of tyrosine hydroxylase was altered to phenylalanine by site-directed mutagenesis. The Y371F protein was fully active in tyrosine hydroxylation, eliminating an essential mechanistic role for this residue. There was no change in the product distribution seen with phenylalanine or 4-methylphenylalanine as a substrate, suggesting that the reactivity of the hydroxylating intermediate was unaffected. However, the KM value for phenylalanine was decreased 10-fold in the mutant protein. These results are interpreted as an indication of greater conformational flexibility in the active site of the mutant protein.     

A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens

Sponges (Porifera) represent the lowest metazoan phylum, characterized by a pronounced plasticity in the determination of cell lineages. In a first approach to elucidate the molecular mechanisms controlling the switch from the cell lineage with a putative indefinite growth capacity to senescent, somatic cells, the activity of the telomerase as an indicator for immortality has been determined. The studies were performed with the marine demosponges Suberites domuncula and Geodia cydonium. It was found that the activity for the telomerase in the tissue of both sponges is high; a quantitative analysis revealed that the extract from S. domuncula contained 10.3 TPG units per 5000 cell equivalents and the one from G. cydonium 8.3 TPG units; hence the activity reached approximately 30-20% of the activity seen in telomerase-positive reference cells. In contrast, dissociated spherulous cells from G. cydonium, after an incubation period of 24 h, contained no detectable telomerase activity. From earlier studies it is known that isolated sponge cells do not proliferate. Based on these findings it is assumed that the separation of the senescent sponge cell lineage from the immortal germ/somatic cell lineage is triggered by the loss of contact with cell adhesion factors. First evidence is included which suggests that the final progress of the senescent, telomerase-negative cells to cell death is caused by apoptosis.     

Deletion mutagenesis of rat PC12 tyrosine hydroxylase regulatory and catalytic domains

The functional organization of rat tyrosine hydroxylase was investigated by deletion mutagenesis of the regulatory and catalytic domains. A series of tyrosine hydroxylase cDNA deletion mutants were amplified by PCR, cloned into the pET3C prokaryotic expression vector, and the mutant proteins were partially purified from E. coli. The results show that the deletion of up to 157 N-terminal amino acids activated the enzyme, but further deletion to position 184 completely destroyed catalytic activity. On the carboxyl end, the removal of 43 amino acids decreased but did not eliminate activity, suggesting that this region may play a different role in the regulation of the enzyme. These findings place the amino end of the catalytic domain between residues 158 and 184 and the carboxyl end at or prior to position 455. Deletions within the first 157 amino acids in the N-terminus caused an increase in hydroxylating activity, a decrease in the apparent Km for tyrosine and phenylalanine substrates, and a substantial increase in the Ki for dopamine inhibition. The results define this region of the N-terminus as the regulatory domain of tyrosine hydroxylase, whose primary functions are to restrict the binding of amino acid substrates and to facilitate catecholamine inhibition. The results also suggest that the well-established role of the regulatory domain in restricting cofactor binding may be secondary to an increase in catecholamine binding, which in turn lowers the affinity for the cofactor. These findings provide new insight into the functional organization and mechanisms of regulation of tyrosine hydroxylase.     

Immunoglobulin-like domain is present in the extracellular part of the receptor tyrosine kinase from the marine sponge Geodia cydonium

We have isolated and characterized two cDNAs from the marine sponge Geodia cydonium coding for a new member of a receptor tyrosine kinase of class II. The deduced amino acid sequence shows two characteristic domains: (i) the tyrosine kinase domain; and (ii) an immunoglobulin-like domain. The latter part shows high homology to the vertebrate C2 type immunoglobulin domain. This result demonstrates that immunoglobulin domains are not recent achievements of higher animals but exist also in those animals which have diverged from other organisms about 800 million years ago.     

