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1.
Dev Dyn ; 239(10): 2722-34, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20737504

ABSTRACT

In mice lacking Plexin B2, a receptor of the axon guidance molecules Semaphorin 4C and Semaphorin 4D, the closure of the neural tube and structural organization of the cerebellum are severely impaired. We cloned two Plexin B2 orthologs, plxnb2a and plxnb2b, in zebrafish, which is a widely used model for the development of the vertebrate central nervous system (CNS). The predicted proteins, Plexin B2a and Plexin B2b, contain all the conserved and functional domains of the plexin B-subfamily. During embryonic development, plxnb2a is expressed, e.g., in pharyngeal arches while plxnb2b expression is more confined to neuronal structures like the cerebellum. However, both plxnb2a and plxnb2b are expressed at the midbrain-hindbrain boundary, in the otic vesicles, facial ganglia, and pectoral fins. Knockdown of both plxnb2a and plxnb2b simultaneously (>95% and 45%, respectively) resulted in normal CNS structure, axon guidance and swimming performance of the morphants.


Subject(s)
Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Gene Expression Regulation, Developmental , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Amino Acid Sequence , Animals , Axons/metabolism , Behavior, Animal/physiology , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/classification , Central Nervous System/embryology , Central Nervous System/metabolism , Cerebellum/embryology , Cerebellum/metabolism , Embryonic Development/genetics , Embryonic Development/physiology , Exons/genetics , Introns/genetics , Mesencephalon/embryology , Mesencephalon/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/classification , Phylogeny , Rhombencephalon/embryology , Rhombencephalon/metabolism , Zebrafish , Zebrafish Proteins/chemistry , Zebrafish Proteins/classification
2.
Proc Natl Acad Sci U S A ; 106(45): 18960-5, 2009 Nov 10.
Article in English | MEDLINE | ID: mdl-19858488

ABSTRACT

LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function.


Subject(s)
Membrane Proteins/genetics , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Models, Molecular , Penicillin-Binding Proteins/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolism , Amino Acid Sequence , Animals , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Immunoblotting , Male , Mass Spectrometry , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Molecular Sequence Data , Plasmids/genetics , Polymers/metabolism , Rats , Rats, Wistar
3.
J Neurochem ; 109(2): 403-15, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19222706

ABSTRACT

Serotonin (or 5-hydroxytryptamine; 5-HT) and monoamine oxidase (MAO) are involved in several physiological functions and pathological conditions. We show that the serotonergic system and its development in zebrafish are similar to those of other vertebrates rendering zebrafish a good model to study them. Development of MAO expression followed a similar time course as the 5-HT system. MAO expression and activity were located in or adjacent to serotonergic nuclei and their targets, especially in the ventral hypothalamus. MAO mRNA was detected in the brain from 24 h post-fertilization and histochemical enzyme activity from 42 h post-fertilization. Deprenyl (100 microM) decreased MAO activity 34-74% depending on the age. Inhibition of MAO by deprenyl strongly increased 5-HT but not dopamine and noradrenaline levels. Deprenyl decreased 5-HT-immunoreactivity in serotonergic neurons and induced novel ectopic 5-HT-immunoreactivity neurons in the diencephalon in a manner dependent on 5-HT uptake. Deprenyl administration decreased locomotion, altered vertical positioning and increased heart rate. Treatment with p-chlorophenylalanine normalized 5-HT levels and rescued the behavioral alteration, indicating that the symptoms were 5-HT dependent. These findings suggest that zebrafish MAO resembles mammalian MAO A more than MAO B, metabolizing mainly 5-HT. Applications of this model of hyperserotonergism include pharmacological and genetic screenings.


Subject(s)
Monoamine Oxidase Inhibitors/pharmacology , Monoamine Oxidase/metabolism , Phenotype , Serotonin/metabolism , Zebrafish Proteins/antagonists & inhibitors , Animals , Brain/drug effects , Brain/enzymology , Brain/growth & development , Larva , Motor Activity/drug effects , Motor Activity/physiology , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , Selegiline/pharmacology , Zebrafish/growth & development , Zebrafish/metabolism , Zebrafish Proteins/chemistry
4.
BMC Evol Biol ; 8: 26, 2008 Jan 28.
Article in English | MEDLINE | ID: mdl-18226203

ABSTRACT

BACKGROUND: Bacterial penicillin-binding proteins and beta-lactamases (PBP-betaLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-betaL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-betaL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-betaL proteins. RESULTS: Metazoan PBP-betaL homologs including LACTB share unique structural features with bacterial class B low molecular weight penicillin-binding proteins. The amino acid residues necessary for enzymatic activity in bacterial PBP-betaL proteins, including the catalytic serine residue, are conserved in all metazoan homologs. Phylogenetic analysis indicated that metazoan PBP-betaL homologs comprise four alloparalogus protein lineages that derive from alpha-proteobacteria. CONCLUSION: While most components of the peptidoglycan synthesis machinery were dumped by early eukaryotes, a few PBP-betaL proteins were conserved and are found in metazoans including humans. Metazoan PBP-betaL homologs are active-site-serine enzymes that probably have distinct functions in the metabolic circuitry. We hypothesize that PBP-betaL proteins in the early eukaryotic cell enabled the degradation of peptidoglycan from ingested bacteria, thereby maximizing the yield of nutrients and streamlining the cell for effective phagocytotic feeding.