Doppler ultrasound of blood flow velocities in ophthalmic and central retinal arteries during the early neonatal period

Dopamine has been implicated as a potential mediating factor in a variety of neurodegenerative disorders. Dopamine can be oxidized to form a reactive dopamine quinone that can covalently modify cellular macromolecules including protein and DNA. This oxidation can be enhanced through various enzymes including tyrosinase and/or prostaglandin H synthase. One of the potential targets in brain for dopamine quinone damage is tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. The present studies demonstrated that dopamine quinone, the formation of which was enhanced through the activity of the melanin biosynthetic enzyme, tyrosinase, covalently modified and inactivated tyrosine hydroxylase. Dihydroxyphenylalanine (DOPA; the catechol-containing precursor of dopamine) also inactivated tyrosine hydroxylase under these conditions. Catecholamine-mediated inactivation occurred with both purified tyrosine hydroxylase as well as enzyme present in crude pheochromocytoma homogenates. Inactivation was associated with covalent incorporation of radiolabelled dopamine into the enzyme as assessed by immunoprecipitation, size exclusion chromatography, and denaturing sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Furthermore, the covalent modification and inactivation of tyrosine hydroxylase was blocked by antioxidant compounds (dithiothreitol, reduced glutathione, or NADH). In addition to kinetic feedback inhibition and the formation of an inhibitory dopamine/Fe+3 complex, these findings suggest that a third mechanism exists by which dopamine (or DOPA) can inhibit tyrosine hydroxylase, adding further complexity to the regulation of catecholamine biosynthesis.     

The role of phenylalanine in structure-function relationships of phenylalanine hydroxylase revealed by radiation target analysis

The activity of rat liver phenylalanine hydroxylase (PAH; phenylalanine 4-monooxygenase, EC 1.14.16.1) is regulated by interaction with its substrate, phenylalanine, and its coenzyme, BH4 [tetrahydrobiopterin (6R-dihydroxypropyl-L-erythro-5,6,7,8-tetrahydropterin)]. The structural changes accompanying these interactions have been studied by radiation target analysis. PAH purified from rat liver was incubated with 2 mM phenylalanine to achieve complete activation of the enzyme. Frozen samples were irradiated with various doses of high energy electrons; samples were subsequently thawed, and several surviving properties of the enzyme were determined. Each parameter decreased as a single exponential function of radiation dose. Radiation target analysis of enzymatic activity yielded a dimeric target size. Similar radiation effects on subunit monomers and on tetrameric structure were observed. Together with results from unactivated enzyme, these data show that phenylalanine increases the interactions between the subunits in a dimer and weakens the interactions between dimers in a tetramer. These alterations prevent the natural cofactor, a tetrahydrobiopterin, from exerting a negative effect on activity.     

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.     

Cloning of sponge (Geodia cydonium) and tunicate (Botryllus schlosseri) proteasome subunit epsilon (PRCE): implications about the vertebrate MHC-encoded homologue LMP7 (PRCC)

Proteasomes are large protein complexes that play a major role in selective degradation of intracellular proteins. Eukaryotes feature seven different alpha and beta subunits. Two of the vertebrate housekeeping beta-subunits have MHC-encoded homologues that can substitute for the housekeeping counterparts upon interferon-gamma induction. In the present study we report the cloning of invertebrate beta-subunit proteasome epsilon (PRCE), from the marine sponge Geodia cydonium and from the colonial tunicate Botryllus schlosseri. Sequence comparisons revealed that the sponge and tunicate proteins are strikingly similar to vertebrate and yeast PRCEs and their MHC-linked counterparts the PRCCs (also termed LMP7), and to a lesser degree also to archaebacterial proteasome subunit beta. Based on this comparison we suggest that all eukaryotic PRCEs and PRCCs feature a cleavable N-terminal propeptide, including the two mammalian PRCEs which appear to have been wrongly predicted from incomplete cDNAs. Our comparative analysis outlines 25 amino acid positions which appear to be unique for PRCCs, distinct from the corresponding residues in metazoan PRCEs.     

Relation between liver amino acid-catabolizing enzymes and free amino acids of liver and plasma in adult cockerels fed diets containing graded levels of protein

Activities of liver amino acid-catabolizing enzymes and liver and plasma free amino acid concentrations in adult cockerels fed diets containing 3% to 21% casein were determined 2 hours after a meal. Liver tryptophan pyrrolase activity and liver tryptophan increased with an increase in dietary casein level, but plasma tryptophan decreased with the increase dietary casein level. As the dietary casein level increased, liver lysine-ketoglutarate reductase activity decreased, but liver and plasma lysine increased proportionally, and the rate of increase was larger in the plasma than in the liver. Liver phenylalanine hydroxylase activity increased with the increase in dietary casein level from 3% to 12%, and thereafter remained unchanged. Reflecting the change of this enzyme, phenylalanine increased in the liver and decreased in the plasma with the increase in dietary casein level from 3% to 12%, and thereafter the rate of increase in liver phenylalanine became small and plasms phenylalanine increased. Liver tyrosine was not influenced by the dietary casein level, whereas plasma tyrosine increased sharply from 3% to 12%, and thereafter the rate of increase decreased. Thus, the difference between liver and plasma free amino acid responses might be due to the changes in the activities of liver amino acid-catabolizing enzymes.     

Novel mutations identified in exon 7 of phenylalanine hydroxylase gene in Chinese

Exon 7 of the phenylalanine hydroxylase (PAH) gene was analyzed in 45 children affected with classic phenylketonuria (PKU) from northern China by using PCR-single strand conformation polymorphism (PCR-SSCP) technique and DNA direct sequencing. Six missense mutations (i.e. R243Q, R241H, G247V, L249H, P254I and G257V) and one silent mutation (V245V) were identified. The latter three missense mutations were demonstrated as novel mutations in comparison with the PAH mutation Database. One missense mutation (R241H) was first documented in Chinese. Our results showed population and regional differences in the PAH mutation distribution and suggest that there is more than one founding population for PKU in China. The finding of novel mutations will enhance our capability in molecular diagnosis of PKU.     

Dopamine-releasing action of 6R-L-erythro-tetrahydrobiopterin: analysis of its action site using sepiapterin

Recently, we reported that 6R-L-erythro-tetrahydrobiopterin (6R-BH4), a natural cofactor for hydroxylases of tyrosine and tryptophan, has a monoamine-releasing action independent of its cofactor activity. Here we attempted to determine whether 6R-BH4 acts inside the cell or from the outside of the cell by using brain microdialysis in the rat striatum. For this purpose, sepiapterin, and immediate precursor of 6R-BH4 in the salvage pathway, was used to selectively increase the intracellular 6R-BH4 levels. Dialytic perfusion of sepiapterin increased tissue levels of reduced biopterin (mainly 6R-BH4) but not the extracellular levels. Administration of sepiapterin increased the extracellular levels of 3,4-dihydroxyphenylalanine (DOPA) (an index of in vivo tyrosine hydroxylase activity) and of dopamine (DA) (an index of in vivo DA release). Either of the increases was eliminated after pretreatment with a tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. Administration of 6R-BH4 increased extracellular levels of reduced biopterin. DOPA, and DA. After pretreatment with alpha-methyl-p-tyrosine, the increase in DOPA levels was abolished, but most of the increase in DA levels persisted. The increase in DA levels also persisted after pretreatment with nitric oxide synthase inhibitors. These data demonstrate that 6R-BH4 stimulates DA release directly, independent of its cofactor action for tyrosine hydroxylase and nitric oxide synthase, by acting from the outside of neurons.     