Subject(s)
Bacteria/genetics , Evolution, Molecular , Penicillin-Binding Proteins/metabolism , beta-Lactamases/metabolism , Amino Acid Sequence , Animals , Bacteria/enzymology , Bacteria/metabolism , Binding Sites , Catalytic Domain , Conserved Sequence , Humans , Membrane Proteins/genetics , Mitochondrial Proteins , Molecular Weight , Ribosomal Proteins/genetics , Sequence Alignment , Sequence Homology
5.
Biochem Pharmacol ; 73(8): 1205-14, 2007 Apr 15.
Article in English | MEDLINE | ID: mdl-17266939

ABSTRACT

Neuronal histamine regulates several functions in the vertebrate brain. The zebrafish brain contains a widespread histaminergic system and H(3) receptor ligand binding has been reported. In this study we provide evidence for the existence of histamine H(1), H(2) and H(3) receptor genes in zebrafish. Single copies of putative histamine H(1), H(2) and H(3) receptors were identified and cloned from the zebrafish brain. Expression analysis suggested that they are expressed in the brain and a few other tissues. Widespread distribution of zebrafish H(2) receptor binding sites was detected with [(125)I]iodoaminopotentidine in brain sections. Zebrafish larvae were exposed to 1, 10 or 100 microM of the H(1) ligand pyrilamine, the H(2) ligand cimetidine and the H(3) ligands thioperamide and immepip for 5 days. Significant decreases in swimming distance were observed with the highest dose of all ligands, whereas cimetidine gave a significant decrease also with 1 and 10 microM doses. These results provide the first molecular biological evidence for the presence of histamine receptors in zebrafish. These histamine receptors resemble those of higher vertebrates and they provide a useful model for pharmacological and behavioral studies for characterizing the functions of histamine in more detail.


Subject(s)
Behavior, Animal/drug effects , Histamine Agonists/pharmacology , Histamine Antagonists/pharmacology , Receptors, Histamine H1/isolation & purification , Receptors, Histamine H2/isolation & purification , Receptors, Histamine H3/isolation & purification , Animals , Behavior, Animal/physiology , Cimetidine/pharmacology , Imidazoles/pharmacology , Piperidines/pharmacology , Zebrafish
6.
J Comp Neurol ; 498(5): 593-610, 2006 Oct 10.
Article in English | MEDLINE | ID: mdl-16917825

ABSTRACT

Monoamine oxidase (MAO) is a mitochondrial flavoprotein involved in the metabolism of, e.g., aminergic neurotransmitters and the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). We have reported earlier MPTP-related alterations of brain catecholaminergic system in zebrafish (Danio rerio) brain. Here we describe the structural and functional properties of zebrafish MAO and the distribution of MAO mRNA and activity in zebrafish brain. The gene is located in chromosome 9 and consists of 15 exons. The amino acid composition of the active center resembles both human MAO-A and MAO-B. The enzyme displayed the highest substrate specificity for tyramine, followed by serotonin, phenylethylamine, MPTP, and dopamine; isoform-specific antagonists blocked the activity of the enzyme with equal potency. Zebrafish MAO mRNA, which was present in several tissues, and enzyme displayed differential distribution in the brain; dopaminergic cell clusters had low to moderate levels of MAO activity, whereas the highest levels of MAO activity were detected in noradrenergic and serotonergic cell groups and the habenulointerpeduncular pathway, including its caudal projection to the medial ventral rhombencephalon. The results of this study confirm the presence of functionally active MAO in zebrafish brain and other tissues and characterize the neural systems that express MAO and areas of intense activity in the brain. They also suggest that MPTP toxicity not related to MAO may affect the zebrafish brain.


Subject(s)
Brain/anatomy & histology , Brain/enzymology , Monoamine Oxidase/metabolism , Zebrafish/metabolism , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Analysis of Variance , Animals , Biogenic Monoamines/pharmacology , Brain/drug effects , Cloning, Molecular/methods , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Female , Gene Expression Regulation/drug effects , Immunohistochemistry/methods , In Situ Hybridization/methods , Male , Molecular Sequence Data , Monoamine Oxidase/chemistry , Monoamine Oxidase/genetics , Neurotoxins/pharmacology , Protein Conformation , RNA, Messenger/metabolism , Time Factors
7.
Protein Expr Purif ; 45(2): 335-42, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16202624

ABSTRACT

LACTB is a mammalian mitochondrial protein sharing sequence similarity to the beta-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione-agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of alpha-helices, beta-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.


Subject(s)
Escherichia coli/metabolism , Membrane Proteins , Mitochondria/enzymology , Recombinant Fusion Proteins , Ribosomal Proteins , Amino Acid Sequence , Animals , Escherichia coli/genetics , Humans , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Protein Structure, Secondary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/isolation & purification , Ribosomal Proteins/metabolism , Sequence Alignment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectroscopy, Fourier Transform Infrared , beta-Lactamases/genetics , beta-Lactamases/metabolism
8.
J Neurochem ; 94(6): 1559-69, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16000146

ABSTRACT

The alpha2-adrenoceptors are G-protein-coupled receptors that mediate many of the physiological effects of norepinephrine and epinephrine. Mammals have three subtypes of alpha2-adrenoceptors, alpha2A, alpha2B and alpha2C. Zebrafish, a teleost fish used widely as a model organism, has five distinct alpha2-adrenoceptor genes. The zebrafish has emerged as a powerful tool to study development and genetics, with many mutations causing diseases reminiscent of human diseases. Three of the zebrafish adra2 genes code for orthologues of the mammalian alpha2-adrenoceptors, while two genes code for alpha2Da- and alpha2Db- adrenoceptors, representing a duplicated, fourth alpha2-adrenoceptor subtype. The three different mammalian alpha2-adrenoceptor subtypes have distinct expression patterns in different organs and tissues, and mediate different physiological functions. The zebrafish alpha2-adrenergic system, with five different alpha2-adrenoceptors, appears more complicated. In order to deduce the physiological functions of the zebrafish alpha2-adrenoceptors, we localized the expression of the five different alpha2-adrenoceptor subtypes using RT-PCR, mRNA in situ hybridization, and receptor autoradiography using the radiolabelled alpha2-adrenoceptor antagonist [ethyl-3H]RS-79948-197. Localization of the alpha2A-, alpha2B- and alpha2C-adrenoceptors in zebrafish shows marked conservation when compared with mammals. The zebrafish alpha2A, alpha2Da, and alpha2Db each partially follow the distribution pattern of the mammalian alpha2A: a possible indication of subfunction partitioning between these subtypes. The alpha2-adrenergic system is functional in zebrafish also in vivo, as demonstrated by marked locomotor inhibition, similarly to mammals, and lightening of skin colour induced by the specific alpha2-adrenoceptor agonist, dexmedetomidine. Both effects were antagonized by the specific alpha2-adrenoceptor antagonist atipamezole.


Subject(s)
Brain/metabolism , Catecholamines/metabolism , RNA, Messenger/metabolism , Receptors, Adrenergic, alpha-2/metabolism , Zebrafish/metabolism , Adrenergic alpha-Agonists/pharmacology , Adrenergic alpha-Antagonists/pharmacology , Animals , Binding, Competitive/physiology , Brain/anatomy & histology , Female , Isoquinolines/metabolism , Male , Mammals/anatomy & histology , Mammals/metabolism , Motor Activity/drug effects , Motor Activity/physiology , Naphthyridines/metabolism , Pigmentation/drug effects , Pigmentation/physiology , RNA, Messenger/drug effects , Radioligand Assay , Receptors, Adrenergic, alpha-2/drug effects , Receptors, Adrenergic, alpha-2/genetics , Viscera/metabolism , Zebrafish/anatomy & histology
9.
J Neurosci ; 24(11): 2678-89, 2004 Mar 17.
Article in English | MEDLINE | ID: mdl-15028760

ABSTRACT

The orexin/hypocretin (ORX) system is involved in physiological processes such as feeding, energy metabolism, and the control of sleep and wakefulness. The ORX system may drive the aminergic and cholinergic activities that control sleep and wakefulness states because of the ORX fiber projections to the aminergic and cholinergic cell clusters. The biological mechanisms and relevance of the interactions between these neurotransmitter systems are poorly understood. We studied these systems in zebrafish, a model organism in which it is possible to simultaneously study these systems and their interactions. We cloned a zebrafish prepro-ORX gene that encodes for the two functional neuropeptides orexin-A (ORX-A) and orexin-B (ORX-B). The prepro-ORX gene of the zebrafish consisted of one exon in contrast to mammals. The sequence of the ORX-A peptide of the zebrafish was less conserved than the ORX-B peptide compared with other vertebrates. By using in situ hybridization and immunohistochemistry, we found that the organization of the ORX system of zebrafish was similar to the ORX system in mammals, including a hypothalamic cell cluster and widespread fiber projections. The ORX system of the zebrafish showed a unique characteristic with an additional putatively ORX-containing cell group. The ORX system innervated several aminergic nuclei, raphe, locus ceruleus, the mesopontine-like area, dopaminergic clusters, and histaminergic neurons. A reciprocal relationship was found between the ORX system and several aminergic systems. Our results suggest that the architecture of these neurotransmitter systems is conserved in vertebrates and that these neurotransmitter systems in zebrafish may be involved in regulation of states of wakefulness and energy homeostasis by similar mechanisms as those in mammals.


Subject(s)
Acetylcholine/metabolism , Carrier Proteins/metabolism , Dopamine/metabolism , Histamine/metabolism , Intracellular Signaling Peptides and Proteins , Neuropeptides/metabolism , Protein Precursors/metabolism , Zebrafish/metabolism , Amino Acid Sequence , Animals , Antibody Specificity , Carrier Proteins/genetics , Cholinergic Fibers/metabolism , Cloning, Molecular , Hypothalamus/metabolism , Immunohistochemistry , Models, Animal , Molecular Sequence Data , Neural Pathways/metabolism , Neurons/cytology , Neurons/metabolism , Neuropeptides/genetics , Neurotransmitter Agents/metabolism , Norepinephrine/metabolism , Orexins , Protein Precursors/genetics , RNA, Messenger/metabolism , Serotonin/metabolism , Tyrosine 3-Monooxygenase/biosynthesis
10.
J Neurochem ; 88(2): 443-53, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14690532

ABSTRACT

Dopaminergic deficiency in the brain of zebrafish was produced by systemic administration of two catecholaminergic neurotoxins, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and the neurochemical and behavioural changes were characterized. The levels of dopamine and noradrenaline decreased significantly after the injection of MPTP and 6-OHDA. Corresponding to these changes, fish exhibited characteristic changes in locomotor behaviour, i.e. the total distance moved and velocity decreased after both neurotoxins. Tyrosine hydroxylase and caspase 3 protein levels were not altered after MPTP or 6-OHDA injections, as studied by immunohistochemistry and western blotting. The catecholaminergic cell clusters suggested to correspond to the mammalian nigrostriatal cell group displayed normal tyrosine hydroxylase immunoreactivity after the toxin treatment and did not show signs of DNA fragmentation that would indicate activation of cascades that lead to cell death. The results show that single systemic injections of MPTP and 6-OHDA induce both biochemical and behavioural changes in zebrafish, albeit failing to produce any significant morphological alteration in catecholaminergic cell clusters at the tested doses. This approach may be used for the screening of chemicals affecting the dopaminergic system. The model may be especially useful for evaluation of the role of novel genes in neurotoxicity, as a large number of zebrafish mutants are becoming available.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Brain/drug effects , Motor Activity/drug effects , Oxidopamine/pharmacology , Zebrafish/metabolism , Animals , Brain/metabolism , Dopamine/metabolism , Female , Injections, Intramuscular , Male , Motor Activity/physiology , Norepinephrine/metabolism , Pilot Projects
11.
J Neurochem ; 86(2): 432-41, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12871584

ABSTRACT

The functional role of histamine (HA) in zebrafish brains was studied. Zebrafish did not display a clear circadian variation in brain HA levels. Loading of zebrafish with l-histidine increased HA concentration in the brain. A single injection of the histidine decarboxylase (HDC) inhibitor, alpha-fluoromethylhistidine (alpha-FMH), gave rise to a rapid reduction in zebrafish brain HA. Low HDC activity in the brain after injections verified the effect of alpha-FMH. A reduction in the number of histaminergic fibres but not neurones and an increased expression of HDC mRNA was evident after alpha-FMH. Automated behavioural analysis after alpha-FMH injection showed no change in swimming activity, but abnormalities were detected in exploratory behaviour examined in a circular tank. No significant behavioural changes were detected after histidine loading. The time spent for performance in the T-maze was significantly increased in the first trial 4 days after alpha-FMH injections, suggesting that lack of HA may impair long-term memory. The rostrodorsal telencephalon, considered to correspond to the mammalian amygdala and hippocampus in zebrafish, is densely innervated by histaminergic fibres. These results suggest that low HA decreases anxiety and/or affects learning and memory in zebrafish, possibly through mechanisms that involve the dorsal forebrain.


Subject(s)
Behavior, Animal/drug effects , Brain/metabolism , Histamine/metabolism , Methylhistidines/pharmacology , Zebrafish/physiology , Animals , Brain/drug effects , Circadian Rhythm/physiology , Enzyme Induction/drug effects , Enzyme Inhibitors/pharmacology , Female , Histidine/pharmacology , Histidine Decarboxylase/antagonists & inhibitors , Histidine Decarboxylase/metabolism , Male , Maze Learning/drug effects , Swimming
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