Reliability of potential clinical measures of muscle tone in the elbows of patients after stroke

In order to establish a genotype-phenotype relationship, we have identified both mutant phenylalanine hydroxylase (PAH) genes in 108 phenylketonuria (PKU) patients (27 different alleles, 54 different genotypes). One major group of patients with very high pretreatment phenylalanine values ("classical" PKU) exclusively comprised homozygotes of the PKU mutations I65T, G272X, F299C, Y356X, R408W, IVS12nt1, and compound heterozygotes of various combinations of these alleles with G46S, R261Q, R252W, A259T, R158Q, D143G, R243X, E280K, or Y204C. A second major group of patients with lower phenylalanine values ("mild" PKU) comprised mutations A300S, R408Q, Y414C in various compound heterozygous states, and R261Q, R408Q, Y414C in homozygotes. The phenylalanine values in these groups were non-overlapping. In addition, a smaller group of patients formed the transition between the two main groups. In sib pairs 4 of 15 had discordant pretreatment phenylalanine values. Conclusion: Our results are consistent with the view that allelic heterogeneity at the PAH locus dominates the biochemical phenotype in PKU and that genotype information is able to predict the metabolic phenotype in PKU patients.     

Role of aromatic L-amino acid decarboxylase for dopamine replacement by genetically modified fibroblasts in a rat model of Parkinson's disease

Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic L-amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic L-amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic L-amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. L-DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic L-amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic L-amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic L-amino acid decarboxylase produced less L-DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic L-amino acid decarboxylase near striatal grafts producing L-DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.     

Substantial production of dopamine in the human gastrointestinal tract

Considerable urinary excretion of dopamine metabolites indicates that large amounts of dopamine are produced in unknown locations of the body. This study assessed the contribution of mesenteric organs (gastrointestinal tract, spleen, and pancreas) to the total body production of dopamine in humans and examined the presence of the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase, in gastrointestinal tissues. Blood sampled from an artery and portal and hepatic veins in eight subjects and from arterial and renal venous sites in other subjects was analyzed for plasma concentrations of dopamine and its metabolites. The activity and distribution of tyrosine hydroxylase was also examined in tissue samples from the stomach and duodenum. Higher concentrations of dopamine and its metabolites in portal venous than arterial plasma indicated substantial production of dopamine by mesenteric organs (12.0 nmol/min) amounting to 42-46% of the renal removal of circulating dopamine metabolites. Tissue samples showed immunoreactive tyrosine hydroxylase in nonneuronal cell bodies and detectable levels of tyrosine hydroxylase in nonneuronal cell bodies and detectable levels of tyrosine hydroxylase enzyme activity. The results show that mesenteric organs produce close to half of the dopamine formed in the body, most of which is unlikely to be derived from sympathetic nerves but may reflect production in a novel nonneuronal dopaminergic system.     

Protection against influenza after annually repeated vaccination: a meta-analysis of serologic and field studies

We report the effects of a tyrosine (and phenylalanine)-free amino acid mixture on tyrosine levels, ex vivo catecholamine synthesis and in vivo catecholamine release in brain regions of the rat. Administration of a tyrosine-free amino acid load reduced tissue levels of tyrosine (-50% after 2 h) in all brain regions examined (frontal cortex, hippocampus, striatum). The tyrosine-free amino acid mixture also reduced DOPA accumulation: this effect was most marked in striatum (-44%) and nucleus accumbens (-34%), areas with a predominantly dopaminergic innervation. Smaller decreases (-20-24%) were detected in other areas (cortex, hippocampus and hypothalamus). The effect on DOPA accumulation was prevented by supplementing the mixture with tyrosine/phenylalanine. The tyrosine-free amino acid mixture did not alter 5-HTP accumulation in any region. In microdialysis experiments, the tyrosine-free amino acid mixture did not consistently alter striatal extracellular dopamine under basal conditions but markedly, and dose-dependently, reduced the release of dopamine induced by amphetamine. In contrast, the tyrosine-free amino acid mixture did not alter either basal or amphetamine-evoked release of noradrenaline in hippocampus. Overall, these studies indicate that administration of a tyrosine-free amino acid mixture to rats depletes brain tyrosine to cause a decrease in regional brain catecholamine synthesis and release. Dopaminergic neurones appear to be more vulnerable to tyrosine depletion than noradrenergic neurones.     

Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli

The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin.