Tyrosine hydroxylase positive nerves and mast cells in the porcine gallbladder

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I. Stefanov

2017-03-01

Full Text Available The aim of this study was to detect the localisation of tyrosine hydroxylase (TH positive nerve fibres (THN and distribution of tyrosine hydroxylase positive mast cells (THMC in the wall of porcine gallbladder. THN were observed as single fibres, nerve fibres forming perivascular plexuses and nerve fibres grouped within the nerve fascicles. In the gallbladder`s fundus, body and neck, the TH+ fibres formed mucosal, muscular and serosal nonganglionated nerve plexuses. Toluidine blue (TB staining was used to confirm that the TH positive cells were mast cells. The number of THMC in the propria of gallbladder`s fundus, body and neck was significantly higher than in the muscular and serosal layers in both genders. The number of mast cells in the musculature was higher than in the serosa. The density and location of the THMC were similar to the TB positive (with gamma meta-chromasia mast cells in both males and females, and statistically significant difference was not established. In conclusion, original data concerning the existence and localisation of catecholaminergic nerves and THMC distribution in the porcine gallbladder’s wall are presented. The results could con-tribute to the body of knowledge of functional communication between autonomic nerves and mast cells in the gallbladder.

Quantitative radioautographic determination of brain tyrosine hydroxylase after direct transfer into nitro-cellulose and immunochemical detection

International Nuclear Information System (INIS)

Weissmann, D.; Labatut, R.; Gillon, J.Y.

1988-01-01

An improved quantitative immuno chemical determination of tyrosine hydroxylase brain concentrations was designed by using direct transfer into nitro-cellulose from 20 μm thick brain sections followed by immuno-detection and quantitative radioautography [fr

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

Science.gov (United States)

Nagatsu, T; Oka, K; Kato, T

1979-07-21

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.

Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

OpenAIRE

Khwanraj, Kawinthra; Phruksaniyom, Chareerut; Madlah, Suriyat; Dharmasaroja, Permphan

2015-01-01

The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson's disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+ and evaluate the differential expression of tyrosine hydroxylase (TH) in undifferentiated and retinoic acid- (RA-) induced differentiated cells. The western blot results showed a gradual decre...

Tyrosine hydroxylase in the ventral tegmental area of rams with high or low libido-A role for dopamine.

Science.gov (United States)

Kramer, A C; Mirto, A J; Austin, K J; Roselli, C E; Alexander, B M

2017-12-01

Dopamine synthesis in the ventral tegmental area (VTA) is necessary for the reinforcement of sexual behavior. The objective of this study determined if sexual stimuli initiates reward, and whether reward is attenuated in sexually inactive rams. Sexually active rams were exposed to urine from estrous (n=4) or ovariectomized (n=3) ewes with inactive rams (n=3) exposed to urine from estrous ewes. Following exposure, rams were exsanguinated and brains perfused. Alternating sections of the VTA were stained for Fos related antigens (FRA), tyrosine hydroxylase, and dopamine beta-hydroxylase activity. Forebrain tissue, mid-sagittal ventral to the anterior corpus callosum, was stained for dopamine D 2 receptors. Concentrations of cortisol was determined prior to and following exposure. Exposure to ovariectomized-ewe urine in sexually active rams did not influence (P=0.6) FRA expression, but fewer (PSexually inactive rams had fewer (Psexually active rams following exposure to estrous ewe urine. VTA neurons staining positive for dopamine beta-hydroxylase did not differ by sexual activity (P=0.44) or urine exposure (P=0.07). Exposure to stimulus did not influence (P=0.46) numbers of forebrain neurons staining positive for dopamine D2 receptors in sexually active rams, but fewer (P=0.04) neurons stain positive in inactive rams. Serum concentrations of cortisol did not differ (P≥0.52) among rams prior to or following stimulus. In conclusion sexual inactivity is unlikely due to stress, but may be partially a result of decreased tyrosine hydroxylase and/or the response to dopamine. Copyright © 2017 Elsevier B.V. All rights reserved.

The expression and significance of tyrosine hydroxylase in the brain tissue of Parkinsons disease rats

OpenAIRE

Chen, Yuan; Lian, Yajun; Ma, Yunqing; Wu, Chuanjie; Zheng, Yake; Xie, Nanchang

2017-01-01

The expression and significance of tyrosine hydroxylase (TH) in brain tissue of rats with Parkinson's disease (PD) were explored and analyzed. A total of 120 clean-grade and healthy adult Wistar rats weighing 180–240 g were randomly divided equally into four groups according to the random number table method. Rats were sacrificed before and after the model establishment for 3, 6 or 8 weeks. The number of revolutions in rats was observed and the relative expression of TH mRNA in brain tissue w...

Heterogeneous expression of cholesterol 7α-hydroxylase and sterol 27- hydroxylase genes in the rat liver lobulus

NARCIS (Netherlands)

Twisk, J.; Hoekman, M.F.M.; Mager, W.H.; Moorman, A.F.M.; Boer, P.A.J. de; Scheja, L.; Princen, H.M.G.; Gebhardt, R.

1995-01-01

We investigated the lobular localization and molecular level of expression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene

Increased expression of tyrosine hydroxylase immunoreactivity in paraventricular and supraoptic neurons in illnesses with prolonged osmotic or nonosmotic stimulation of vasopressin release

NARCIS (Netherlands)

Panayotacopoulou, Maria T.; Malidelis, Yiannis I.; Fliers, Eric; Bouras, Constantin; Ravid, Rivka; Swaab, Dick F.

2002-01-01

Our previous studies indicated that in the human para-ventricular (PVN) and supraoptic (SON) nuclei, tyrosine hydroxylase (TH) - the first and rate-limiting enzyme in catecholamine synthesis - is localized mainly in magnocellular neurons and that antemortem factors regulate its expression. The

Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (USA))

1988-11-01

Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

International Nuclear Information System (INIS)

Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C.

1988-01-01

Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human α 1 -antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the α 1 antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes

Excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in experimental approaches of Parkinsonism.

Science.gov (United States)

Fernández-Espejo, Emilio; Bis-Humbert, Cristian

2018-06-06

3-iodo-l-tyrosine might play a role in Parkinson's disease since this molecule is able, at high concentration, to inhibit tyrosine-hydroxylase activity, the rate-limiting enzyme in dopamine biosynthesis. The possible Parkinson-like effects of 3-iodo-l-tyrosine were tested on three experimental approaches in mice: cultured substantia nigra neurons, the enteric nervous system of the jejunum after intra-peritoneal infusions, and the nigrostriatal system following unilateral intrabrain injections. 3-iodo-l-tyrosine, a physiological molecule, was used at concentrations higher than its serum levels in humans. Parkinson-like signs were evaluated through abnormal aggregation of α-synuclein and tyrosine-hydroxylase, loss of tyrosine-hydroxylase-expressing and striatum-projecting neurons and fibers, reduced tyrosine-hydroxylase density, and Parkinson-like motor and non-motor deficits. The retrograde tracer FluoroGold was used in the brain model. The findings revealed that excess amounts of 3-iodo-l-tyrosine induce Parkinson-like effects in the three experimental approaches. Thus, culture neurons of substantia nigra show, after 3-iodo-l-tyrosine exposure, intracytoplasmic inclusions that express α-synuclein and tyrosine-hydroxylase. Intra-peritoneal infusions of 3-iodo-l-tyrosine cause, in the long-term, α-synuclein aggregation, thicker α-synuclein-positive fibers, and loss of tyrosine-hydroxylase-positive cells and fibers in intramural plexuses and ganglia of the jejunum. Infusion of 3-iodo-l-tyrosine into the left dorsal striata of mice damages the nigrostriatal system, as revealed through lower striatal tyrosine-hydroxylase density, reduced number of tyrosine-hydroxylase-expressing and striatum-projecting neurons in the left substantia nigra, as well as the emergence of Parkinson-like behavioral deficits such as akinesia, bradykinesia, motor disbalance, and locomotion directional bias. In conclusion, excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in

Tyrosine hydroxylase-immunoreactivity and its relations with gonadotropin-releasing hormone and neuropeptide Y in the preoptic area of the guinea pig.

Science.gov (United States)

Bogus-Nowakowska, Krystyna; Równiak, Maciej; Hermanowicz-Sobieraj, Beata; Wasilewska, Barbara; Najdzion, Janusz; Robak, Anna

2016-12-01

The present study examines the distribution of tyrosine hydroxylase (TH) immunoreactivity and its morphological relationships with neuropeptide Y (NPY)- and gonadoliberin (GnRH)-immunoreactive (IR) structures in the preoptic area (POA) of the male guinea pig. Tyrosine hydroxylase was expressed in relatively small population of perikarya and they were mostly observed in the periventricular preoptic nucleus and medial preoptic area. The tyrosine hydroxylase-immunoreactive (TH-IR) fibers were dispersed troughout the whole POA. The highest density of these fibers was observed in the median preoptic nucleus, however, in the periventricular preoptic nucleus and medial preoptic area they were only slightly less numerous. In the lateral preoptic area, the density of TH-IR fibers was moderate. Two morphological types of TH-IR fibers were distinguished: smooth and varicose. Double immunofluorescence staining showed that TH and GnRH overlapped in the guinea pig POA but they never coexisted in the same structures. TH-IR fibers often intersected with GnRH-IR structures and many of them touched the GnRH-IR perikarya or dendrites. NPY wchich was abundantly present in the POA only in fibers showed topographical proximity with TH-IR structures. Althoug TH-IR perikarya and fibers were often touched by NPY-IR fibers, colocalization of TH and NPY in the same structures was very rare. There was only a small population of fibers which contained both NPY and TH. In conclusion, the morphological evidence of contacts between TH- and GnRH-IR nerve structures may be the basis of catecholaminergic control of GnRH release in the preoptic area of the male guinea pig. Moreover, TH-IR neurons were conatcted by NPY-IR fibers and TH and NPY colocalized in some fibers, thus NPY may regulate catecholaminergic neurons in the POA. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of runway training on rat brain tyrosine hydroxylase: differential effect of continuous and partial reinforcement schedules.

Science.gov (United States)

Boarder, M R; Feldon, J; Gray, J A; Fillenz, M

1979-12-01

Previous experiments have implicated ascending noradrenergic systems in the development of the behavioural responses to different patterns of reward. In this report food deprived male Sprague--Dawley rats were trained to run a straight alley for good reward on a continuous reinforcement (CRF) or a partial reinforcement (PRF) schedule. Tyrosine hydroxylase measured in a partially solubilized preparation from hippocampus and hypothalamus at the end of acquisition was not different from controls, indicating that enzyme induction does not occur during either training schedules. However, hippocampal synaptosomal tyrosine hydroxylation rates from the CRF group was significantly higher than from either the PRF group or the handled controls. This indicates that at the end of the acquisition schedule the noradrenergic projection to hippocampus was more active in the CRF group than with the PRF group or the handled control.

Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons.

Science.gov (United States)

Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

2016-01-01

The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson's disease.

Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

Directory of Open Access Journals (Sweden)

Kawinthra Khwanraj

2015-01-01

Full Text Available The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson’s disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+ and evaluate the differential expression of tyrosine hydroxylase (TH in undifferentiated and retinoic acid- (RA- induced differentiated cells. The western blot results showed a gradual decrease in TH in undifferentiated cells and a gradual increase in TH in differentiated cells from days 4 to 10 after cell plating. Immunostaining revealed a gradual increase in TH along with neuritic outgrowth in differentiated cells on days 4 and 7 of RA treatment. For the study on cell susceptibility to MPP+ and the expression of apoptosis-related genes, MTT assay showed a decrease in cell viability to approximately 50% requiring 500 and 1000 μM of MPP+ for undifferentiated and RA-differentiated cells, respectively. Using real-time RT-PCR, treatment with 500 μM MPP+ led to significant increases in the Bax/Bcl-2 ratio, p53, and caspase-3 in undifferentiated cells but was without significance in differentiated cells. In conclusion, differentiated cells may be more suitable, and the shorter duration of RA differentiation may make the SH-SY5Y cell model more accessible.

The alpha(2)-adrenoceptors do not modify the activity of tyrosine hydroxylase, corticoliberine, and neuropeptide Y producing hypothalamic magnocellular neurons ion the Long Evans and Brattleboro rats

DEFF Research Database (Denmark)

Bundzikova, J; Pirnik, Z; Zelena, D

2010-01-01

The hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei are activated by body salt-fluid variations. Stimulation of alpha(2)-adrenoceptors by an agonist-xylazine (XYL) activates oxytocinergic but not vasopressinergic magnocellular neurons. In this study, tyrosine hydroxylase (TH), cort...

Putaminal mosaic visualized by tyrosine hydroxylase immunohistochemistry in the human neostriatum.

Directory of Open Access Journals (Sweden)

Ryoma eMorigaki

2016-04-01

Full Text Available Among the basal ganglia-thalamocortical circuits, the putamen plays a critical role in the ‘motor’ circuits that control voluntary movements and motor learning. The human neostriatum comprises two functional subdivisions known as the striosome (patch and matrix compartments. Accumulating evidence suggests that compartment-specific dysregulations of dopamine activity might be involved in the disease-specific pathology and symptoms of human striatal diseases including movement disorders. This study was undertaken to examine whether or how striatal dopaminergic innervations are organized into the compartmentalized architecture found in the putamen of adult human brains. For this purpose, we used a highly sensitive immunohistochemistry technique to identify tyrosine hydroxylase (TH, EC 1.14.16.2, a marker for striatal dopaminergic axons and terminals, in formalin-fixed paraffin-embedded tissues obtained from autopsied human brains. Herein, we report that discrete compartmentalization of TH-labeled innervations occurs in the putamen, as in the caudate nucleus, with a higher density of TH labeling in the matrix compared to the striosomes. Our results provide anatomical evidence to support the hypothesis that compartment-specific dysfunction of the striosome-matrix dopaminergic systems might contribute to the genesis of movement disorders.

Early fetal acquisition of the chromaffin and neuronal immunophenotype by human adrenal medullary cells. An immunohistological study using monoclonal antibodies to chromogranin A, synaptophysin, tyrosine hydroxylase, and neuronal cytoskeletal proteins.

NARCIS (Netherlands)

Molenaar, W M; Lee, V M; Trojanowski, J Q

1990-01-01

The development of chromaffin and neuronal features in the adrenal medulla was studied in normal human fetuses with gestational ages (GAs) of 6-34 weeks. Monoclonal antibodies specific for chromogranin A, synaptophysin, and tyrosine hydroxylase; for different subunits and phosphoisoforms of

Mobilisation of store Ca2+ activates tyrosine hydroxylase in bovine adrenal chromaffin cells

International Nuclear Information System (INIS)

McKenzie, S.; Marley, P.D.

2001-01-01

Full text: Many receptor agonists are able to activate tyrosine hydroxylase (TOH) in bovine adrenal chromaffin cells. The majority of these are dependent on extracellular Ca 2+ for this action. Entry of extracellular Ca 2+ through voltage-operated Ca 2+ channels is very effective at activating TOH. The contribution of the intracellular Ca 2+ stores to TOH activation however is not known. Previous studies have shown that mobilisation of intracellular Ca 2+ stores is effective at increasing phosphorylation of TOH, but its effect on TOH activity has not been studied. Therefore, in the present study, the effect of mobilisation of store Ca 2+ on TOH activity was investigated using primary cultures of bovine adrenal chromaffin cells. Cells were prepared from abattoir tissue and cultured for 3-6 days. TOH activity was determined over 10 minutes, measuring the 14 CO 2 produced following the hydroxylation and rapid decarboxylation of 14 C-tyrosine offered to intact cells. Caffeine increased TOH activity in a concentration-dependent manner with a maximum response of 100% increase at 20mM. This effect was not due to osmolarity since 20mM sucrose had no effect.Nor was it due to inhibition of phosphodiesterases, since the effect of caffeine was still seen in the presence of 1mM IBMX. However,caffeine-induced TOH activation was substantially reduced in the absence of extracellular Ca 2+ . The results suggest that TOH activity can be increased by mobilising intracellular Ca 2+ stores, but that this effect involves extracellular Ca 2+ influx, possibly through store-operated channels. Copyright (2001) Australian Neuroscience Society

Characterization of carotenoid hydroxylase gene promoter in Haematococcus pluvialis.

Science.gov (United States)

Meng, C X; Wei, W; Su, Z- L; Qin, S

2006-10-01

Astaxanthin, a high-value ketocarotenoid is mainly used in fish aquaculture. It also has potential in human health due to its higher antioxidant capacity than beta-carotene and vitamin E. The unicellular green alga Haematococcus pluvialis is known to accumulate astaxanthin in response to environmental stresses, such as high light intensity and salt stress. Carotenoid hydroxylase plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, we report the characterization of a promoter-like region (-378 to -22 bp) of carotenoid hydroxylase gene by cloning, sequence analysis and functional verification of its 919 bp 5'-flanking region in H. pluvialis. The 5'-flanking region was characterized using micro-particle bombardment method and transient expression of LacZ reporter gene. Results of sequence analysis showed that the 5'-flanking region might have putative cis-acting elements, such as ABA (abscisic acid)-responsive element (ABRE), C-repeat/dehydration responsive element (C-repeat/DRE), ethylene-responsive element (ERE), heat-shock element (HSE), wound-responsive element (WUN-motif), gibberellin-responsive element (P-box), MYB-binding site (MBS) etc., except for typical TATA and CCAAT boxes. Results of 5' deletions construct and beta-galactosidase assays revealed that a highest promoter-like region might exist from -378 to -22 bp and some negative regulatory elements might lie in the region from -919 to -378 bp. Results of site-directed mutagenesis of a putative C-repeat/DRE and an ABRE-like motif in the promoter-like region (-378 to -22 bp) indicated that the putative C-repeat/DRE and ABRE-like motif might be important for expression of carotenoid hydroxylase gene.

Transient knockdown of tyrosine hydroxylase during development has persistent effects on behaviour in adult zebrafish (Danio rerio.

Directory of Open Access Journals (Sweden)

Isabel Formella

Full Text Available Abnormal dopamine (DA signaling is often suggested as causative in schizophrenia. The other prominent hypothesis for this disorder, largely driven by epidemiological data, is that certain adverse events during the early stages of brain development increase an individual's risk of developing schizophrenia later in life. However, the clinical and preclinical literature consistently implicates behavioural, cognitive, and pharmacological abnormalities, implying that DA signaling is abnormal in the adult brain. How can we reconcile these two major hypotheses underlying much of the clinical and basic research into schizophrenia? In this study we have transiently knocked down tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis gene expression in the early stages of brain development in zebrafish using morpholinos. We show that by adulthood, TH and DA levels have returned to normal and basic DA-mediated behaviours, such as locomotion, are also normal. However, when they were exposed to a novel environment the levels of freezing and immediate positioning in deeper zones were significantly reduced in these adult fish. The neurochemistry underlying these behaviours is complex, and the exact mechanisms for these abnormal behaviours remains unknown. This study demonstrates that early transient alterations in DA ontogeny can produce persistent alterations in adult brain function and suggests that the zebrafish may be a promising model animal for future studies directed at clarifying the basic neurodevelopmental mechanisms behind complex psychiatric disease.

Characterisation of tryptic peptides of phosphorylated tyrosine hydroxylase by high-pressure liquid chromatography electrospray ionisation mass spectrometry

International Nuclear Information System (INIS)

Graham, Mark E.; Dickson, Phillip W.; Dunkley, Peter R.; Nagy-Felsobuki, Ellak I. von

2005-01-01

Tyrosine hydroxylase (TH) is involved in the biosynthesis of catecholamines and is activated by phosphorylation. Phosphorylated TH was analysed using high-pressure liquid chromatography combined with electrospray mass spectrometry (HPLC ESI-MS). Two mass scanning methods were used to detect tryptic cleavage products of TH. In the positive electrospray ionisation mode (ESI+), the peptides that contain the phosphorylation sites of TH were identified. In the alternative method, a phosphopeptide was detected in the negative electrospray ionisation mode (ESI-) using single ion monitoring in combination with a sequential ESI+ switching experiment. A raised baseline interfered with detection of hydrophilic peptides in ESI-, with the signal-to-noise ratio indicating that the method was operating near the limit of detection for a conventional electrospray source. The switching method improved the certainty of identification of phosphopeptides

Effect of ghrelin receptor agonist and antagonist on the activity of arcuate nucleus tyrosine hydroxylase containing neurons in C57BL/6 male mice exposed to normal or high fat diet

Czech Academy of Sciences Publication Activity Database

Pirník, Z.; Majerčíková, Z.; Holubová, Martina; Pirník, R.; Železná, Blanka; Maletínská, Lenka; Kiss, A.

2014-01-01

Roč. 65, č. 4 (2014), s. 477-486 ISSN 0867-5910 Institutional support: RVO:61388963 Keywords : growth hormone secretagogue receptor * ghrelin receptor agonist * ghrelin receptor antagonist * high fat diet * tyrosine hydroxylase * arcuate nucleus * food intake Subject RIV: CE - Biochemistry Impact factor: 2.386, year: 2014

AAV-mediated gene transfer of the obesity-associated gene Etv5 in rat midbrain does not affect energy balance or motivated behavior.

Directory of Open Access Journals (Sweden)

Arjen J Boender

Full Text Available Several genome-wide association studies have implicated the transcription factor E-twenty- six version 5 (Etv5 in the regulation of body mass index. Further substantiating the role of Etv5 in feeding behavior are the findings that targeted disruption of Etv5 in mice leads to decreased body weight gain and that expression of Etv5 is decreased in the ventral tegmental area and substantia nigra pars compacta (VTA/SNpc after food restriction. As Etv5 has been suggested to influence dopaminergic neurotransmission by driving the expression of genes that are responsible for the synthesis and release of dopamine, we investigated if expression levels of Etv5 are dependent on nutritional state and subsequently influence the expression levels of tyrosine hydroxylase. While it was shown that Etv5 expression in the VTA/SNpc increases after central administration of leptin and that Etv5 was able to drive expression of tyrosine hydroxylase in vitro, AAV-mediated gene transfer of Etv5 into the VTA/SNpc of rats did not alter expression of tyrosine hydroxylase in vivo. Moreover, AAV-mediated gene transfer of Etv5 in the VTA/SNpc did not affect measures of energy balance or performances in a progressive ratio schedule. Thus, these data do not support a role for increased expression of Etv5 in the VTA/SNpc in the regulation of feeding behavior.

FoxO1 in dopaminergic neurons regulates energy homeostasis and targets tyrosine hydroxylase

Science.gov (United States)

Doan, Khanh V.; Kinyua, Ann W.; Yang, Dong Joo; Ko, Chang Mann; Moh, Sang Hyun; Shong, Ko Eun; Kim, Hail; Park, Sang-Kyu; Kim, Dong-Hoon; Kim, Inki; Paik, Ji-Hye; DePinho, Ronald A.; Yoon, Seul Gi; Kim, Il Yong; Seong, Je Kyung; Choi, Yun-Hee; Kim, Ki Woo

2016-01-01

Dopaminergic (DA) neurons are involved in the integration of neuronal and hormonal signals to regulate food consumption and energy balance. Forkhead transcriptional factor O1 (FoxO1) in the hypothalamus plays a crucial role in mediation of leptin and insulin function. However, the homoeostatic role of FoxO1 in DA system has not been investigated. Here we report that FoxO1 is highly expressed in DA neurons and mice lacking FoxO1 specifically in the DA neurons (FoxO1 KODAT) show markedly increased energy expenditure and interscapular brown adipose tissue (iBAT) thermogenesis accompanied by reduced fat mass and improved glucose/insulin homoeostasis. Moreover, FoxO1 KODAT mice exhibit an increased sucrose preference in concomitance with higher dopamine and norepinephrine levels. Finally, we found that FoxO1 directly targets and negatively regulates tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of the catecholamine synthesis, delineating a mechanism for the KO phenotypes. Collectively, these results suggest that FoxO1 in DA neurons is an important transcriptional factor that directs the coordinated control of energy balance, thermogenesis and glucose homoeostasis. PMID:27681312

Tyrosine hydroxylase regulatory domain as indicator of enzyme sensitivity to irradiation

International Nuclear Information System (INIS)

Mustafayeva, N.N.; Alieva, I.N.; Aliev, Ds.I.

2002-01-01

Full text: At the present time contra dictionary and variously kind opinions concern to effect of different level of irradiation on the structure and functional activity of the tyrosine hydroxylase (TH), the key a rate-limiting enzyme in the biosynthesis of catecholamines are discussed in this study. To date, the effect of the irradiation on the both catalytic and N-terminal regulatory domains of TH localized in the different parts of the brain has been established. Th is responsible for dopamine, noradrenaline and adrenaline catecholamines neuro mediators biosynthesis, so a number of pathological changes in an organism has been induced by the structural reorganization different parts of the TH domains under pathological effect of environment. The available conformational states of the human TH type 1 (hTH1) regulatory domain, the activity of which is regulated by the feedback inhibition of the catecholamine products including dopamine has been established by the method of molecular mechanics. It is shown that N-terminal sequence Met30-Ser40 of hTH1 located between the two a-helices (residues 16-29 and residues 41-59) has a number of low-energy conformational states. The most available structures consists of b-turn type II on the pentapeptide fragment of hTH1. This fragment distortion under pathological factors effect, i.e. irradiation may lead to global reorganization in enzyme structure as well as at the enzyme catalytic and regulatory functions

The metabolism of L-phenylalanine and L-tyrosine by liver cells isolated from adrenalectomized rats and from streptozotocin-diabetic rats.

OpenAIRE

Stanley, J C; Fisher, M J; Pogson, C I

1985-01-01

Flux through, and maximal activities of, key enzymes of phenylalanine and tyrosine degradation were measured in liver cells prepared from adrenalectomized rats and from streptozotocin-diabetic rats. Adrenalectomy decreased the phenylalanine hydroxylase flux/activity ratio; this was restored by steroid treatment in vivo. Changes in the phosphorylation state of the hydroxylase may mediate these effects; there was no significant change in the maximal activity of the hydroxylase. Tyrosine metabol...

Expression of tyrosine hydroxylase in CD4+ T cells contributes to alleviation of Th17/Treg imbalance in collagen-induced arthritis.

Science.gov (United States)

Wang, Xiao-Qin; Liu, Yan; Cai, Huan-Huan; Peng, Yu-Ping; Qiu, Yi-Hua

2016-12-01

Tyrosine hydroxylase (TH), a rate-limiting enzyme for the synthesis of catecholamines, is expressed in T lymphocytes. However, the role of T cell-expressed TH in rheumatoid arthritis (RA) is less clear. Herein, we aimed to show the contribution of TH expression by CD4 + T cells to alleviation of helper T (Th)17/regulatory T (Treg) imbalance in collagen-induced arthritis (CIA), a mouse model of RA. CIA was prepared by intradermal injection of collagen type II (CII) at tail base of DBA1/J mice. Expression of TH in the spleen and the ankle joints was measured by real-time polymerase chain reaction and Western blot analysis. Percentages of TH-expressing Th17 and Treg cells in splenic CD4 + T cells were determined by flow cytometry. Overexpression and knockdown of TH gene in CD4 + T cells were taken to evaluate effects of TH on Th17 and Treg cells in CIA. TH expression was upregulated in both the inflamed tissues (spleen and ankle joints) and the CD4 + T cells of CIA mice. In splenic CD4 + T cells, the cells expressing TH were increased during CIA. These cells that expressed more TH in CIA were mainly Th17 cells rather than Treg cells. TH gene overexpression in CD4 + T cells from CIA mice reduced Th17 cell percentage as well as Th17-related transcription factor and cytokine expression and secretion, whereas TH gene knockdown enhanced the Th17 cell activity. In contrast, TH gene overexpression increased Treg-related cytokine expression and secretion in CD4 + T cells of CIA mice, while TH gene knockdown decreased the Treg cell changes. Collectively, these findings show that CIA induces TH expression in CD4 + T cells, particularly in Th17 cells, and suggest that the increased TH expression during CIA represents an anti-inflammatory mechanism.

Determination of Phenylalanine and Tyrosine by High Performance Liquid Chromatography-Tandem Mass Spectrometry.

Science.gov (United States)

Peat, Judy; Garg, Uttam

2016-01-01

Hyperphenylalaninemia/phenylketonuria (PKU) is one of the most common inborn errors of amino acid metabolism affecting about 1:15,000 infants in the United States. PKU is an autosomal recessive disorder that if untreated results in mental retardation. The most common cause of PKU is deficiency of the enzyme phenylalanine hydroxylase that converts phenylalanine to tyrosine. Tyrosine deficiency results in impaired synthesis of catecholamines and thyroxine. Less commonly, it can result from defects in the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor for the enzyme phenylalanine hydroxylase. Increased phenylalanine and decreased tyrosine in blood are used in the diagnosis and follow-up of patients with PKU. LC/MS/MS method is described for the quantification of phenylalanine and tyrosine.

Expression Profiling of Tyrosine Kinase Genes

National Research Council Canada - National Science Library

Weier, Heinz

2000-01-01

... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

A novel therapeutic approach to 6-OHDA-induced Parkinson's disease in rats via supplementation of PTD-conjugated tyrosine hydroxylase.

Science.gov (United States)

Wu, Shao Ping; Fu, Ai Ling; Wang, Yu Xia; Yu, Lei Ping; Jia, Pei Yuan; Li, Qian; Jin, Guo Zhang; Sun, Man Ji

2006-07-21

The present study aimed to evaluate whether the protein transduction domain (PTD)-conjugated human tyrosine hydroxylase (TH) fusion protein was effective on the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model rats. An expression vector pET-PTD-TH harbouring the PTD-TH gene was constructed and transformed to the Escherichia coli BL21 cells for expression. The expressed recombinant PTD-TH with a molecular weight of 61 kD was successfully transduced (1 microM) into the dopaminergic SH-sy5y human neuroblastoma cells in vitro and visualized by immunohistochemical assay. An in vivo experiment in rats showed that the iv administered PTD-TH protein (8 mg/kg) permeated across the blood-brain barrier, penetrated into the striatum and midbrain, and peaked at 5-8 h after the injection. The behavioral effects of PTD-TH on the apomorphine-induced rotations in the PD model rats 8 weeks after the 6-OHDA lesion showed that a single bolus of PTD-TH (8 mg/kg) iv injection caused a decrement of 60% of the contralateral turns on day 1 and 40% on days 5-17. The results imply that iv delivery of PTD-TH is therapeutically effective on the 6-OHDA-induced PD in rats, the PTD-mediated human TH treatment opening a promising therapeutic direction in treatment of PD.

A novel therapeutic approach to 6-OHDA-induced Parkinson's disease in rats via supplementation of PTD-conjugated tyrosine hydroxylase

International Nuclear Information System (INIS)

Wu Shaoping; Fu Ailing; Wang Yuxia; Yu Leiping; Jia Peiyuan; Li Qian; Jin Guozhang; Sun Manji

2006-01-01

The present study aimed to evaluate whether the protein transduction domain (PTD)-conjugated human tyrosine hydroxylase (TH) fusion protein was effective on the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model rats. An expression vector pET-PTD-TH harbouring the PTD-TH gene was constructed and transformed to the Escherichia coli BL21 cells for expression. The expressed recombinant PTD-TH with a molecular weight of 61 kD was successfully transduced (1 μM) into the dopaminergic SH-sy5y human neuroblastoma cells in vitro and visualized by immunohistochemical assay. An in vivo experiment in rats showed that the iv administered PTD-TH protein (8 mg/kg) permeated across the blood-brain barrier, penetrated into the striatum and midbrain, and peaked at 5-8 h after the injection. The behavioral effects of PTD-TH on the apomorphine-induced rotations in the PD model rats 8 weeks after the 6-OHDA lesion showed that a single bolus of PTD-TH (8 mg/kg) iv injection caused a decrement of 60% of the contralateral turns on day 1 and 40% on days 5-17. The results imply that iv delivery of PTD-TH is therapeutically effective on the 6-OHDA-induced PD in rats, the PTD-mediated human TH treatment opening a promising therapeutic direction in treatment of PD

Elevated blood plasma levels of epinephrine, norepinephrine, tyrosine hydroxylase, TGFβ1, and TNFα associated with high-altitude pulmonary edema in Indian population

Directory of Open Access Journals (Sweden)

Pandey P

2016-08-01

Full Text Available Priyanka Pandey,1,2 Zahara Ali,1,2 Ghulam Mohammad,3 MA Qadar Pasha1,2 1Functional Genomics Unit, CSIR-Institute of Genomics and Integrative Biology, Delhi, 2Department of Biotechnology, Savitribai Phule Pune University, Pune, 3Department of Medicine, SNM Hospital, Ladakh, Jammu and Kashmir, India Abstract: Biomarkers are essential to unravel the locked pathophysiology of any disease. This study investigated the role of biomarkers and their interactions with each other and with the clinical parameters to study the physiology of high-altitude pulmonary edema (HAPE in HAPE-patients (HAPE-p against adapted highlanders (HLs and healthy sojourners, HAPE-controls (HAPE-c. For this, seven circulatory biomarkers, namely, epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor beta 1, tumor necrosis factor alpha (TNFα, platelet-derived growth factor beta beta, and C-reactive protein (CRP, were measured in blood plasma of the three study groups. All the subjects were recruited at ~3,500 m, and clinical features such as arterial oxygen saturation (SaO2, body mass index, and mean arterial pressure were measured. Increased levels of epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor-beta 1, and TNFα were observed in HAPE-p against the healthy groups, HAPE-c, and HLs (P<0.0001. CRP levels were decreased in HAPE-p against HAPE-c and HLs (P<0.0001. There was no significant difference or very marginal difference in the levels of these biomarkers in HAPE-c and HLs (P>0.01. Correlation analysis revealed a negative correlation between epinephrine and norepinephrine (P=4.6E-06 in HAPE-p and positive correlation in HAPE-c (P=0.004 and HLs (P=9.78E-07. A positive correlation was observed between TNFα and CRP (P=0.004 in HAPE-p and a negative correlation in HAPE-c (P=4.6E-06. SaO2 correlated negatively with platelet-derived growth factor beta beta (HAPE-p; P=0.05, norepinephrine (P=0.01, and TNFα (P=0.005 and

The conversion of phenylalanine to tyrosine in man. Direct measurement by continuous intravenous tracer infusions of L-[ring-2H5]phenylalanine and L-[1-13C] tyrosine in the postabsorptive state

International Nuclear Information System (INIS)

Clarke, J.T.; Bier, D.M.

1982-01-01

Steady state phenylalanine and tyrosine turnover and the rate of conversion of phenylalanine of tyrosine in vivo were determined in 6 healthy postabsorptive adult volunteers. Continuous infusions of tracer amounts of L-[ring- 2 H5]phenylalanine were determined intravenously for 13-14 hr. After 9-10 hr, a priming dose followed by a continuous infusion of L-[1- 13 C]tyrosine was added and maintained, along with the [ 2 H5]phenylalanine infusion, for 4 hr. Venous plasma samples were obtained before the initiation of each infusion and every 30 min during the course of the combined [ 2 H5]phenylalanine and [ 13 C]tyrosine infusion for determination of isotopic enrichments of [ 2 H5]phenylalanine, [ 13 C]tyrosine, and [ 2 H4]tyrosine by gas chromatograph-mass spectrometric analysis of the N-trifluoroacetyl-, methyl ester derivatives of the amino acids. Calculated from the observed enrichments, free phenylalanine and tyrosine turnover rates were 36.1 +/- 5.1 mumole . kg-1 . h-1 and 39.8 +/- 3.5 mumole . kg-1 . h-1, respectively. Phenylalanine was converted to tyrosine at the rate of 5.83 +/- 0.59 mumole . kg-1 . h-1, accounting for approximately 16% of either the phenylalanine or the tyrosine flux. The results indicate that the normal basal steady state phenylalanine hydroxylase activity in vivo in man is lower than that obtained from phenylalanine loading studies. This supports the existence of some type of substance activation of the enzyme as reflected in the previously reported exponential relationship between phenylalanine concentration and phenylalanine hydroxylase activity in vitro. The use of continuous simultaneous infusions of tracer amounts of stable isotope-labeled phenylalanine and tyrosine provides a direct means for studying physiological regulation of phenylalanine hydroxylase activity in vivo

Tyrosine hydroxylase immunoreactivity and [3H]WIN 35,428 binding to the dopamine transporter in a hamster model of idiopathic paroxysmal dystonia

International Nuclear Information System (INIS)

Nobrega, J.N.; Gernert, M.; Loescher, W.; Raymond, R.; Belej, T.; Richter, A.

1999-01-01

Recent pharmacological studies and receptor analyses have suggested that dopamine neurotransmission is enhanced in mutant dystonic hamsters (dt sz ), a model of idiopathic paroxysmal dystonia which displays attacks of generalized dystonia in response to mild stress. In order to further characterize the nature of dopamine alterations, the present study investigated possible changes in the number of dopaminergic neurons, as defined by tyrosine hydroxylase immunohistochemistry, as well as binding to the dopamine transporter labelled with [ 3 H]WIN 35,428 in dystonic hamsters. No differences in the number of tyrosine hydroxylase-immunoreactive neurons were found within the substantia nigra and ventral tegmental area of mutant hamsters compared to non-dystonic control hamsters. Similarly, under basal conditions, i.e. in the absence of a dystonic episode, no significant changes in [ 3 H]WIN 35,428 binding were detected in dystonic brains. However, in animals killed during the expression of severe dystonia, significant decreases in dopamine transporter binding became evident in the nucleus accumbens and ventral tegmental area in comparison to controls exposed to the same external stimulation. Since stimulation tended to increase [ 3 H]WIN 35,428 binding in control brains, the observed decrease in the ventral tegmental area appeared to be due primarily to the fact that binding was increased less in dystonic brains than in similarly stimulated control animals.This finding could reflect a diminished ability of the dopamine transporter to undergo adaptive changes in response to external stressful stimulation in mutant hamsters. The selective dopamine uptake inhibitor GBR 12909 (20 mg/kg) aggravated dystonia in mutant hamsters, further suggesting that acute alterations in dopamine transporter function during stimulation may be an important component of dystonia in this model. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

Molecular characterization of ferulate 5-hydroxylase gene from kenaf (Hibiscus cannabinus L.)

Science.gov (United States)

The purpose of this research was to clone and characterize the expression pattern of a kenaf (Hibiscus cannabinus L.) F5H gene that encodes ferulate 5-hydroxylase in the phenylpropanoid pathway. Kenaf is well known as a fast growing dicotyledonous plant, which makes it a valuable biomass plant. The ...

A Tyrosine-Hydroxylase Characterization of Dopaminergic Neurons in the Honey Bee Brain

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Stevanus R. Tedjakumala

2017-07-01

Full Text Available Dopamine (DA plays a fundamental role in insect behavior as it acts both as a general modulator of behavior and as a value system in associative learning where it mediates the reinforcing properties of unconditioned stimuli (US. Here we aimed at characterizing the dopaminergic neurons in the central nervous system of the honey bee, an insect that serves as an established model for the study of learning and memory. We used tyrosine hydroxylase (TH immunoreactivity (ir to ensure that the neurons detected synthesize DA endogenously. We found three main dopaminergic clusters, C1–C3, which had been previously described; the C1 cluster is located in a small region adjacent to the esophagus (ES and the antennal lobe (AL; the C2 cluster is situated above the C1 cluster, between the AL and the vertical lobe (VL of the mushroom body (MB; the C3 cluster is located below the calyces (CA of the MB. In addition, we found a novel dopaminergic cluster, C4, located above the dorsomedial border of the lobula, which innervates the visual neuropils of the bee brain. Additional smaller processes and clusters were found and are described. The profuse dopaminergic innervation of the entire bee brain and the specific connectivity of DA neurons, with visual, olfactory and gustatory circuits, provide a foundation for a deeper understanding of how these sensory modules are modulated by DA, and the DA-dependent value-based associations that occur during associative learning.

Species differences in the regulation of tyrosine hydroxylase in Cnemidophorus whiptail lizards.

Science.gov (United States)

Woolley, Sarah C; Crews, David

2004-09-05

Evolution of behavioral phenotype involves changes in the underlying neural substrates. Cnemidophorus whiptail lizards enable the study of behavioral and neural evolution because ancestral species involved in producing unisexual, hybrid species still exist. Catecholaminergic systems modulate the expression of social behaviors in a number of vertebrates, including whiptails, and therefore we investigated how changes in catecholamine production correlated with evolutionary changes in behavioral phenotype by measuring the size and number of catecholamine producing (tyrosine hydroxylase-immunoreactive, or TH-ir) cells across the reproductive cycle in females from two related whiptail species. Cnemidophorusuniparens is a triploid, parthenogenetic species that arose from hybridization events involving the diploid, sexual species C. inornatus. Prior to ovulation, females from both species display femalelike receptive behaviors. However, after ovulation, only parthenogenetic individuals display malelike mounting behavior. In all nuclei measured, we found larger TH-ir cells in the parthenogen, a difference consistent with species differences in ploidy. In contrast, species differences in the number of TH-ir cells were nucleus specific. In the preoptic area and anterior hypothalamus, parthenogens had fewer TH-ir cells than females of the sexual species. Reproductive state only affected TH-ir cell number in the substantia nigra pars compacta (SNpc), and C. uniparens individuals had more TH-ir cells after ovulation than when previtellogenic. Thus, species differences over the reproductive cycle in the SNpc are correlated with species differences in behavior, and it appears that the process of speciation may have produced a novel neural and behavioral phenotype in the parthenogen.

Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex

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Lin Yuheng

2012-04-01

Full Text Available Abstract Background Caffeic acid (3,4-dihydroxycinnamic acid is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. Results We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H, is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. Conclusion We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis

Tyrosine hydroxylase immunoreactivity and [{sup 3}H]WIN 35,428 binding to the dopamine transporter in a hamster model of idiopathic paroxysmal dystonia

Energy Technology Data Exchange (ETDEWEB)

Nobrega, J.N. [Neuroimaging Research Section, Clarke Institute of Psychiatry, Toronto (Canada); Gernert, M.; Loescher, W. [Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Buenteweg 17, D-30559 Hannover (Germany); Raymond, R.; Belej, T. [Neuroimaging Research Section, Clarke Institute of Psychiatry, Toronto (Canada); Richter, A. [Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Buenteweg 17, D-30559 Hannover (Germany)

1999-08-01

Recent pharmacological studies and receptor analyses have suggested that dopamine neurotransmission is enhanced in mutant dystonic hamsters (dt{sup sz}), a model of idiopathic paroxysmal dystonia which displays attacks of generalized dystonia in response to mild stress. In order to further characterize the nature of dopamine alterations, the present study investigated possible changes in the number of dopaminergic neurons, as defined by tyrosine hydroxylase immunohistochemistry, as well as binding to the dopamine transporter labelled with [{sup 3}H]WIN 35,428 in dystonic hamsters. No differences in the number of tyrosine hydroxylase-immunoreactive neurons were found within the substantia nigra and ventral tegmental area of mutant hamsters compared to non-dystonic control hamsters. Similarly, under basal conditions, i.e. in the absence of a dystonic episode, no significant changes in [{sup 3}H]WIN 35,428 binding were detected in dystonic brains. However, in animals killed during the expression of severe dystonia, significant decreases in dopamine transporter binding became evident in the nucleus accumbens and ventral tegmental area in comparison to controls exposed to the same external stimulation. Since stimulation tended to increase [{sup 3}H]WIN 35,428 binding in control brains, the observed decrease in the ventral tegmental area appeared to be due primarily to the fact that binding was increased less in dystonic brains than in similarly stimulated control animals.This finding could reflect a diminished ability of the dopamine transporter to undergo adaptive changes in response to external stressful stimulation in mutant hamsters. The selective dopamine uptake inhibitor GBR 12909 (20 mg/kg) aggravated dystonia in mutant hamsters, further suggesting that acute alterations in dopamine transporter function during stimulation may be an important component of dystonia in this model. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved000.

Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes

International Nuclear Information System (INIS)

Schallreuter, Karin U.; Wazir, Umar; Kothari, Sonal; Gibbons, Nicholas C.J.; Moore, Jeremy; Wood, John M.

2004-01-01

Epidermal phenylalanine hydroxylase (PAH) produces L-tyrosine from the essential amino acid L-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I-VI (Fitzpatrick classification) and also increase up to 24 h after UVB light with only one minimal erythemal dose. Since UVB generates also H 2 O 2 , we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate L-phenylalanine (1 x 10 -3 M), the V max for enzyme activity increased 4-fold by H 2 O 2 (>2.0 x 10 -3 M). Lineweaver-Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp 120 residue to 5-OH-Trp 120 by H 2 O 2 causing a conformational change in the regulatory domain. PAH was still activated by H 2 O 2 in the presence of the electron donor/cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H 2 O 2 by UVB can activate epidermal PAH leading to an increased L-tyrosine pool for melanogenesis

Tumor necrosis factor-alpha-independent downregulation of hepatic cholesterol 7alpha-hydroxylase gene in mice treated with lead nitrate.

Science.gov (United States)

Kojima, Misaki; Sekikawa, Kenji; Nemoto, Kiyomitsu; Degawa, Masakuni

2005-10-01

We previously reported that lead nitrate (LN), an inducer of hepatic tumor necrosis factor-alpha (TNF-alpha), downregulated gene expression of cholesterol 7alpha-hydroxylase. Herein, to clarify the role of TNF-alpha in LN-induced downregulation of cholesterol 7alpha-hydroxylase, effects of LN on gene expression of hepatic cholesterol 7alpha-hydroxylase (Cyp7a1) in TNF-alpha-knockout (KO) and TNF-alpha-wild-type (WT) mice were comparatively examined. Gene expression of hepatic Cyp7a1 in both WT and KO mice decreased to less than 5% of the corresponding controls at 6-12 h after treatment with LN (100 mumol/kg body weight, iv). Levels of hepatic TNF-alpha protein in either WT or KO mice were below the detection limit, although expression levels of the TNF-alpha gene markedly increased at 6 h in WT mice by LN treatment, but not in KO mice. In contrast, in both WT and KO mice, levels of hepatic IL-1beta protein, which is known to be a suppressor of the cholesterol 7alpha-hydroxylase gene in hamsters, were significantly increased 3-6 h after LN treatment. Furthermore, LN-induced downregulation of the Cyp7a1 gene did not necessarily result from altered gene expression of hepatic transcription factors, including positive regulators (liver X receptor alpha, retinoid X receptor alpha, fetoprotein transcription factor, and hepatocyte nuclear factor 4alpha) and a negative regulator small heterodimer partner responsible for expression of the Cyp7a1 gene. The present findings indicated that LN-induced downregulation of the Cyp7a1 gene in mice did not necessarily occur through a TNF-alpha-dependent pathway and might occur mainly through an IL-1beta-dependent pathway.

Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease

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Margarita Chumarina

2017-03-01

Full Text Available Mouse embryonic stem cell (mESC lines were derived by crossing heterozygous transgenic (tg mice expressing green fluorescent protein (GFP under the control of the rat tyrosine hydroxylase (TH promoter, with homozygous alpha-synuclein (aSYN mice expressing human mutant SNCAA53T under the control of the mouse Prion promoter (MoPrP, or wildtype (WT mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons.

Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

Science.gov (United States)

Bezem, Maria T.; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I.

2016-01-01

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

International Nuclear Information System (INIS)

Lichter-Konecki, U.; Konecki, D.S.; DiLella, A.G.; Brayton, K.; Marvit, J.; Hahn, T.M.; Trefz, E.K.; Woo, S.L.C.

1988-01-01

A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAD cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM - phenotype. Together with the other mutations recently reported in the PAH gene,the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level

Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

Energy Technology Data Exchange (ETDEWEB)

Lichter-Konecki, U.; Konecki, D.S.; DiLella, A.G.; Brayton, K.; Marvit, J.; Hahn, T.M.; Trefz, E.K.; Woo, S.L.C.

1988-04-19

A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAD cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM/sup -/ phenotype. Together with the other mutations recently reported in the PAH gene,the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.

Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

National Research Council Canada - National Science Library

Weier, Heinz-Ulrich

2001-01-01

... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

National Research Council Canada - National Science Library

Weier, Heinz-Ulrich

2002-01-01

... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency

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Guerra-Júnior Gil

2010-06-01

Full Text Available Abstract Background Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in CYP21A2 gene. The human gene is located at 6p21.3 within a locus containing the genes for putative serine/threonine Kinase RP, complement C4, steroid 21-hydroxylase CYP21 tenascin TNX, normally, in a duplicated cluster known as RCCX module. The CYP21 extra copy is a pseudogene (CYP21A1P. In Brazil, 30-kb deletion forming monomodular alleles that carry chimeric CYP21A1P/A2 genes corresponds to ~9% of disease-causing alleles. Such alleles are considered to result from unequal crossovers within the bimodular C4/CYP21 locus. Depending on the localization of recombination breakpoint, different alleles can be generated conferring the locus high degree of allelic variability. The purpose of the study was to investigate the variability of deleted alleles in patients with 21-hydroxylase deficiency. Methods We used different techniques to investigate the variability of 30-kb deletion alleles in patients with 21-hydroxylase deficiency. Alleles were first selected after Southern blotting. The composition of CYP21A1P/A2 chimeric genes was investigated by ASO-PCR and MLPA analyses followed by sequencing to refine the location of recombination breakpoints. Twenty patients carrying at least one allele with C4/CYP21 30-kb deletion were included in the study. Results An allele carrying a CYP21A1P/A2 chimeric gene was found unusually associated to a C4B/C4A Taq I 6.4-kb fragment, generally associated to C4B and CYP21A1P deletions. A novel haplotype bearing both p.P34L and p.H62L, novel and rare mutations, respectively, was identified in exon 1, however p.P30L, the most frequent pseudogene-derived mutation in this exon, was absent. Four unrelated patients showed this haplotype. Absence of p.P34L in CYP21A1P of normal controls indicated that it is not derived from pseudogene. In addition, the combination of different

Amino acids in health and disease: New perspectives

Energy Technology Data Exchange (ETDEWEB)

Kaufman, S.

1987-01-01

This book contains 33 selections. Some of the titles are: Regulation of Adrenal Tyrosine Hydroxylase Gene Expression During Cold Stress; The Molecular Genetics of Phenylketonuria; Prospects for Somatic Gene Therapy of Phenylketonuria; Behavioral Effects of Sugar; Effects of Tyrosine and Tryptophan on Blood Pressure in the Rat; and The Enzymology of the Aromatic Amino Acid Hydroxylases.

Abnormal tyrosine and phenylalanine metabolism in patients with tyrosyluria and phenylketonuria; gas-liquid chromatographic analysis of urinary metabolites

NARCIS (Netherlands)

Wadman, S.K.; Heiden, C. van der; Ketting, D.; Sprang, F.J. van

Gas-liquid chromatographic methods have been developed for the analysis of: urinary phenylalanine metabolites (I) in patients with phenylketonuria, tyrosine metabolites (II) in patients with a disturbed tyrosine metabolism at the level of p-hydroxyphenylpyruvate hydroxylase, and homogentisic acid in

Cinnamic acid 4-hydroxylase of sorghum [Sorghum biocolor (L.) Moench] gene SbC4H1 restricts lignin synthesis in Arabidopsis

Science.gov (United States)

Cinnamic acid 4-hydroxylase (C4H) is the first hydroxylase enzyme of the phenylpropanoid pathway, and its content and activity affects the lignin synthesis. In this study, we isolated a C4H gene SbC4H1 from the suppression subtractive hybridization library of brown midrib (bmr) mutants of Sorghum b...

Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene

Science.gov (United States)

Sheng, Yanmin; Wang, Yingdian; Capell, Teresa; Shi, Lianxuan; Ni, Xiuzhen; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

2015-01-01

The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity. PMID:26030746

The mechanism of the tyrosine transporter TyrP supports a proton motive tyrosine decarboxylation pathway in Lactobacillus brevis

NARCIS (Netherlands)

Wolken, WAM; Lucas, PM; Lonvaud-Funel, A; Lolkema, JS; Wolken, Wout A.M.; Lucas, Patrick M.

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L.

Association between A218C polymorphism of the tryptophan-hydroxylase-1 gene, harm avoidance and binge eating behavior in bulimia nervosa.

Science.gov (United States)

Monteleone, Palmiero; Tortorella, Alfonso; Martiadis, Vassilis; Serino, Ismene; Di Filippo, Carmela; Maj, Mario

2007-06-21

Genes involved in serotonin transmission are likely involved in the biological predisposition to bulimia nervosa. We investigated whether the A218C polymorphism of the tryptophan-hydroxylase-1 gene was associated to bulimia nervosa and/or to some phenotypic aspects of the disorder. One hundred eighty Caucasian women (91 patients with bulimia nervosa and 89 healthy controls) were enrolled into the study. They underwent a blood sample collection for A218C polymorphism of the tryptophan-hydroxylase-1 genotyping and a clinical evaluation assessing comorbidity for Axis I and II psychiatric disorders, harm avoidance personality dimension and bulimic symptoms. The distribution of both tryptophan-hydroxylase-1 A218C genotypes and alleles did not significantly differ between patients and controls. Bulimic women with the AA genotype exhibited a more severe binge eating behavior and higher harm avoidance scores than those with CC genotype. These findings support the idea that tryptophan-hydroxylase-1 A218C polymorphism does not play a part in the genetic susceptibility to bulimia nervosa, but it seems to be involved in predisposing bulimic patients to a more disturbed eating behavior and higher harm avoidance.

Identification and characterization of phenol hydroxylase from phenol-degrading Candida tropicalis strain JH8.

Science.gov (United States)

Long, Yan; Yang, Sheng; Xie, Zhixiong; Cheng, Li

2014-09-01

The gene phhY encoding phenol hydroxylase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The gene phhY contained an open reading frame of 2130 bp encoding a polypeptide of 709 amino acid residues. From its sequence analysis, it is a member of a family of flavin-containing aromatic hydroxylases and shares 41% amino acid identity with phenol hydroxylase from Trichosporon cutaneum. The recombinant phenol hydroxylase exists as a homotetramer structure with a native molecular mass of 320 kDa. Recombinant phenol hydroxylase was insensitive to pH treatment; its optimum pH was at 7.6. The optimum temperature for the enzyme was 30 °C, and its activity was rapidly lost at temperatures above 60 °C. Under the optimal conditions with phenol as substrate, the K(m) and V(max) of recombinant phenol hydroxylase were 0.21 mmol·L(-1) and 0.077 μmol·L(-1)·min(-1), respectively. This is the first paper presenting the cloning and expression in E. coli of the phenol hydroxylase gene from C. tropicalis and the characterization of the recombinant phenol hydroxylase.

Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

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Martini, Sabrina V; Silva, Adriana L; Ferreira, Debora; Rabelo, Rafael; Ornellas, Felipe M; Gomes, Karina; Rocco, Patricia R M; Petrs-Silva, Hilda; Morales, Marcelo M

2016-01-01

Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy. © 2016 The Author(s) Published by S. Karger AG, Basel.

Involvement of tryptophan hydroxylase 2 gene polymorphisms in susceptibility to tic disorder in Chinese Han population

OpenAIRE

Zheng, Ping; Li, Erzhen; Wang, Jianhua; Cui, Xiaodai; Wang, Liwen

2013-01-01

Abstract Background Tryptophan hydroxylase-2 (TPH2) is a potential candidate gene for screening tic disorder (TD). Methods A case–control study was performed to examine the association between the TPH2 gene and TD. The Sequenom® Mass ARRAY iPLEX GOLD System was used to genotype two single nucleotide polymorphisms (SNPs) of the TPH2 gene in 149 TD children and in 125 normal controls. Results For rs4565946, individuals with the TT genotype showed a significantly higher risk of TD than those wit...

Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia

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Boer, Judith M.; Steeghs, Elisabeth M.P.; Marchante, João R.M.; Boeree, Aurélie; Beaudoin, James J.; Berna Beverloo, H.; Kuiper, Roland P.; Escherich, Gabriele; van der Velden, Vincent H.J.; van der Schoot, C. Ellen; de Groot-Kruseman, Hester A.; Pieters, Rob; den Boer, Monique L.

2017-01-01

Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome. PMID:27894077

Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells

Energy Technology Data Exchange (ETDEWEB)

Ledley, F.D.; Grenett, H.E.; McGinnis-Shelnutt, M.; Woo, S.L.C.

1986-01-01

Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into Psi2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected Psi2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylaline hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normal synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.

Tyrosine kinase chromosomal translocations mediate distinct and overlapping gene regulation events

International Nuclear Information System (INIS)

Kim, Hani; Gillis, Lisa C; Jarvis, Jordan D; Yang, Stuart; Huang, Kai; Der, Sandy; Barber, Dwayne L

2011-01-01

Leukemia is a heterogeneous disease commonly associated with recurrent chromosomal translocations that involve tyrosine kinases including BCR-ABL, TEL-PDGFRB and TEL-JAK2. Most studies on the activated tyrosine kinases have focused on proximal signaling events, but little is known about gene transcription regulated by these fusions. Oligonucleotide microarray was performed to compare mRNA changes attributable to BCR-ABL, TEL-PDGFRB and TEL-JAK2 after 1 week of activation of each fusion in Ba/F3 cell lines. Imatinib was used to control the activation of BCR-ABL and TEL-PDGFRB, and TEL-JAK2-mediated gene expression was examined 1 week after Ba/F3-TEL-JAK2 cells were switched to factor-independent conditions. Microarray analysis revealed between 800 to 2000 genes induced or suppressed by two-fold or greater by each tyrosine kinase, with a subset of these genes commonly induced or suppressed among the three fusions. Validation by Quantitative PCR confirmed that eight genes (Dok2, Mrvi1, Isg20, Id1, gp49b, Cxcl10, Scinderin, and collagen Vα1(Col5a1)) displayed an overlapping regulation among the three tested fusion proteins. Stat1 and Gbp1 were induced uniquely by TEL-PDGFRB. Our results suggest that BCR-ABL, TEL-PDGFRB and TEL-JAK2 regulate distinct and overlapping gene transcription profiles. Many of the genes identified are known to be involved in processes associated with leukemogenesis, including cell migration, proliferation and differentiation. This study offers the basis for further work that could lead to an understanding of the specificity of diseases caused by these three chromosomal translocations

A Novel Mutation in the CYP11B1 Gene Causes Steroid 11β-Hydroxylase Deficient Congenital Adrenal Hyperplasia with Reversible Cardiomyopathy

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Mohammad A. Alqahtani

2015-01-01

Full Text Available Congenital adrenal hyperplasia (CAH due to steroid 11β-hydroxylase deficiency is the second most common form of CAH, resulting from a mutation in the CYP11B1 gene. Steroid 11β-hydroxylase deficiency results in excessive mineralcorticoids and androgen production leading to hypertension, precocious puberty with acne, enlarged penis, and hyperpigmentation of scrotum of genetically male infants. In the present study, we reported 3 male cases from a Saudi family who presented with penile enlargement, progressive darkness of skin, hypertension, and cardiomyopathy. The elder patient died due to heart failure and his younger brothers were treated with hydrocortisone and antihypertensive medications. Six months following treatment, cardiomyopathy disappeared with normal blood pressure and improvement in the skin pigmentation. The underlying molecular defect was investigated by PCR-sequencing analysis of all coding exons and intron-exon boundary of the CYP11B1 gene. A novel biallelic mutation c.780 G>A in exon 4 of the CYP11B1 gene was found in the patients. The mutation created a premature stop codon at amino acid 260 (p.W260∗, resulting in a truncated protein devoid of 11β-hydroxylase activity. Interestingly, a somatic mutation at the same codon (c.779 G>A, p.W260∗ was reported in a patient with papillary thyroid cancer (COSMIC database. In conclusion, we have identified a novel nonsense mutation in the CYP11B1 gene that causes classic steroid 11β-hydroxylase deficient CAH. Cardiomyopathy and cardiac failure can be reversed by early diagnosis and treatment.

Renal denervation decreases blood pressure and renal tyrosine hydroxylase but does not augment the effect of hypotensive drugs.

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Skrzypecki, Janusz; Gawlak, Maciej; Huc, Tomasz; Szulczyk, Paweł; Ufnal, Marcin

2017-01-01

The effect of renal denervation on the efficacy of antihypertensive drugs has not yet been elucidated. Twenty-week-old spontaneously hypertensive rats were treated with metoprolol, losartan, indapamide, or saline (controls) and assigned to renal denervation or a sham procedure. Acute hemodynamic measurements were performed ten days later. Series showing a significant interaction between renal denervation and the drugs were repeated with chronic telemetry measurements. In the saline series, denervated rats showed a significantly lower mean arterial blood pressure (blood pressure) than the sham-operated rats. In contrast, in the metoprolol series denervated rats showed a significantly higher blood pressure than sham rats. There were no differences in blood pressure between denervated and sham rats in the losartan and indapamide series. In chronic studies, a 4-week treatment with metoprolol caused a decrease in blood pressure. Renal denervation and sham denervation performed 10 days after the onset of metoprolol treatment did not affect blood pressure. Denervated rats showed markedly reduced renal nerve tyrosine hydroxylase levels. In conclusion, renal denervation decreases blood pressure in hypertensive rats. The hypotensive action of metoprolol, indapamide, and losartan is not augmented by renal denervation, suggesting the absence of synergy between renal denervation and the drugs investigated in this study.

Cytoplasm-predominant Pten associates with increased region-specific brain tyrosine hydroxylase and dopamine D2 receptors in mouse model with autistic traits.

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He, Xin; Thacker, Stetson; Romigh, Todd; Yu, Qi; Frazier, Thomas W; Eng, Charis

2015-01-01

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by impairment in social communication/interaction and inflexible/repetitive behavior. Several lines of evidence support genetic factors as a predominant cause of ASD. Among those autism susceptibility genes that have been identified, the PTEN tumor suppressor gene, initially identified as predisposing to Cowden heritable cancer syndrome, was found to be mutated in a subset of ASD patients with extreme macrocephaly. However, the ASD-relevant molecular mechanism mediating the effect of PTEN mutations remains elusive. We developed a Pten knock-in murine model to study the effects of Pten germline mutations, specifically altering subcellular localization, in ASD. Proteins were isolated from the hemispheres of the male littermates, and Western blots were performed to determine protein expression levels of tyrosine hydroxylase (TH). Immunohistochemical stains were carried out to validate the localization of TH and dopamine D2 receptors (D2R). PC12 cells ectopically expressing either wild-type or missense mutant PTEN were then compared for the differences in TH expression. Mice carrying Pten mutations have high TH and D2R in the striatum and prefrontal cortex. They also have increased phosphorylation of cAMP response element-binding protein (CREB) and TH. Mechanistically, PTEN downregulates TH production in PC12 cells via inhibiting the phosphoinositide 3-kinase (PI3K)/CREB signaling pathway, while PTEN reduces TH phosphorylation via suppressing MAPK pathway. Unlike wild-type PTEN but similar to the mouse knock-in mutant Pten, three naturally occurring missense mutations of PTEN that we previously identified in ASD patients, H93R, F241S, and D252G, were not able to suppress TH when overexpressed in PC12 cells. In addition, two other PTEN missense mutations, C124S (pan phosphatase dead) and G129E (lipid phosphatase dead), failed to suppress TH when ectopically expressed in PC12 cells

Alkane Hydroxylase Gene (alkB Phylotype Composition and Diversity in Northern Gulf of Mexico Bacterioplankton

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Conor Blake Smith

2013-12-01

Full Text Available Natural and anthropogenic activities introduce alkanes into marine systems where they are degraded by alkane hydroxylases expressed by phylogenetically diverse bacteria. Partial sequences for alkB, one of the structural genes of alkane hydroxylase, have been used to assess the composition of alkane-degrading communities, and to determine their responses to hydrocarbon inputs. We present here the first spatially extensive analysis of alkB in bacterioplankton of the northern Gulf of Mexico (nGoM, a region that experiences numerous hydrocarbon inputs. We have analyzed 401 partial alkB gene sequences amplified from genomic extracts collected during March 2010 from 17 water column samples that included surface waters and bathypelagic depths. Previous analyses of 16S rRNA gene sequences for these and related samples have shown that nGoM bacterial community composition and structure stratify strongly with depth, with distinctly different communities above and below 100 m. Although we hypothesized that alkB gene sequences would exhibit a similar pattern, PCA analyses of operational protein units (OPU indicated that community composition did not vary consistently with depth or other major physical-chemical variables. We observed 22 distinct OPUs, one of which was ubiquitous and accounted for 57% of all sequences. This OPU clustered with alkB sequences from known hydrocarbon oxidizers (e.g., Alcanivorax and Marinobacter. Some OPUs could not be associated with known alkane degraders, however, and perhaps represent novel hydrocarbon-oxidizing populations or genes. These results indicate that the capacity for alkane hydrolysis occurs widely in the nGoM, but that alkane degrader diversity varies substantially among sites and responds differently than bulk communities to physical-chemical variables.

Effect of long-term actual spaceflight on the expression of key genes encoding serotonin and dopamine system

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Popova, Nina; Shenkman, Boris; Naumenko, Vladimir; Kulikov, Alexander; Kondaurova, Elena; Tsybko, Anton; Kulikova, Elisabeth; Krasnov, I. B.; Bazhenova, Ekaterina; Sinyakova, Nadezhda

The effect of long-term spaceflight on the central nervous system represents important but yet undeveloped problem. The aim of our work was to study the effect of 30-days spaceflight of mice on Russian biosatellite BION-M1 on the expression in the brain regions of key genes of a) serotonin (5-HT) system (main enzymes in 5-HT metabolism - tryptophan hydroxylase-2 (TPH-2), monoamine oxydase A (MAO A), 5-HT1A, 5-HT2A and 5-HT3 receptors); b) pivotal enzymes in DA metabolism (tyrosine hydroxylase, COMT, MAO A, MAO B) and D1, D2 receptors. Decreased expression of genes encoding the 5-HT catabolism (MAO A) and 5-HT2A receptor in some brain regions was shown. There were no differences between “spaceflight” and control mice in the expression of TPH-2 and 5-HT1A, 5-HT3 receptor genes. Significant changes were found in genetic control of DA system. Long-term spaceflight decreased the expression of genes encoding the enzyme in DA synthesis (tyrosine hydroxylase in s.nigra), DA metabolism (MAO B in the midbrain and COMT in the striatum), and D1 receptor in hypothalamus. These data suggested that 1) microgravity affected genetic control of 5-HT and especially the nigrostriatal DA system implicated in the central regulation of muscular tonus and movement, 2) the decrease in the expression of genes encoding key enzyme in DA synthesis, DA degradation and D1 receptor contributes to the movement impairment and dyskinesia produced by the spaceflight. The study was supported by Russian Foundation for Basic Research grant No. 14-04-00173.

Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum-contaminated soils

OpenAIRE

Tsuboi, Shun; Yamamura, Shigeki; Nakajima-Kambe, Toshiaki; Iwasaki, Kazuhiro

2015-01-01

The study investigated the diversity and genotypic features of alkane hydroxylase genes on rhizoplanes of grasses planted in artificial petroleum-contaminated soils to acquire new insights into the bacterial communities responsible for petroleum degradation in phytoremediation. Four types of grass (Cynodon dactylon, two phenotypes of Zoysia japonica, and Z. matrella) were used. The concentrations of total petroleum hydrocarbon effectively decreased in the grass-planted systems compared with t...

Sphingolipid base modifying enzymes in sunflower (Helianthus annuus): cloning and characterization of a C4-hydroxylase gene and a new paralogous Δ8-desaturase gene.

Science.gov (United States)

Moreno-Pérez, Antonio J; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

2011-05-15

Sphingolipids are components of plant cell membranes that participate in the regulation of important physiological processes. Unlike their animal counterparts, plant sphingolipids are characterized by high levels of base C4-hydroxylation. Moreover, desaturation at the Δ8 position predominates over the Δ4 desaturation typically found in animal sphingolipids. These modifications are due to the action of C4-hydroxylases and Δ8-long chain base desaturases, and they are important for complex sphingolipids finally becoming functional. The long chain bases of sunflower sphingolipids have high levels of hydroxylated and unsaturated moieties. Here, a C4-long chain base hydroxylase was functionally characterized in sunflower plant, an enzyme that could complement the sur2Δ mutation when heterologously expressed in this yeast mutant deficient in hydroxylation. This hydroxylase was ubiquitously expressed in sunflower, with the highest levels found in the developing cotyledons. In addition, we identified a new Δ8-long base chain desaturase gene that displays strong homology to a previously reported desaturase gene. This desaturase was also expressed in yeast and was able to change the long chain base composition of the transformed host. We studied the expression of this desaturase and compared it with that of the other isoform described in sunflower. The desaturase form studied in this paper displayed higher expression levels in developing seeds. Copyright © 2010 Elsevier GmbH. All rights reserved.

Differential regulation of catecholamine synthesis and transport in rat adrenal medulla by fluoxetine treatment.

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Spasojevic, Natasa; Jovanovic, Predrag; Dronjak, Sladjana

2015-03-01

We have recently shown that chronic fluoxetine treatment acted significantly increasing plasma norepinephrine and epinephrine concentrations both in control and chronically stressed adult male rats. However, possible effects of fluoxetine on catecholamine synthesis and re-uptake in adrenal medulla have been largely unknown. In the present study the effects of chronic fluoxetine treatment on tyrosine hydroxylase, a rate-limiting enzyme in catecholamine synthesis, as well as a norepinephrine transporter and vesicular monoamine transporter 2 gene expressions in adrenal medulla of animals exposed to chronic unpredictable mild stress (CUMS) for 4 weeks, were investigated. Gene expression analyses were performed using a real-time quantitative reverse transcription-PCR. Chronically stressed animals had increased tyrosine hydroxylase mRNA levels and decreased expression of both transporters. Fluoxetine increased tyrosine hydroxylase and decreased norepinephrine transporter gene expression in both unstressed and CUMS rats. These findings suggest that chronic fluoxetine treatment increased plasma catecholamine levels by affecting opposing changes in catecholamine synthesis and uptake.

Differential regulation of catecholamine synthesis and transport in rat adrenal medulla by fluoxetine treatment

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NATASA SPASOJEVIC

2015-03-01

Full Text Available We have recently shown that chronic fluoxetine treatment acted significantly increasing plasma norepinephrine and epinephrine concentrations both in control and chronically stressed adult male rats. However, possible effects of fluoxetine on catecholamine synthesis and re-uptake in adrenal medulla have been largely unknown. In the present study the effects of chronic fluoxetine treatment on tyrosine hydroxylase, a rate-limiting enzyme in catecholamine synthesis, as well as a norepinephrine transporter and vesicular monoamine transporter 2 gene expressions in adrenal medulla of animals exposed to chronic unpredictable mild stress (CUMS for 4 weeks, were investigated. Gene expression analyses were performed using a real-time quantitative reverse transcription-PCR. Chronically stressed animals had increased tyrosine hydroxylase mRNA levels and decreased expression of both transporters. Fluoxetine increased tyrosine hydroxylase and decreased norepinephrine transporter gene expression in both unstressed and CUMS rats. These findings suggest that chronic fluoxetine treatment increased plasma catecholamine levels by affecting opposing changes in catecholamine synthesis and uptake.

Childhood asthma and spirometric indices are associated with polymorphic markers of two vitamin D 25-hydroxylase genes.

Science.gov (United States)

Leung, Ting Fan; Wang, Susan Shuxin; Tang, Man Fung; Kong, Alice Pik-Shan; Sy, Hing Yee; Hon, Kam Lun; Chan, Juliana Chung-ngor; Wong, Gary Wing-kin

2015-06-01

Polymorphic markers of vitamin D pathway genes have been associated with asthma traits in different White populations. This study investigated the relationship between asthma phenotypes and single nucleotide polymorphisms (SNPs) of vitamin D receptor (VDR), vitamin D binding protein (GC), two 25-hydroxylases (CYP2R1 and CYP27A1), and 1α-hydroxylase (CYP27B1) in Hong Kong Chinese children. 23 SNPs of the five vitamin D pathway genes were successfully genotyped in 914 asthmatic children and 1231 non-allergic controls. Genotypic and haplotypic associations with asthma phenotypes (diagnosis, spirometric indices, total IgE, and eosinophil percentage) were analyzed by multivariate regression. Generalized multifactor dimensionality reduction was used to detect epistatic interactions between SNPs for asthma phenotypes. Several SNPs of CYP27A1, CYP27B1, GC, and CYP2R1 were associated with asthma or spirometric indices, although only the association between FEV1 and CYP2R1 rs7935792 passed Bonferroni correction (p = 2.73 × 10(-4) ). Patients with CC genotype of rs7935792 had higher FEV1 than those with the other two genotypes. Asthma was also associated with TT haplotype of CYP27A1 and AGGATA haplotype of CYP2R1 (p = 0.021 and 0.024, respectively). Besides, strong association was found between FEV1 and GATAG of CYP2R1 (β = 13.37, p = 4.83 × 10(-4) ). GMDR failed to identify any 2-locus to 4-locus interaction that modulated asthma or spirometric indices. Several SNPs and haplotypes of CYP2R1 are associated with asthma diagnosis and FEV1 in children. Asthma is also modestly associated with a CYP27A1 haplotype. These two 25-hydroxylase genes may be genetic determinants for asthma phenotypes in children. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Functional analysis of Antirrhinum kelloggii flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes; critical role in flower color and evolution in the genus Antirrhinum.

Science.gov (United States)

Ishiguro, Kanako; Taniguchi, Masumi; Tanaka, Yoshikazu

2012-05-01

The enzymes flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) play an important role in flower color by determining the B-ring hydroxylation pattern of anthocyanins, the major floral pigments. F3'5'H is necessary for biosynthesis of the delphinidin-based anthocyanins that confer a violet or blue color to most plants. Antirrhinum majus does not produce delphinidin and lacks violet flower colour while A. kelloggii produces violet flowers containing delphinidin. To understand the cause of this inter-specific difference in the Antirrhinum genus, we isolated one F3'H and two F3'5'H homologues from the A. kelloggii petal cDNA library. Their amino acid sequences showed high identities to F3'Hs and F3'5'Hs of closely related species. Transgenic petunia expressing these genes had elevated amounts of cyanidin and delphinidin respectively, and flower color changes in the transgenics reflected the type of accumulated anthocyanidins. The results indicate that the homologs encode F3'H and F3'5'H, respectively, and that the ancestor of A. majus lost F3'5'H activity after its speciation from the ancestor of A. kelloggii.

Regulation of gene expression by dietary Ca2+ in kidneys of 25-hydroxyvitamin D3-1 alpha-hydroxylase knockout mice.

NARCIS (Netherlands)

Hoenderop, J.G.J.; Chon, H.; Gkika, D.; Bluyssen, H.A.; Holstege, F.C.; St. Arnaud, R.; Braam, B.; Bindels, R.J.M.

2004-01-01

BACKGROUND: Pseudovitamin D deficiency rickets (PDDR) is an autosomal disease, characterized by undetectable levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), rickets and secondary hyperparathyroidism. Mice in which the 25-hydroxyvitamin D3-1 alpha-hydroxylase (1 alpha-OHase) gene was inactivated,

The Effects of Insulin-Induced Hypoglycaemia on Tyrosine Hydroxylase Phosphorylation in Rat Brain and Adrenal Gland.

Science.gov (United States)

Senthilkumaran, Manjula; Johnson, Michaela E; Bobrovskaya, Larisa

2016-07-01

In this study we investigated the effects of insulin-induced hypoglycaemia on tyrosine hydroxylase (TH) protein and TH phosphorylation in the adrenal gland, C1 cell group, locus coeruleus (LC) and midbrain dopaminergic cell groups that are thought to play a role in response to hypoglycaemia and compared the effects of different concentrations of insulin in rats. Insulin (1 and 10 U/kg) treatment caused similar reductions in blood glucose concentration (from 7.5-9 to 2-3 mmol/L); however, plasma adrenaline concentration was increased 20-30 fold in response to 10 U/kg insulin and only 14 fold following 1 U/kg. Time course studies (at 10 U/kg insulin) revealed that in the adrenal gland, Ser31 phosphorylation was increased between 30 and 90 min (4-5 fold), implying that TH was activated to increase catecholamine synthesis in adrenal medulla to replenish the stores. In the brain, Ser19 phosphorylation was limited to certain dopaminergic groups in the midbrain, while Ser31 phosphorylation was increased in most catecholaminergic regions at 60 min (1.3-2 fold), suggesting that Ser31 phosphorylation may be an important mechanism to maintain catecholamine synthesis in the brain. Comparing the effects of 1 and 10 U/kg insulin revealed that Ser31 phosphorylation was increased to similar extent in the adrenal gland and C1 cell group in response to both doses whereas Ser31 and Ser19 phosphorylation were only increased in response to 1 U/kg insulin in LC and in response to 10 U/kg insulin in most midbrain regions. Thus, the adrenal gland and some catecholaminergic brain regions become activated in response to insulin administration and brain catecholamines may be important for initiation of physiological defences against insulin-induced hypoglycaemia.

Human albumin prevents 6-hydroxydopamine-induced loss of tyrosine hydroxylase in in vitro and in vivo.

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Li-Juan Zhang

Full Text Available Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH expression of dopaminergic (DA neurons induced by 6-hydroxydopamine (6-OHDA toxicity that is most commonly used to create a rat model of Parkinson's disease (PD. In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.

Human Albumin Prevents 6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and In Vivo

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Zhang, Li-Juan; Xue, Yue-Qiang; Yang, Chun; Yang, Wei-Hua; Chen, Long; Zhang, Qian-Jin; Qu, Ting-Yu; Huang, Shile; Zhao, Li-Ru; Wang, Xiao-Min; Duan, Wei-Ming

2012-01-01

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis. PMID:22815976

Vitamin D regulates tyrosine hydroxylase expression: N-cadherin a possible mediator.

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Cui, X; Pertile, R; Liu, P; Eyles, D W

2015-09-24

Vitamin D is a neuroactive steroid. Its genomic actions are mediated via the active form of vitamin D, 1,25(OH)2D3, binding to the vitamin D receptor (VDR). The VDR emerges in the rat mesencephalon at embryonic day 12, representing the peak period of dopaminergic cell birth. Our prior studies reveal that developmental vitamin D (DVD)-deficiency alters the ontogeny of dopaminergic neurons in the developing mesencephalon. There is also consistent evidence from others that 1,25(OH)2D3 promotes the survival of dopaminergic neurons in models of dopaminergic toxicity. In both developmental and toxicological studies it has been proposed that 1,25(OH)2D3 may modulate the differentiation and maturation of dopaminergic neurons; however, to date there is lack of direct evidence. The aim of the current study is to investigate this both in vitro using a human SH-SY5Y cell line transfected with rodent VDR and in vivo using a DVD-deficient model. Here we show that in VDR-expressing SH-SY5Y cells, 1,25(OH)2D3 significantly increased production of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. This effect was dose- and time-dependent, but was not due to an increase in TH-positive cell number, nor was it due to the production of trophic survival factors for dopamine neurons such as glial-derived neurotrophic factor (GDNF). In accordance with 1,25(OH)2D3's anti-proliferative actions in the brain, 1,25(OH)2D3 reduced the percentage of dividing cells from approximately 15-10%. Given the recently reported role of N-cadherin in the direct differentiation of dopaminergic neurons, we examined here whether it may be elevated by 1,25(OH)2D3. We confirmed this in vitro and more importantly, we showed DVD-deficiency decreases N-cadherin expression in the embryonic mesencephalon. In summary, in our in vitro model we have shown 1,25(OH)2D3 increases TH expression, decreases proliferation and elevates N-cadherin, a potential factor that mediates these processes

Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

International Nuclear Information System (INIS)

Zhang Danhui; Kanthasamy, Arthi; Anantharam, Vellareddy; Kanthasamy, Anumantha

2011-01-01

Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKCδ) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKCδ negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here, we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKCδ-PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3-10 μM) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKCδ specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1-1 μM Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKCδ kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKCδ inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKCδ and PP2A activity.

Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

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Shuhei Noda

Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

Lipoprotein cholesterol uptake mediates upregulation of bile acid synthesis by increasing cholesterol 7a-hydroxylase but not sterol 27- hydroxylase gene expression in cultured rat hepatocytes.

NARCIS (Netherlands)

Post, S.M.; Twisk, J.W.R.; van der Fits, L.T.E.; Wit, E.C.M.; Hoekman, M.F.M.; Mager, W.H.; Princen, H.M.G.

1999-01-01

Lipoproteins may supply substrate for the formation of bile acids, and the amount of hepatic cholesterol can regulate bile-acid synthesis and increase cholesterol 7α-hydroxylase expression. However, the effect of lipoprotein cholesterol on sterol 27-hydroxylase expression and the role of different

Chronic desipramine treatment alters tyrosine hydroxylase but not norepinephrine transporter immunoreactivity in norepinephrine axons in the rat prefrontal cortex

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Erickson, Susan L.; Gandhi, Anjalika R.; Asafu-Adjei, Josephine K.; Sampson, Allan R.; Miner, LeeAnn; Blakely, Randy D.; Sesack, Susan R.

2011-01-01

Pharmacological blockade of norepinephrine (NE) reuptake is clinically effective in treating several mental disorders. Drugs that bind to the NE transporter (NET) alter both protein levels and activity of NET and also the catecholamine synthetic enzyme tyrosine hydroxylase (TH). We examined the rat prefrontal cortex (PFC) by electron microscopy to determine whether the density and subcellular distribution of immunolabeling for NET and colocalization of NET with TH within individual NE axons were altered by chronic treatment with the selective NE uptake inhibitor desipramine (DMI). Following DMI treatment (21 days, 15 mg/kg/day), NET-immunoreactive (-ir) axons were significantly less likely to colocalize TH. This finding is consistent with reports of reduced TH levels and activity in the locus coeruleus after chronic DMI and indicates a reduction of NE synthetic capacity in the PFC. Measures of NET expression and membrane localization, including the number of NET-ir profiles per tissue area sampled, the number of gold particles per NET-ir profile area, and the proportion of gold particles associated with the plasma membrane, were similar in DMI and vehicle treated rats. These findings were verified using two different antibodies directed against distinct epitopes of the NET protein. The results suggest that chronic DMI treatment does not reduce NET expression within individual NE axons in vivo or induce an overall translocation of NET protein away from the plasma membrane in the PFC as measured by ultrastructural immunogold labeling. Our findings encourage consideration of possible postranslational mechanisms for regulating NET activity in antidepressant-induced modulation of NE clearance. PMID:21208501

Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

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Viggor, Signe; Jõesaar, Merike; Vedler, Eve; Kiiker, Riinu; Pärnpuu, Liis; Heinaru, Ain

2015-12-30

Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sequence variation at the phenylalanine hydroxylase gene in the British Isles

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Tyfield, L.A. [Southmead Hospital, Bristol (United Kingdom)]|[Univ. of Bristol (United Kingdom); Stephenson, A. [Southmead Hospital, Bristol (United Kingdom); Cockburn, F. [Royal Hospital for Sick Children, Glasgow (United Kingdom)] [and others

1997-02-01

Using mutation and haplotype analysis, we have examined the phenylalanine hydroxylase gene in the phenylketonuria populations of four geographical areas of the British Isles: the west of Scotland, southern Wales, and southwestern and southeastern England. The enormous genetic diversity of this locus within the British Isles is demonstrated in the large number of different mutations characterized and in the variety of genetic backgrounds on which individual mutations are found. Allele frequencies of the more common mutations exhibited significant nonrandom distribution in a north/south differentiation. Differences between the west of Scotland and southwestern England may be related to different events in the recent and past histories of their respective populations. Similarities between southern Wales and southeastern England are likely to reflect the heterogeneity that is seen in and around two large capital cities. Finally, comparison with more recently colonized areas of the world corroborates the genealogical origin by range expansion of several mutations. 38 refs., 2 tabs.

Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11β-hydroxylase deficiency.

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Khattab, Ahmed; Haider, Shozeb; Kumar, Ameet; Dhawan, Samarth; Alam, Dauood; Romero, Raquel; Burns, James; Li, Di; Estatico, Jessica; Rahi, Simran; Fatima, Saleel; Alzahrani, Ali; Hafez, Mona; Musa, Noha; Razzghy Azar, Maryam; Khaloul, Najoua; Gribaa, Moez; Saad, Ali; Charfeddine, Ilhem Ben; Bilharinho de Mendonça, Berenice; Belgorosky, Alicia; Dumic, Katja; Dumic, Miroslav; Aisenberg, Javier; Kandemir, Nurgun; Alikasifoglu, Ayfer; Ozon, Alev; Gonc, Nazli; Cheng, Tina; Kuhnle-Krahl, Ursula; Cappa, Marco; Holterhus, Paul-Martin; Nour, Munier A; Pacaud, Daniele; Holtzman, Assaf; Li, Sun; Zaidi, Mone; Yuen, Tony; New, Maria I

2017-03-07

Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1 , a gene encoding 11β-hydroxylase, represents a rare autosomal recessive Mendelian disorder of aberrant sex steroid production. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where consanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not frequently measured, is the most robust biochemical marker for diagnosing 11β-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11β-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, genetic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11β-hydroxylase deficiency CAH.

Production of tyrosine through phenylalanine hydroxylation bypasses the intrinsic feedback inhibition in Escherichia coli.

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Huang, Jin; Lin, Yuheng; Yuan, Qipeng; Yan, Yajun

2015-04-01

Tyrosine is a proteinogenic aromatic amino acid that is often used as a supplement of food and animal feed, as well as a (bio-)synthetic precursor to various pharmaceutically or industrially important molecules. Extensive metabolic engineering efforts have been made towards the efficient and cost-effective microbial production of tyrosine. Conventional strategies usually focus on eliminating intrinsic feedback inhibition and redirecting carbon flux into the shikimate pathway. In this study, we found that continuous conversion of phenylalanine into tyrosine by the action of tetrahydromonapterin (MH4)-utilizing phenylalanine 4-hydroxylase (P4H) can bypass the feedback inhibition in Escherichia coli, leading to tyrosine accumulation in the cultures. First, expression of the P4H from Xanthomonas campestris in combination with an MH4 recycling system in wild-type E. coli allowed the strain to accumulate tyrosine at 262 mg/L. On this basis, enhanced expression of the key enzymes associated with the shikimate pathway and the MH4 biosynthetic pathway resulted in the elevation of tyrosine production up to 401 mg/L in shake flasks. This work demonstrated a novel approach to tyrosine production and verified the possibility to alleviate feedback inhibition by creating a phenylalanine sink.

  A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity

DEFF Research Database (Denmark)

Huebener, Nicole; Fest, Stefan; Strandsby, Anne Bystrup

2008-01-01

Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we show for the first time effective therapeutic vaccination followed by suppression of established spontaneous neuroblastoma metastases using a tyrosine...... show effective therapeutic vaccination against neuroblastoma with a novel rationally designed TH minigene vaccine without induction of autoimmunity providing an important baseline for future clinical application of this strategy....

A novel homozygous mutation IVS6+5G>T in CYP11B1 gene in a Vietnamese patient with 11β-hydroxylase deficiency.

Science.gov (United States)

Nguyen, Thi Phuong Mai; Nguyen, Thu Hien; Ngo, Diem Ngoc; Vu, Chi Dung; Nguyen, Thi Kim Lien; Nong, Van Hai; Nguyen, Huy Hoang

2015-07-10

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is characterized by a deficiency of one of the enzymes involved in the synthesis of cortisol from cholesterol by the adrenal cortex. CAH cases arising from impaired 11β-hydroxylase are the second most common form. Mutations in the CYP11B1 gene are the cause of 11β-hydroxylase deficiency. This study was performed on a patient with congenital adrenal hyperplasia and with premature development such as enlarged penis, muscle development, high blood pressure, and bone age equivalent of 5 years old at 2 years of chronological age. Biochemical tests for steroids confirmed the diagnosis of CAH. We used PCR and sequencing to screen for mutations in CYP11B1 gene. Results showed that the patient has a novel homozygous mutation of guanine (G) to thymine (T) in intron 6 (IVS6+5G>T). The analysis of this mutation by MaxEntScan boundary software indicated that this mutant could affect the gene splicing during transcription. Copyright © 2015 Elsevier B.V. All rights reserved.

Serotonin and Early Cognitive Development: Variation in the Tryptophan Hydroxylase 2 Gene Is Associated with Visual Attention in 7-Month-Old Infants

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Leppanen, Jukka M.; Peltola, Mikko J.; Puura, Kaija; Mantymaa, Mirjami; Mononen, Nina; Lehtimaki, Terho

2011-01-01

Background: Allelic variation in the promoter region of a gene that encodes tryptophan hydroxylase isoform 2 (TPH2), a rate-limiting enzyme of serotonin synthesis in the central nervous system, has been associated with variations in cognitive function and vulnerability to affective spectrum disorders. Little is known about the effects of this gene…

Tyrosine Hydroxylase (TH)- and Aromatic-L-Amino Acid Decarboxylase (AADC)-Immunoreactive Neurons of the Common Marmoset (Callithrix jacchus) Brain: An Immunohistochemical Analysis

Science.gov (United States)

Karasawa, Nobuyuki; Hayashi, Motoharu; Yamada, Keiki; Nagatsu, Ikuko; Iwasa, Mineo; Takeuchi, Terumi; Uematsu, Mitsutoshi; Watanabe, Kazuko; Onozuka, Minoru

2007-01-01

From the perspective of comparative morphology, the distribution of non-monoaminergic neurons in the common marmoset (Callithrix jacchus) was investigated using an immunohistochemical method with specific antibodies to tyrosine hydroxylase (TH) and aromatic-L-amino acid decarboxylase (AADC). TH-immunoreactive (IR) neurons (but not AADC-IR) neurons were observed in the olfactory tubercle, preoptic suprachiasmatic nucleus, periventricular hypothalamic nucleus, arcuate nucleus, paraventricular nucleus, periaqueductal gray matter, medial longitudinal fasciculus, substantia nigra, and nucleus solitaris. In contrast, AADC-IR (but not TH-IR), small, oval and spindle-shaped neurons were sparsely distributed in the following areas: the hypothalamus from the anterior nucleus to the lateral nucleus, the dorsomedial nucleus, the dorsomedial area of the medial mammillary nucleus and the arcuate nucleus; the midbrain, including the stria medullaris and substantia nigra; and the medulla oblongata, including the dorsal area of the nucleus solitaris and the medullary reticular nucleus. The distribution of AADC-IR neurons was not as extensive in the marmoset as it is in rats. However, these neurons were located in the marmoset, but not the rat substantia nigra. Furthermore, AADC-IR neurons that are present in the human striatum were absent in that of the marmoset. The present results indicate that the distribution of non-monoaminergic neurons in the brain of the common marmoset is unique and different from that in humans and rodents. PMID:17653300

Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco

Science.gov (United States)

Blee, K.; Choi, J. W.; O'Connell, A. P.; Jupe, S. C.; Schuch, W.; Lewis, N. G.; Bolwell, G. P.

2001-01-01

A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.

Renal albumin excretion: twin studies identify influences of heredity, environment, and adrenergic pathway polymorphism

DEFF Research Database (Denmark)

Rao, Fangwen; Wessel, Jennifer; Wen, Gen

2007-01-01

biosynthesis (tyrosine hydroxylase), catabolism (monoamine oxidase A), storage/release (chromogranin A), receptor target (dopamine D1 receptor), and postreceptor signal transduction (sorting nexin 13 and rho kinase). Epistasis (gene-by-gene interaction) occurred between alleles at rho kinase, tyrosine...... hydroxylase, chromogranin A, and sorting nexin 13. Dopamine D1 receptor polymorphism showed pleiotropic effects on both albumin and dopamine excretion. These studies establish new roles for heredity and environment in albumin excretion. Urinary excretions of albumin and catecholamines are highly heritable......, and their parallel suggests adrenergic mediation of early glomerular permeability alterations. Albumin excretion is influenced by multiple adrenergic pathway genes and is, thus, polygenic. Such functional links between adrenergic activity and glomerular injury suggest novel approaches to its prediction, prevention...

Genesis by meiotic unequal crossover of a de novo deletion that contributes to steroid 21-hydroxylase deficiency

International Nuclear Information System (INIS)

Sinnott, P.; Collier, S.; Dyer, P.A.; Harris, R.; Strachan, T.; Costigan, C.

1990-01-01

The HLA-linked human steroid 21-hydroxylase gene CYP21B and its closely homologous pseudogene CYP21A are each normally located centromeric to a fourth component of complement (C4) gene, C4B and C4A, respectively, in an organization suggesting tandem duplication of a ca. 30-kilobase DNA unit containing a CYP21 gene and a C4 gene. Such an organization has been considered to facilitate gene deletion and addition events by unequal crossover between the tandem repeats. The authors have identified a steroid 21-hydroxylase deficiency patient who has a maternally inherited disease haplotype that carries a de novo deletion of a ca. 30-kilobase repeat unit including the CYP21B gene and associated C4B gene. This disease haplotype appears to have been generated as a result of meiotic unequal crossover between maternal homologous chromosomes. One of the maternal haplotypes is the frequently occurring HLA-DR3,B8,A1 haplotype that normally carries a deletion of a ca. 30-kilobase unit including the CYP21A gene and C4A gene. Haplotypes of this type may possible act as premutations, increasing the susceptibility of developing a 21-hydroxylase deficiency mutation by facilitating unequal chromosome pairing

Effects of methyl jasmonate, on stevioside and rebaudioside A content and expression of the ent-Kaurenoic acid 13-hydroxylase gene in Stevia rebaudiana Bert. in vitro

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Mehrdad Behmanesh

2014-08-01

Full Text Available Glycosides are a form of secondary metabolites that consist variety compounds and in some cases can play a role in primary metabolism. Steviol is lipophilic skeleton of Stevioside and Rebaudioside A, two main glycosides of Stevia rebuadiana. Steviol glycosides which are synthesized in S.rebaudiana have important medical and nutritional values as high intensity natural sweeteners. Steviol is synthesized from Kaurenoic acid in chloroplastic Terpenoid pathway that mediated by Kaurenoic acid 13-hydroxylase. In this study, HPLC method and RT-PCR were performed for quantification of glycosides and gene expression (ent-Kaurenoic acid 13-hydroxylase respectively. Methyl jasmonate treatment (at 20 micromolar in vitro induced glycoside biosynthesis significantly (P≤0.05 whereas higher concentration of Methyl jasmonate (100 µM caused a decrease in glycoside production and growth. The most glycoside content of the plant was three days after treatment. Also Methyl jasmonate treatment caused an increase in ent-Kaurenoic 13-hydroxylase gene expression from 6 hours to 48 hours (after treatment Results showed that biosynthesis of Stevia glycosides was probably a defense mechanism against pathogens and herbivore insects. Also we found that different concentrations of Methyl jasmonate, alter the ratio between glycosides rather than the increase in glycoside contents.

Molecular phenotyping of transient postnatal tyrosine hydroxylase neurons in the rat bed nucleus of the stria terminalis.

Science.gov (United States)

Carter, David A

2017-07-01

The bed nucleus of the stria terminalis (BNST) is a complex integrative centre in the forebrain, composed of multiple sub-nuclei, each with discrete populations of neurons. Progress in understanding BNST function, both in the adult and during postnatal maturation, is dependent upon a more complete characterization of neuronal phenotypes in the BNST. The aim of the current study was to define the molecular phenotype of one postnatal BNST neuronal population, in order to identify molecular factors that may underlie both (protein marker-related) immaturity, and secondly, the transience of this phenotype. This BNST population was originally identified by high, but transient expression of the EGR1 transcription factor (TF) in postnatal rat lateral intermediate BNST (BNSTLI). The current results confirm a high level of Egr1 activation in postnatal day 10 (PN10) male BNSTLI that is lost at PN40, and now demonstrate a similar pattern of transient activation in female brains. Apparent cellular immaturity in this population, as indicated by low levels of the adult neuronal marker NeuN/RBFOX3, was found to be uncorrelated with both key neuronal regulator protein expression (SOX2 and REST), and also RBFOX2 protein levels. The BNSTLI neurons have a partial catecholaminergic phenotype (tyrosine hydroxylase-positive/dopa decarboxylase-negative; TH+ve/DDC-ve) that is lost at PN40. In contrast, the co-expressed neuropeptide, somatostatin, is maintained, albeit at lower levels, at PN40. The transcriptional basis of the transient and partial catecholaminergic phenotype was investigated by analysing TFs known to maintain adult dopaminergic (TH+ve/DDC+ve) neuronal phenotypes. The BNSTLI neurons were shown to lack forkhead TFs including FOXA1, FOXA2 and FOXO1. In addition, the BNSTLI neurons had low, primarily cytoplasmic, expression of NR4A2/NURR1, an orphan nuclear receptor that is critical for adult maintenance of midbrain dopamine neurons. These results detail the molecular features

Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU)

Science.gov (United States)

Harding, Cary O.; Winn, Shelley R.; Gibson, K. Michael; Arning, Erland; Bottiglieri, Teodoro; Grompe, Markus

2014-01-01

Summary Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis. Dietary supplementation with large neutral amino acids (LNAA) including Tyr and Trp has been recommended for individuals with chronically elevated blood Phe in an attempt to restore amino acid and monoamine homeostasis in brain. As a potential alternative treatment approach, we demonstrate that pharmacologic inhibition of Tyr degradation through oral administration of nitisinone (NTBC) yielded sustained increases in blood and brain Tyr, decreased blood and brain Phe, and consequently increased dopamine synthesis in a murine model of PKU. Our results suggest that Phe-mediated inhibition of TH activity is the likely mechanism of impaired dopamine synthesis in PKU. Pharmacologic inhibition of Tyr degradation may be a promising adjunct therapy for CNS monoamine neurotransmitter deficiency in hyperphenylalaninemic individuals with PKU. PMID:24487571

The Tyrosine Aminomutase TAM1 Is Required for β-Tyrosine Biosynthesis in Rice

Science.gov (United States)

Yan, Jian; Aboshi, Takako; Teraishi, Masayoshi; Strickler, Susan R.; Spindel, Jennifer E.; Tung, Chih-Wei; Takata, Ryo; Matsumoto, Fuka; Maesaka, Yoshihiro; McCouch, Susan R.; Okumoto, Yutaka; Mori, Naoki; Jander, Georg

2015-01-01

Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-β-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with β-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated β-tyrosine production in Nipponbare. Rice cultivars that do not produce β-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although β-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant β-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 μM. As β-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice. PMID:25901084

Novel vitamin D 1α-hydroxylase gene mutations in a Chinese ...

Indian Academy of Sciences (India)

2011-08-19

Aug 19, 2011 ... known as vitamin D 1α-hydroxylase deficiency or pseu- dovitamin D ... amplicons of the 378 bp were digested with restriction enzyme PvuI and ... have no enzymatic activity; a missense mutation c.473T>C. (p.L158P) in the ...

Organisation and tyrosine hydroxylase and calretinin immunoreactivity in the main olfactory bulb of paca (Cuniculus paca): a large caviomorph rodent.

Science.gov (United States)

Sasahara, Tais Harumi de Castro; Leal, Leonardo Martins; Spillantini, Maria Grazia; Machado, Márcia Rita Fernandes

2015-04-01

The majority of neuroanatomical and chemical studies of the olfactory bulb have been performed in small rodents, such as rats and mice. Thus, this study aimed to describe the organisation and the chemical neuroanatomy of the main olfactory bulb (MOB) in paca, a large rodent belonging to the Hystricomorpha suborder and Caviomorpha infraorder. For this purpose, histological and immunohistochemical procedures were used to characterise the tyrosine hydroxylase (TH) and calretinin (CR) neuronal populations and their distribution. The paca MOB has eight layers: the olfactory nerve layer (ONL), the glomerular layer (GL), the external plexiform layer (EPL; subdivided into the inner and outer sublayers), the mitral cell layer (MCL), the internal plexiform layer (IPL), the granule cell layer (GCL), the periventricular layer and the ependymal layer. TH-ir neurons were found mostly in the GL, and moderate numbers of TH-ir neurons were scattered in the EPL. Numerous varicose fibres were distributed in the IPL and in the GCL. CR-ir neurons concentrated in the GL, around the base of the olfactory glomeruli. Most of the CR-ir neurons were located in the MCL, IPL and GCL. Some of the granule cells had an apical dendrite with a growth cone. The CR immunoreactivity was also observed in the ONL with olfactory nerves strongly immunostained. This study has shown that the MOB organisation in paca is consistent with the description in other mammals. The characterisation and distribution of the population of TH and CR in the MOB is not exclusively to this species. This large rodent shares common patterns to other caviomorph rodent, as guinea pig, and to the myomorph rodents, as mice, rats and hamsters.

A novel mutation in the lysyl hydroxylase 1 gene causes decreased lysyl hydroxylase activity in an ehlers-danlos VIA patient

NARCIS (Netherlands)

Walker, L.C.; Overstreet, M.A.; Siddiqui, A.; Paepe, A. de; Ceylaner, G.; Malfait, F.; Symoens, S.; Atsawasuwan, P.; Yamauchi, M.; Ceylaner, S.; Bank, R.A.; Yeowell, H.N.

2005-01-01

The clinical diagnosis of a patient with the phenotype of Ehlers-Danlos syndrome type VI was confirmed biochemically by the severely diminished level of lysyl hydroxylase (LH) activity in the patient's skin fibroblasts. A novel homozygous mutation, a single base change of T1360 → G in exon 13 of the

Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

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Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

2015-02-01

Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

The Dopamine D2 Receptor Gene in Lamprey, Its Expression in the Striatum and Cellular Effects of D2 Receptor Activation

Science.gov (United States)

Robertson, Brita; Huerta-Ocampo, Icnelia; Ericsson, Jesper; Stephenson-Jones, Marcus; Pérez-Fernández, Juan; Bolam, J. Paul; Diaz-Heijtz, Rochellys; Grillner, Sten

2012-01-01

All basal ganglia subnuclei have recently been identified in lampreys, the phylogenetically oldest group of vertebrates. Furthermore, the interconnectivity of these nuclei is similar to mammals and tyrosine hydroxylase-positive (dopaminergic) fibers have been detected within the input layer, the striatum. Striatal processing is critically dependent on the interplay with the dopamine system, and we explore here whether D2 receptors are expressed in the lamprey striatum and their potential role. We have identified a cDNA encoding the dopamine D2 receptor from the lamprey brain and the deduced protein sequence showed close phylogenetic relationship with other vertebrate D2 receptors, and an almost 100% identity within the transmembrane domains containing the amino acids essential for dopamine binding. There was a strong and distinct expression of D2 receptor mRNA in a subpopulation of striatal neurons, and in the same region tyrosine hydroxylase-immunoreactive synaptic terminals were identified at the ultrastructural level. The synaptic incidence of tyrosine hydroxylase-immunoreactive boutons was highest in a region ventrolateral to the compact layer of striatal neurons, a region where most striatal dendrites arborise. Application of a D2 receptor agonist modulates striatal neurons by causing a reduced spike discharge and a diminished post-inhibitory rebound. We conclude that the D2 receptor gene had already evolved in the earliest group of vertebrates, cyclostomes, when they diverged from the main vertebrate line of evolution (560 mya), and that it is expressed in striatum where it exerts similar cellular effects to that in other vertebrates. These results together with our previous published data (Stephenson-Jones et al. 2011, 2012) further emphasize the high degree of conservation of the basal ganglia, also with regard to the indirect loop, and its role as a basic mechanism for action selection in all vertebrates. PMID:22563388

Effect of dioxins on regulation of tyrosine hydroxylase gene expression by aryl hydrocarbon receptor: a neurotoxicology study

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Akahoshi Eiichi

2009-06-01

Full Text Available Abstract Background Dioxins and related compounds are suspected of causing neurological disruption. Epidemiological studies indicated that exposure to these compounds caused neurodevelopmental disturbances such as learning disability and attention deficit hyperactivity disorder, which are thought to be closely related to dopaminergic dysfunction. Although the molecular mechanism of their actions has not been fully investigated, a major participant in the process is aryl hydrocarbon receptor (AhR. This study focused on the effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD exposure on the regulation of TH, a rate-limiting enzyme of dopamine synthesis, gene expression by AhR. Methods N2a-Rβ cells were established by transfecting murine neuroblastoma Neuro2a with the rat AhR cDNA. TH expression induced by TCDD was assessed by RT-PCR and Western blotting. Participation of AhR in TCDD-induced TH gene expression was confirmed by suppressing AhR expression using the siRNA method. Catecholamines including dopamine were measured by high-performance liquid chromatography. A reporter gene assay was used to identify regulatory motifs in the promoter region of TH gene. Binding of AhR with the regulatory motif was confirmed by an electrophoretic mobility shift assay (EMSA. Results Induction of TH by TCDD through AhR activation was detected at mRNA and protein levels. Induced TH protein was functional and its expression increased dopamine synthesis. The reporter gene assay and EMSA indicated that AhR directly regulated TH gene expression. Regulatory sequence called aryl hydrocarbon receptor responsive element III (AHRE-III was identified upstream of the TH gene from -285 bp to -167 bp. Under TCDD exposure, an AhR complex was bound to AHRE-III as well as the xenobiotic response element (XRE, though AHRE-III was not identical to XRE, the conventional AhR-binding motif. Conclusion Our results suggest TCDD directly regulate the dopamine system by TH gene

Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

DEFF Research Database (Denmark)

Jacob, K K; Sap, J; Stanley, F M

1998-01-01

A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

Expression and functional analysis of citrus carotene hydroxylases: unravelling the xanthophyll biosynthesis in citrus fruits.

Science.gov (United States)

Ma, Gang; Zhang, Lancui; Yungyuen, Witchulada; Tsukamoto, Issei; Iijima, Natsumi; Oikawa, Michiru; Yamawaki, Kazuki; Yahata, Masaki; Kato, Masaya

2016-06-29

Xanthophylls are oxygenated carotenoids and fulfill critical roles in plant growth and development. In plants, two different types of carotene hydroxylases, non-heme di-iron and heme-containing cytochrome P450, were reported to be involved in the biosynthesis of xanthophyll. Citrus fruits accumulate a high amount of xanthophylls, especially β,β-xanthophylls. To date, however, the roles of carotene hydroxylases in regulating xanthophyll content and composition have not been elucidated. In the present study, the roles of four carotene hydroxylase genes (CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C) in the biosynthesis of xanthophyll in citrus fruits were investigated. Phylogenetic analysis showed that the four citrus carotene hydroxylases presented in four distinct clusters which have been identified in higher plants. CitHYb was a non-heme di-iron carotene hydroxylase, while CitCYP97A, CitCYP97B, and CitCYP97C were heme-containing cytochrome P450-type carotene hydroxylases. Gene expression results showed that the expression of CitHYb increased in the flavedo and juice sacs during the ripening process, which was well consistent with the accumulation of β,β-xanthophyll in citrus fruits. The expression of CitCYP97A and CitCYP97C increased with a peak in November, which might lead to an increase of lutein in the juice sacs during the ripening process. The expression level of CitCYP97B was much lower than that of CitHYb, CitCYP97A, and CitCYP97C in the juice sacs during the ripening process. Functional analysis showed that the CitHYb was able to catalyze the hydroxylation of the β-rings of β-carotene and α-carotene in Escherichia coli BL21 (DE3) cells. Meanwhile, when CitHYb was co-expressed with CitCYP97C, α-carotene was hydroxylated on the β-ring and ε-ring sequentially to produce lutein. CitHYb was a key gene for β,β-xanthophyll biosynthesis in citrus fruits. CitCYP97C functioned as an ε-ring hydroxylase to produce lutein using zeinoxanthin as a substrate

Regional mapping of the phenylalanine hydroxylase gene and the phenylketonuria locus in the human genome

Energy Technology Data Exchange (ETDEWEB)

Lidsky, A.S.; Law, M.L.; Morse, H.G.; Kao, F.T.; Rabin, M.; Ruddle, F.H.; Woo, S.L.C.

1985-09-01

Phenylketonuria (PKU) is an autosomal recessive disorder of amino acid metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. To define the regional map position of the disease locus and the PAH gene on human chromosome 12, DNA was isolated from human-hamster somatic cell hybrids with various deletions of human chromosome 12 and was analyzed by Southern blot analysis using the human cDNA PAH clone as a hybridization probe. From these results, together with detailed biochemical and cytogenetic characterization of the hybrid cells, the region on chromosome 12 containing the human PAH gene has been defined as 12q14.3..-->..qter. The PAH map position on chromosome 12 was further localized by in situ hybridization of /sup 125/I-labeled human PAH cDNA to chromosomes prepared from a human lymphoblastoid cell line. Results of these experiments demonstrated that the region on chromosome 12 containing the PAH gene and the PKU locus in man is 12q22..-->..12q24.1. These results not only provide a regionalized map position for a major human disease locus but also can serve as a reference point for linkage analysis with other DNA markers on human chromosome 12.

Regional mapping of the phenylalanine hydroxylase gene and the phenylketonuria locus in the human genome

International Nuclear Information System (INIS)

Lidsky, A.S.; Law, M.L.; Morse, H.G.; Kao, F.T.; Rabin, M.; Ruddle, F.H.; Woo, S.L.C.

1985-01-01

Phenylketonuria (PKU) is an autosomal recessive disorder of amino acid metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. To define the regional map position of the disease locus and the PAH gene on human chromosome 12, DNA was isolated from human-hamster somatic cell hybrids with various deletions of human chromosome 12 and was analyzed by Southern blot analysis using the human cDNA PAH clone as a hybridization probe. From these results, together with detailed biochemical and cytogenetic characterization of the hybrid cells, the region on chromosome 12 containing the human PAH gene has been defined as 12q14.3→qter. The PAH map position on chromosome 12 was further localized by in situ hybridization of 125 I-labeled human PAH cDNA to chromosomes prepared from a human lymphoblastoid cell line. Results of these experiments demonstrated that the region on chromosome 12 containing the PAH gene and the PKU locus in man is 12q22→12q24.1. These results not only provide a regionalized map position for a major human disease locus but also can serve as a reference point for linkage analysis with other DNA markers on human chromosome 12

Increasing proportions of tyrosine hydroxylase-immunoreactive interneurons colocalize with choline acetyltransferase or vasoactive intestinal peptide in the developing rat cerebral cortex

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Asmus, Stephen E.; Cocanougher, Benjamin T.; Allen, Donald L.; Boone, John B.; Brooks, Elizabeth A.; Hawkins, Sarah M.; Hench, Laura A.; Ijaz, Talha; Mayfield, Meredith N.

2011-01-01

Cortical interneurons are critical for information processing, and their dysfunction has been implicated in neurological disorders. One subset of this diverse cell population expresses tyrosine hydroxylase (TH) during postnatal rat development. Cortical TH-immunoreactive neurons appear at postnatal day (P) 16. The number of TH cells sharply increases between P16 and P20 and subsequently decreases to adult values. The absence of apoptotic markers in these cells suggests that the reduction in cell number is not due to cell death but is due to a decline in TH production. Cortical TH cells lack all additional catecholaminergic enzymes, and many coexpress GABA and calretinin, but little else is known about their phenotype or function. Because interneurons containing choline acetyltransferase (ChAT) or vasoactive intestinal peptide (VIP) share characteristics with cortical TH neurons, the coexpression of TH with ChAT or VIP was examined throughout the neocortex at P16, P20, and P30. The proportions of TH cell profiles double-labeled for ChAT or VIP significantly increased between P16 and P30. Based on their proximity to blood vessels, intrinsic cholinergic and VIPergic cells have been hypothesized to regulate cortical microcirculation. Labeling with the gliovascular marker aquaporin-4 revealed that at least half of the TH cells were apposed to microvessels at these ages, and many of these cells contained ChAT or VIP. Cortical TH neurons did not coproduce nitric oxide synthase. These results suggest that increasing proportions of cortical TH neurons express ChAT or VIP developmentally and that a subset of these TH neurons may regulate local blood flow. PMID:21295554

Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae).

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Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M; Zhang, Shuyi

2014-01-01

Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

Mutation pattern in the Bruton's tyrosine kinase gene in 26 unrelated patients with X-linked agammaglobulinemia

DEFF Research Database (Denmark)

Vorechovský, I; Luo, L; Hertz, Jens Michael

1997-01-01

Mutation pattern was characterized in the Bruton's tyrosine kinase gene (BTK) in 26 patients with X-linked agammaglobulinemia, the first described immunoglobulin deficiency, and was related to BTK expression. A total of 24 different mutations were identified. Most BTK mutations were found to result...

No association of peptide tyrosine-tyrosine (PYY) gene R72T variant with obesity in the Kampar Health Clinic cohort, Malaysia.

Science.gov (United States)

Chan, P M; Fan, S H; Say, Y H

2011-08-01

Peptide Tyrosine-Tyrosine (PYY) is a 36-amino acid peptide hormone released post-prandially from the endocrine cells in the intestinal tract to suppress pancreatic secretions and eventually reduce appetite. The R72T variant in the PYY gene (rs1058046) has been associated with increased susceptibility to obesity. Therefore, the objective of this study was to investigate the association of this variant with obesity and its related anthropometric measurements among the Kampar Health Clinic cohort, Malaysia. A total of 197 (78 males, 119 females; 98 non-obese, 99 obese) subjects were recruited by convenience sampling and anthropometric measurements were taken. Genotyping was performed using StuI Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), revealing 61 RR, 94 RT and 42 TT subjects. Most of the obese subjects had the RT genotype (50.5%), while only 18.2% were TT. PYY R72T genotypes and alleles had no association with obesity (p = 0.535; 0.074, respectively), gender (p = 0.767; p = 0.100, respectively) but were associated with ethnicity (p = 0.003; p = 0.002, respectively). Among the 13 anthropometric measurements taken, significant difference was only found in Waist Circumference (WC) and Visceral Fat Level (VFL) among the alleles, suggesting that subjects with T allele will have an increment of 1.82 cm in WC and 1.32% in VFL. The R72T variant in PYY gene was not associated with obesity and most of its related anthropometric measurements. This suggests that other genes and/or environmental factors like dietary habits and lifestyle factors may be the contributors of obesity.

Intracellular Growth Is Dependent on Tyrosine Catabolism in the Dimorphic Fungal Pathogen Penicillium marneffei

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Boyce, Kylie J.; McLauchlan, Alisha; Schreider, Lena; Andrianopoulos, Alex

2015-01-01

During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host’s defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells. PMID:25812137

Phenylalanine hydroxylase from Legionella pneumophila is a thermostable enzyme with a major functional role in pyomelanin synthesis.

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Marte I Flydal

Full Text Available Legionella pneumophila is a pathogenic bacterium that can cause Legionnaires' disease and other non-pneumonic infections in humans. This bacterium produces a pyomelanin pigment, a potential virulence factor with ferric reductase activity. In this work, we have investigated the role of phenylalanine hydroxylase from L. pneumophila (lpPAH, the product of the phhA gene, in the synthesis of the pyomelanin pigment and the growth of the bacterium in defined compositions.Comparative studies of wild-type and phhA mutant corroborate that lpPAH provides the excess tyrosine for pigment synthesis. phhA and letA (gacA appear transcriptionally linked when bacteria were grown in buffered yeast extract medium at 37°C. phhA is expressed in L. pneumophila growing in macrophages. We also cloned and characterized lpPAH, which showed many characteristics of other PAHs studied so far, including Fe(II requirement for activity. However, it also showed many particular properties such as dimerization, a high conformational thermal stability, with a midpoint denaturation temperature (T(m = 79 ± 0.5°C, a high specific activity at 37°C (10.2 ± 0.3 µmol L-Tyr/mg/min and low affinity for the substrate (K(m (L-Phe = 735 ± 50 µM.lpPAH has a major functional role in the synthesis of pyomelanin and promotes growth in low-tyrosine media. The high thermal stability of lpPAH might reflect the adaptation of the enzyme to withstand relatively high survival temperatures.

Effect of hyperthyroidism on circulating prolactin and hypothalamic expression of tyrosine hydroxylase, prolactin signaling cascade members and estrogen and progesterone receptors during late pregnancy and lactation in the rat.

Science.gov (United States)

Pennacchio, Gisela E; Neira, Flavia J; Soaje, Marta; Jahn, Graciela A; Valdez, Susana R

2017-02-15

Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae.

Directory of Open Access Journals (Sweden)

Bin Shen

Full Text Available Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae and two New World fruit bats (Phyllostomidae. Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

Isolation and characterization of biosurfactant-producing Alcanivorax strains: hydrocarbon accession strategies and alkane hydroxylase gene analysis.

Science.gov (United States)

Olivera, Nelda L; Nievas, Marina L; Lozada, Mariana; Del Prado, Guillermo; Dionisi, Hebe M; Siñeriz, Faustino

2009-01-01

Biosurfactant-producing bacteria belonging to the genera Alcanivorax, Cobetia and Halomonas were isolated from marine sediments with a history of hydrocarbon exposure (Aristizábal and Gravina Peninsulas, Argentina). Two Alcanivorax isolates were found to form naturally occurring consortia with strains closely related to Pseudomonas putida and Microbacterium esteraromaticum. Alkane hydroxylase gene analysis in these two Alcanivorax strains resulted in the identification of two novel alkB genes, showing 86% and 60% deduced amino acid sequence identity with those of Alcanivorax sp. A-11-3 and Alcanivorax dieselolei P40, respectively. In addition, a gene homologous to alkB2 from Alcanivorax borkumensis was present in one of the strains. The consortium formed by this strain, Alcanivorax sp. PA2 (98.9% 16S rRNA gene sequence identity with A. borkumensis SK2(T)) and P. putida PA1 was characterized in detail. These strains form cell aggregates when growing as mixed culture, though only PA2 was responsible for biosurfactant activity. During exponential growth phase of PA2, cells showed high hydrophobicity and adherence to hydrocarbon droplets. Biosurfactant production was only detectable at late growth and stationary phases, suggesting that it is not involved in initiating oil degradation and that direct interfacial adhesion is the main hydrocarbon accession mode of PA2. This strain could be useful for biotechnological applications due to its biosurfactant production, catabolic and aggregation properties.

Seventeen Alpha-hydroxylase Deficiency

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Siew-Lee Wong

2006-01-01

Full Text Available Seventeen a-hydroxylase deficiency (17OHD is a rare form of congenital adrenal hyperplasia in which defects in the biosynthesis of cortisol and sex steroid result in mineralocorticoid excess, hypokalemic hypertension and sexual abnormalities such as pseudohermaphroditism in males, and sexual infantilism in females. The disease is inherited in an autosomal recessive pattern, and is caused by mutations in the gene encoding cytochrome P450c17 (CYP17, which is the single polypeptide that mediates both 17α-hydroxylase and 17,20-lyase activities. We report the case of a 15-year-old patient with 17OHD who had a female phenotype but male karyotype (46,XY. The diagnosis was made based on classical clinical features, biochemical data and molecular genetic study. Two mutations were identified by polymerase chain reaction amplification and sequencing, including a S106P point mutation in exon 2 and a 9-bp (GACTCTTTC deletion from nucleotide position 1519 in exon 8 of CYP17. The first of these mutations was found in the father and the second in the mother, and both have been previously reported in Asia. The patient's hypertension and hypokalemia resolved after glucocorticoid replacement and treatment with potassium-sparing diuretics. Sex hormone replacement was prescribed for induction of sexual development and reduction of the final height. Prophylactic gonadectomy was scheduled. In summary, 17OHD should be suspected in patients with hypokalemic hypertension and lack of secondary sexual development so that appropriate therapy can be implemented.

Genetics Home Reference: 21-hydroxylase deficiency

Science.gov (United States)

... adrenal hyperplasias that impair hormone production and disrupt sexual development. 21-hydroxylase deficiency is responsible for about 95 ... excess production of androgens leads to abnormalities of sexual development in people with 21-hydroxylase deficiency . A lack ...

Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice.

NARCIS (Netherlands)

Hoenderop, J.G.J.; Dardenne, O.; Abel, M. van; Kemp, J.W.C.M. van der; Os, C.H. van; Arnaud, R. St.; Bindels, R.J.M.

2002-01-01

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal disease characterized by hyperparathyroidism, rickets, and undetectable levels of 1,25-dihydroxyvitaminD3 (1,25(OH)2D3). Mice in which the 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) gene was inactivated presented the same clinical

Subthalamic hGAD65 Gene Therapy and Striatum TH Gene Transfer in a Parkinson’s Disease Rat Model

Science.gov (United States)

Zheng, Deyu; Jiang, Xiaohua; Zhao, Junpeng; Duan, Deyi; Zhao, Huanying; Xu, Qunyuan

2013-01-01

The aim of the present study is to detect a combination method to utilize gene therapy for the treatment of Parkinson’s disease (PD). Here, a PD rat model is used for the in vivo gene therapy of a recombinant adeno-associated virus (AAV2) containing a human glutamic acid decarboxylase 65 (rAAV2-hGAD65) gene delivered to the subthalamic nucleus (STN). This is combined with the ex vivo gene delivery of tyrosine hydroxylase (TH) by fibroblasts injected into the striatum. After the treatment, the rotation behavior was improved with the greatest efficacy in the combination group. The results of immunohistochemistry showed that hGAD65 gene delivery by AAV2 successfully led to phenotypic changes of neurons in STN. And the levels of glutamic acid and GABA in the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr) were obviously lower than the control groups. However, hGAD65 gene transfer did not effectively protect surviving dopaminergic neurons in the SNc and VTA. This study suggests that subthalamic hGAD65 gene therapy and combined with TH gene therapy can alleviate symptoms of the PD model rats, independent of the protection the DA neurons from death. PMID:23738148

Use of deuterated tyrosine and phenylalanine in the study of catecholamine and aromatic acid metabolism

International Nuclear Information System (INIS)

Curtius, H.C.; Redweik, U.; Steinmann, B.; Leimbacher, W.; Wegmann, H.

1975-01-01

Deuterated tyrosine and phenylalanine have been used for the study of their respective metabolism in patients with phenylketonuria (PKU) and in healthy persons. Urinary excretion of dopamine and its metabolites was studied by GC-MS after oral administration of deuterated L-tyrosine in 2 patients with PKU and in normal controls at low and high plasma phenylalanine levels. From these studies it seemed that the in vivo tyrosine 3-hydroxylase activity and thus the formation of L-dopa depend on the phenylalanine concentration in plasma and also in tissues. After loading 3 mentally retarded patients with 3,5-[ 2 H 2 ]-4-hydroxyphenylalanine, we found, among others, excretion of deuterated m-hydroxyphenyl-hydracrylic acid, p-hydroxymandelic acid, p-hydroxybenzoic acid, p-hydroxyhippuric acid, benzoic acid and hippuric acid. An intramolecular rearrangement is postulated. Deuterated phenylalanine was used to investigate phenylalanine and dopa metabolism in PKU. In addition, one untreated person with PKU of normal intelligence and normal excretion of catecholamines at high plasma phenylalanine concentration was investigated in order to see whether there exists an alternative metabolic pathway from phenylalanine to dopa formation

The ducky(2J) mutation in Cacna2d2 results in reduced spontaneous Purkinje cell activity and altered gene expression.

Science.gov (United States)

Donato, Roberta; Page, Karen M; Koch, Dietlind; Nieto-Rostro, Manuela; Foucault, Isabelle; Davies, Anthony; Wilkinson, Tonia; Rees, Michele; Edwards, Frances A; Dolphin, Annette C

2006-11-29

The mouse mutant ducky and its allele ducky(2J) represent a model for absence epilepsy characterized by spike-wave seizures and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the alpha2delta-2 calcium channel subunit. Of relevance to the ataxic phenotype, alpha2delta-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2(du2J) mutation results in a 2 bp deletion in the coding region and a complete loss of alpha2delta-2 protein. Here we show that du(2J)/du(2J) mice have a 30% reduction in somatic calcium current and a marked fall in the spontaneous PC firing rate at 22 degrees C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34 degrees C, du(2J)/du(2J) PCs show no spontaneous intrinsic activity. Du(2J)/du(2J) mice also have alterations in the cerebellar expression of several genes related to PC function. At postnatal day 21, there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du(2J)/+ mice have a marked reduction in alpha2delta-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tyrosine hydroxylase gene expression. However, du(2J)/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in alpha2delta-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of alpha2delta-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma.

CRYSTAL STRUCTURE OF HUMAN DOPAMINE BETA-HYDROXYLASE

DEFF Research Database (Denmark)

2017-01-01

A crystalline form of dopamine β-hydroxylase is provided. X-ray crystallography reveals the space group and cell dimensions, as well as the atomic coordinates. The information can be used for identifying one or more modulators of dopamine β-hydroxylase, which can then be chemically synthesised...... and used in treatment. A process for preparing the crystalline form of human dopamine β-hydroxylase is also provided....

Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner

Energy Technology Data Exchange (ETDEWEB)

Fitzpatrick, Susan F.; Fábián, Zsolt; Schaible, Bettina; Lenihan, Colin R.; Schwarzl, Thomas [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Rodriguez, Javier [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Zheng, Xingnan; Li, Zongwei [Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC (United States); Tambuwala, Murtaza M. [School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, BT52 1SA, Northern Ireland (United Kingdom); Higgins, Desmond G.; O' Meara, Yvonne [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Slattery, Craig [School of Biomolecular and Biomedical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Manresa, Mario C. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Fraisl, Peter; Bruning, Ulrike [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Baes, Myriam [Laboratory for Cell Metabolism, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven (Belgium); Carmeliet, Peter; Doherty, Glen [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Kriegsheim, Alex von [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Cummins, Eoin P. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); and others

2016-06-03

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1{sup −/−} hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease. -- Highlights: •Genetic ablation of PHD1 upregulates NF-kappaB (NF-κB) in hepatocytes. •Activation of NF-κB leads to differential DNA-binding of p50/p65 and results in differential regulation of apoptotic genes. •We identified proline 191 in the beta subunit of the I-kappaB kinase as a target for PHD1-mediated hydroxylation. •Blockade of prolyl-4-hydroxylases has been found cytoprotective in liver cells.

Light response and potential interacting proteins of a grape flavonoid 3'-hydroxylase gene promoter.

Science.gov (United States)

Sun, Run-Ze; Pan, Qiu-Hong; Duan, Chang-Qing; Wang, Jun

2015-12-01

Flavonoid 3'-hydroxylase (F3'H), a member of cytochrome P450 protein family, introduces B-ring hydroxyl group in the 3' position of the flavonoid. In this study, the cDNA sequence of a F3'H gene (VviF3'H), which contains an open reading frame of 1530 bp encoding a polypeptide of 509 amino acids, was cloned and characterized from Vitis vinifera L. cv. Cabernet Sauvignon. VviF3'H showed high homology to known F3'H genes, especially F3'Hs from the V. vinifera reference genome (Pinot Noir) and lotus. Expression profiling analysis using real-time PCR revealed that VviF3'H was ubiquitously expressed in all tested tissues including berries, leaves, flowers, roots, stems and tendrils, suggesting its important physiological role in plant growth and development. Moreover, the transcript level of VviF3'H gene in grape berries was relatively higher at early developmental stages and gradually decreased during véraison, and then increased in the mature phase. In addition, the promoter of VviF3'H was isolated by using TAIL-PCR. Yeast one-hybrid screening of the Cabernet Sauvignon cDNA library and subsequent in vivo/vitro validations revealed the interaction between VviF3'H promoter and several transcription factors, including members of HD-Zip, NAC, MYB and EIN families. A transcriptional regulation mechanism of VviF3'H expression is proposed for the first time. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Putative tyrosine kinases expressed in K-562 human leukemia cells

International Nuclear Information System (INIS)

Partanen, J.; Maekelae, T.P.; Lehvaeslaiho, H.; Alitalo, K.; Alitalo, R.

1990-01-01

Tyrosine phosphorylation is important in the transmission of growth and differentiation signals; known tyrosine kinases include several oncoproteins and growth factor receptors. Interestingly, some differentiated cell types, such as erythrocytes and platelets contain high amounts of phosphotyrosine. The authors analyzed tyrosine kinases expressed in the K-562 chronic myelogenous leukemia cell line, which has a bipotential erythroid and megakaryoblastoid differentiation capacity. Analysis of 359 polymerase chain reaction-amplified cDNA clones led to the identification of 14 different tyrosine kinase-related sequences (JTK1-14). Two of the clones (JTK2 and JTK4) represent unusual members of the fibroblast growth factor receptor gene family, and the clones JTK5, JTK11, and JTK14 may also belong to the family of receptor tyrosine kinases but lack a close relationship to any known tyrosine kinase. Each of these different genes has its own characteristic expression pattern in K-562 cells and several other human tumor cell lines. In addition, the JTK11 and JTK14 mRNAs are induced during the megakaryoblastoid differentiation of K-562 cells. These tyrosine kinases may have a role in the differentiation of megakaryoblasts or in the physiology of platelets

Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene

Energy Technology Data Exchange (ETDEWEB)

Goltsov, A.A.; Eisensmith, R.C.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (United States)); Konecki, D.S.; Lichter-Konecki, U.

1992-09-01

The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiples of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between theses alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations. 32 refs., 3 figs., 3 tabs.

Gypsy Phenylketonuria: A point mutation of the phenylalanine hydroxylase gene in Gypsy families from Slovakia

Energy Technology Data Exchange (ETDEWEB)

Kalanin, J. [Institute for Clinical and Experical Medicine, Praha (Czechoslovakia); Takarada, Y. [Toyobo Research Center, Shiga (Japan); Kagawa, S.; Yamashita, K.; Ohtsuka, N.; Matsuoka, A. [Hyogo College of Medicine, Nishinomiya (Japan)

1994-01-15

A direct mutational analysis of the phenylalanine hydroxylase gene (PAH) in Gypsy families with phenylketonuria (PKU) has not yet been presented. However, they obviously represent a group at high risk for this inherited disease. The authors analyzed the PAH loci of 65 Gypsies originating from Eastern Slovakia by a combination of PCR amplification, direct sequencing and ASO hybridization. These studies uncovered 10 {open_quotes}classical PKU{close_quotes} patients to be homozygous for a R252W (CGG-TGG) transition, and 29 heterozygous carriers of this mutation. Fifteen control Caucasoid PKU patients from the Czech and Slovak Republics were selected. In this group they detected R252W mutation in two subjects (6.67% of all mutant alleles). Both were compound heterozygous for two different mutations. Previous haplotype studies of Welsh Gypsies with PKU were uninformative in the determination of heterozygosity. ASO hybridization served effectively for the consequent analyses in Gypsy PKU-related families and to identify the carriers among the unrelated subjects. 19 refs., 2 figs.

Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco.

Science.gov (United States)

Mahajan, Monika; Yadav, Sudesh Kumar

2014-08-01

Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.

Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

DEFF Research Database (Denmark)

Bauer, Matthias; Meyer, Morten; Brevig, Thomas

2002-01-01

Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM...... tissue grafts to PD patients indicate that one reason for the poor outcome of neural transplantation is the low survival and differentiation of grafted dopaminergic neurons. To improve dopaminergic cell survival through a gene-therapeutic approach we have established and report here results of lipid-mediated...... numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo...

Treadmill exercise does not change gene expression of adrenal catecholamine biosynthetic enzymes in chronically stressed rats

Directory of Open Access Journals (Sweden)

LJUBICA GAVRILOVIC

2013-09-01

Full Text Available ABSTRACT Chronic isolation of adult animals represents a form of psychological stress that produces sympatho-adrenomedullar activation. Exercise training acts as an important modulator of sympatho-adrenomedullary system. This study aimed to investigate physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes (tyrosine hydroxylase, dopamine-ß-hydroxylase and phenylethanolamine N-methyltransferase and cyclic adenosine monophosphate response element-binding (CREB in the adrenal medulla, concentrations of catecholamines and corticosterone (CORT in the plasma and the weight of adrenal glands of chronically psychosocially stressed adult rats exposed daily to 20 min treadmill running for 12 weeks. Also, we examined how additional acute immobilization stress changes the mentioned parameters. Treadmill running did not result in modulation of gene expression of catecholamine synthesizing enzymes and it decreased the level of CREB mRNA in the adrenal medulla of chronically psychosocially stressed adult rats. The potentially negative physiological adaptations after treadmill running were recorded as increased concentrations of catecholamines and decreased morning CORT concentration in the plasma, as well as the adrenal gland hypertrophy of chronically psychosocially stressed rats. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. Treadmill exercise does not change the activity of sympatho-adrenomedullary system of chronically psychosocially stressed rats.

Mutations of the phenylalanine hydroxylase gene in patients with phenylketonuria in Shanxi, China

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Yong-An Zhou

2012-01-01

Full Text Available The variation in mutations in exons 3, 6, 7, 11 and 12 of the phenylalanine hydroxylase (PAH gene was investigated in 59 children with phenylketonuria (PKU and 100 normal children. Three single nucleotide polymorphisms were detected by sequence analysis. The mutational frequencies of cDNA 696, cDNA 735 and cDNA 1155 in patients were 96.2%, 76.1% and 7.6%, respectively, whereas in healthy children the corresponding frequencies were 97.0%, 77.3% and 8.3%. In addition, 81 mutations accounted for 61.0% of the mutant alleles. R111X, H64 > TfsX9 and S70 del accounted for 5.1%, 0.8% and 0.8% mutation of alleles in exon 3, whereas EX6-96A > G accounted for 10.2% mutation of alleles in exon 6. R243Q had the highest incidence in exon 7 (12.7%, followed by Ivs7 +2T>A (5.1% and T278I (2.5%. G247V, R252Q, L255S, R261Q and E280K accounted for 0.8% while Y356X and V399V accounted for 5.9% and 5.1%, respectively, in exon 11. R413P and A434D accounted for 5.9% and 2.5%, respectively, in exon 12. Seventy-two variant alleles accounted for the 16 mutations observed here. The mutation characteristics and distributions demonstrated that EX6-96A > G and R243Q were the hot regions for mutations in the PAH gene in Shanxi patients with PKU.

Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors.

Science.gov (United States)

Aprelikova, Olga; Chandramouli, Gadisetti V R; Wood, Matthew; Vasselli, James R; Riss, Joseph; Maranchie, Jodi K; Linehan, W Marston; Barrett, J Carl

2004-06-01

Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation. Copyright 2004 Wiley-Liss, Inc.

A novel mutation in the sterol 27-hydroxylase gene of a woman with autosomal recessive cerebrotendinous xanthomatosis

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Garuti Rita

2010-10-01

Full Text Available Article abstract Mutations of the gene encoding the mitochondrial enzyme sterol 27-hydroxylase (CYP27A1 gene cause defects in the cholesterol pathway to bile acids that lead to the storage of cholestanol and cholesterol in tendons, lenses and the central nervous system. This disorder is the cause of a clinical syndrome known as cerebrotendinous xanthomatosis (CTX. Since 1991 several mutations of the CYP27A1 gene have been reported. We diagnosed the clinical features of CTX in a caucasian woman. Serum levels of cholestanol and 7α-hydroxycholesterol were elevated and the concentration of 27-hydroxycholesterol was reduced. Bile alcohols in the urine and faeces were increased. The analysis of the CYP27A1 gene showed that the patient was a compound heterozygote carrying two mutations both located in exon 8. One mutation is a novel four nucleotide deletion (c.1330-1333delTTCC that results in a frameshift and the occurrence of a premature stop codon leading to the formation of a truncated protein of 448 amino acids. The other mutation, previously reported, is a C - > T transition (c. c.1381C > T that converts the glutamine codon at position 461 into a termination codon (p.Q461X. These truncated proteins are expected to have no biological function being devoid of the cysteine residue at position 476 of the normal enzyme that is crucial for heme binding and enzyme activity.

Aquatic contaminants alter genes involved in neurotransmitter synthesis and gonadotropin release in largemouth bass

Energy Technology Data Exchange (ETDEWEB)

Martyniuk, Christopher J. [Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611 (United States); Sanchez, Brian C. [Department of Forestry and Natural Resources and School of Civil Engineering, 195 Marsteller St., Purdue University, West Lafayette, IN 47907 (United States); Szabo, Nancy J.; Denslow, Nancy D. [Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611 (United States); Sepulveda, Maria S., E-mail: mssepulv@purdue.edu [Department of Forestry and Natural Resources and School of Civil Engineering, 195 Marsteller St., Purdue University, West Lafayette, IN 47907 (United States)

2009-10-19

Many aquatic contaminants potentially affect the central nervous system, however the underlying mechanisms of how toxicants alter normal brain function are not well understood. The objectives of this study were to compare the effects of emerging and prevalent environmental contaminants on the expression of brain transcripts with a role in neurotransmitter synthesis and reproduction. Adult male largemouth bass (Micropterus salmoides) were injected once for a 96 h duration with control (water or oil) or with one of two doses of a single chemical to achieve the following body burdens ({mu}g/g): atrazine (0.3 and 3.0), toxaphene (10 and 100), cadmium (CdCl{sub 2}) (0.000067 and 0.00067), polychlorinated biphenyl (PCB) 126 (0.25 and 2.5), and phenanthrene (5 and 50). Partial largemouth bass gene segments were cloned for enzymes involved in neurotransmitter (glutamic acid decarboxylase 65, GAD65; tyrosine hydroxylase) and estrogen (brain aromatase; CYP19b) synthesis for real-time PCR assays. In addition, neuropeptides regulating feeding (neuropeptide Y) and reproduction (chicken GnRH-II, cGnRH-II; salmon GnRH, sGnRH) were also investigated. Of the chemicals tested, only cadmium, PCB 126, and phenanthrene showed any significant effects on the genes tested, while atrazine and toxaphene did not. Cadmium (0.000067 {mu}g/g) significantly increased cGnRH-II mRNA while PCB 126 (0.25 {mu}g/g) decreased GAD65 mRNA. Phenanthrene decreased GAD65 and tyrosine hydroxylase mRNA levels at the highest dose (50 {mu}g/g) but increased cGnRH-II mRNA at the lowest dose (5 {mu}g/g). CYP19b, NPY, and sGnRH mRNA levels were unaffected by any of the treatments. A hierarchical clustering dendrogram grouped PCB 126 and phenanthrene more closely than other chemicals with respect to the genes tested. This study demonstrates that brain transcripts important for neurotransmitter synthesis neuroendocrine function are potential targets for emerging and prevalent aquatic contaminants.

Aquatic contaminants alter genes involved in neurotransmitter synthesis and gonadotropin release in largemouth bass

International Nuclear Information System (INIS)

Martyniuk, Christopher J.; Sanchez, Brian C.; Szabo, Nancy J.; Denslow, Nancy D.; Sepulveda, Maria S.

2009-01-01

Many aquatic contaminants potentially affect the central nervous system, however the underlying mechanisms of how toxicants alter normal brain function are not well understood. The objectives of this study were to compare the effects of emerging and prevalent environmental contaminants on the expression of brain transcripts with a role in neurotransmitter synthesis and reproduction. Adult male largemouth bass (Micropterus salmoides) were injected once for a 96 h duration with control (water or oil) or with one of two doses of a single chemical to achieve the following body burdens (μg/g): atrazine (0.3 and 3.0), toxaphene (10 and 100), cadmium (CdCl 2 ) (0.000067 and 0.00067), polychlorinated biphenyl (PCB) 126 (0.25 and 2.5), and phenanthrene (5 and 50). Partial largemouth bass gene segments were cloned for enzymes involved in neurotransmitter (glutamic acid decarboxylase 65, GAD65; tyrosine hydroxylase) and estrogen (brain aromatase; CYP19b) synthesis for real-time PCR assays. In addition, neuropeptides regulating feeding (neuropeptide Y) and reproduction (chicken GnRH-II, cGnRH-II; salmon GnRH, sGnRH) were also investigated. Of the chemicals tested, only cadmium, PCB 126, and phenanthrene showed any significant effects on the genes tested, while atrazine and toxaphene did not. Cadmium (0.000067 μg/g) significantly increased cGnRH-II mRNA while PCB 126 (0.25 μg/g) decreased GAD65 mRNA. Phenanthrene decreased GAD65 and tyrosine hydroxylase mRNA levels at the highest dose (50 μg/g) but increased cGnRH-II mRNA at the lowest dose (5 μg/g). CYP19b, NPY, and sGnRH mRNA levels were unaffected by any of the treatments. A hierarchical clustering dendrogram grouped PCB 126 and phenanthrene more closely than other chemicals with respect to the genes tested. This study demonstrates that brain transcripts important for neurotransmitter synthesis neuroendocrine function are potential targets for emerging and prevalent aquatic contaminants.

Aquatic contaminants alter genes involved in neurotransmitter synthesis and gonadotropin release in largemouth bass.

Science.gov (United States)

Martyniuk, Christopher J; Sanchez, Brian C; Szabo, Nancy J; Denslow, Nancy D; Sepúlveda, Maria S

2009-10-19

Many aquatic contaminants potentially affect the central nervous system, however the underlying mechanisms of how toxicants alter normal brain function are not well understood. The objectives of this study were to compare the effects of emerging and prevalent environmental contaminants on the expression of brain transcripts with a role in neurotransmitter synthesis and reproduction. Adult male largemouth bass (Micropterus salmoides) were injected once for a 96 h duration with control (water or oil) or with one of two doses of a single chemical to achieve the following body burdens (microg/g): atrazine (0.3 and 3.0), toxaphene (10 and 100), cadmium (CdCl(2)) (0.000067 and 0.00067), polychlorinated biphenyl (PCB) 126 (0.25 and 2.5), and phenanthrene (5 and 50). Partial largemouth bass gene segments were cloned for enzymes involved in neurotransmitter (glutamic acid decarboxylase 65, GAD65; tyrosine hydroxylase) and estrogen (brain aromatase; CYP19b) synthesis for real-time PCR assays. In addition, neuropeptides regulating feeding (neuropeptide Y) and reproduction (chicken GnRH-II, cGnRH-II; salmon GnRH, sGnRH) were also investigated. Of the chemicals tested, only cadmium, PCB 126, and phenanthrene showed any significant effects on the genes tested, while atrazine and toxaphene did not. Cadmium (0.000067 microg/g) significantly increased cGnRH-II mRNA while PCB 126 (0.25 microg/g) decreased GAD65 mRNA. Phenanthrene decreased GAD65 and tyrosine hydroxylase mRNA levels at the highest dose (50 microg/g) but increased cGnRH-II mRNA at the lowest dose (5 microg/g). CYP19b, NPY, and sGnRH mRNA levels were unaffected by any of the treatments. A hierarchical clustering dendrogram grouped PCB 126 and phenanthrene more closely than other chemicals with respect to the genes tested. This study demonstrates that brain transcripts important for neurotransmitter synthesis neuroendocrine function are potential targets for emerging and prevalent aquatic contaminants.

Altered gene expression in pulmonary tissue of tryptophan hydroxylase-1 knockout mice: implications for pulmonary arterial hypertension.

Directory of Open Access Journals (Sweden)

Richard B Rothman

Full Text Available The use of fenfluramines can increase the risk of developing pulmonary arterial hypertension (PAH in humans, but the mechanisms responsible are unresolved. A recent study reported that female mice lacking the gene for tryptophan hydroxylase-1 (Tph1(-/- mice were protected from PAH caused by chronic dexfenfluramine, suggesting a pivotal role for peripheral serotonin (5-HT in the disease process. Here we tested two alternative hypotheses which might explain the lack of dexfenfluramine-induced PAH in Tph1(-/- mice. We postulated that: 1 Tph1(-/- mice express lower levels of pulmonary 5-HT transporter (SERT when compared to wild-type controls, and 2 Tph1(-/- mice display adaptive changes in the expression of non-serotonergic pulmonary genes which are implicated in PAH. SERT was measured using radioligand binding methods, whereas gene expression was measured using microarrays followed by quantitative real time PCR (qRT-PCR. Contrary to our first hypothesis, the number of pulmonary SERT sites was modestly up-regulated in female Tph1(-/- mice. The expression of 51 distinct genes was significantly altered in the lungs of female Tph1(-/- mice. Consistent with our second hypothesis, qRT-PCR confirmed that at least three genes implicated in the pathogenesis of PAH were markedly up-regulated: Has2, Hapln3 and Retlna. The finding that female Tph1(-/- mice are protected from dexfenfluramine-induced PAH could be related to compensatory changes in pulmonary gene expression, in addition to reductions in peripheral 5-HT. These observations emphasize the intrinsic limitation of interpreting data from studies conducted in transgenic mice that are not fully characterized.

Co-localization patterns of neurotensin receptor 1 and tyrosine hydroxylase in brain regions involved in motivation and social behavior in male European starlings.

Science.gov (United States)

Merullo, Devin P; Spool, Jeremy A; Zhao, Changjiu; Riters, Lauren V

2018-04-01

Animals communicate in distinct social contexts to convey information specific to those contexts, such as sexual or agonistic motivation. In seasonally-breeding male songbirds, seasonal changes in day length and increases in testosterone stimulate sexually-motivated song directed at females for courtship and reproduction. Dopamine and testosterone may act in the same brain regions to stimulate sexually-motivated singing. The neuropeptide neurotensin, acting at the neurotensin receptor 1 (NTR1), can strongly influence dopamine transmission. The goal of this study was to gain insight into the degree to which seasonal changes in physiology modify interactions between neurotensin and dopamine to adjust context-appropriate communication. Male European starlings were examined in physiological conditions that stimulate season-typical forms of communication: late summer/early fall non-breeding condition (low testosterone; birds sing infrequently), late fall non-breeding condition (low testosterone; birds produce non-sexually motivated song), and spring breeding condition (high testosterone; males produce sexually-motivated song). Double fluorescent immunolabeling was performed to detect co-localization patterns between tyrosine hydroxylase (TH; the rate-limiting enzyme in dopamine synthesis) and NTR1 in brain regions implicated in motivation and song production (the ventral tegmental area, medial preoptic nucleus, periaqueductal gray, and lateral septum). Co-localization between TH and NTR1 was present in the ventral tegmental area for all physiological conditions, and the number of co-localized cells did not differ across conditions. Immunolabeling for TH and NTR1 was also present in the other examined regions, although no co-localization was seen. These results support the hypothesis that interactions between NTR1 and dopamine in the ventral tegmental area may modulate vocalizations, but suggest that testosterone- or photoperiod-induced changes in NTR1/TH co

Associations between polymorphic variants of the tryptophan hydroxylase 2 gene and obsessive-compulsive disorder Associação entre polimorfismos do gene da triptofano hidroxilase 2 e o transtorno obsessivo-compulsivo

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Felipe Filardi da Rocha

2011-01-01

Full Text Available OBJECTIVE: A substantial body of evidence suggests that obsessive-compulsive disorder has a genetic component, and substantial candidate genes for the disorder have been investigated through association analyses. A particular emphasis has been placed on genes related to the serotonergic system, which is likely to play an important role in the pathogenesis of obsessive-compulsive disorder. The gene for tryptophan hydroxylase 2, which is a rate limiting enzyme in serotonin synthesis is considered an important candidate gene associated with psychiatric disorders. METHOD: Our sample consisted of 321 subjects (107 diagnosed with obsessive-compulsive disorder and 214 healthy controls, which were genotyped for eight tagSNPs (rs4448731, rs4565946, rs11179000, rs7955501, rs10506645, rs4760820, rs1487275 and rs10879357 covering the entire human tryptophan hydroxylase 2 gene. Statistical analyses were performed using UNPHASED, version 3.0.12, and Haploview ((R. RESULTS: Single markers, genotype analysis did not show a significant genetic association with obsessive-compulsive disorder. A significant association between the T-C-T (rs4448731, rs4565946, rs10506645 and C-A-T (rs4565946, rs7955501, rs10506645 haplotypes and obsessive-compulsive disorder was observed, as well as a strong linkage disequilibrium between SNPs rs4448731 and rs4565946, and SNPs rs10506645 and 4760820. DISCUSSION: Our research has not demonstrated the existence of associations between the eight SNPs of TPH2 and obsessive-compulsive disorder. However, two LD and two haplotypes areas were demonstrated, thus suggesting that more studies in TPH2 are needed to investigate the role of tryptophan hydroxylase 2 variants in obsessive-compulsive disorder.OBJETIVO: Diversos estudos demonstram que o transtorno obsessivo-compulsivo apresenta considerável contribuição genética, com diversos genes candidatos tendo sido estudados por meio de estudos de associação. Como alterações do sistema

Associations between polymorphic variants of the tryptophan hydroxylase 2 gene and obsessive-compulsive disorder Associação entre polimorfismos do gene da triptofano hidroxilase 2 e o transtorno obsessivo-compulsivo

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Felipe Filardi da Rocha

2011-06-01

Full Text Available OBJECTIVE: A substantial body of evidence suggests that obsessive-compulsive disorder has a genetic component, and substantial candidate genes for the disorder have been investigated through association analyses. A particular emphasis has been placed on genes related to the serotonergic system, which is likely to play an important role in the pathogenesis of obsessive-compulsive disorder. The gene for tryptophan hydroxylase 2, which is a rate limiting enzyme in serotonin synthesis, is considered an important candidate gene associated with psychiatric disorders. METHOD: Our sample consisted of 321 subjects (107 diagnosed with obsessive-compulsive disorder and 214 healthy controls, which were genotyped for eight tagSNPs (rs4448731, rs4565946, rs11179000, rs7955501, rs10506645, rs4760820, rs1487275 and rs10879357 covering the entire human tryptophan hydroxylase 2 gene. Statistical analyses were performed using UNPHASED, version 3.0.12, and Haploview®. RESULTS: Single markers, genotype analysis did not show a significant genetic association with obsessive-compulsive disorder. A significant association between the T-C-T (rs4448731, rs4565946, rs10506645 and C-A-T (rs4565946, rs7955501, rs10506645 haplotypes and obsessive-compulsive disorder was observed, as well as a strong linkage disequilibrium between SNPs rs4448731 and rs4565946, and SNPs rs10506645 and 4760820. DISCUSSION: Our research has not demonstrated the existence of associations between the eight SNPs of TPH2 and obsessive-compulsive disorder. However, two LD and two haplotypes areas were demonstrated, thus suggesting that more studies in TPH2 are needed to investigate the role of tryptophan hydroxylase 2 variants in obsessive-compulsive disorder.OBJETIVO: Diversos estudos demonstram que o transtorno obsessivo-compulsivo apresenta considerável contribuição genética, com diversos genes candidatos tendo sido estudados por meio de estudos de associação. Como alterações do sistema

Tyrosine phosphorylation in signal transduction

International Nuclear Information System (INIS)

Roberts, T.M.; Kaplan, D.; Morgan, W.; Keller, T.; Mamon, H.; Piwnica-Worms, H.; Druker, B.; Whitman, M.; Morrison, D.; Cohen, B.; Schaffhausen, B.; Cantley, L.; Rapp, U.

1988-01-01

Recent work has focused on the elucidation of the mechanisms by which membrane-bound tyrosine kinases transmit signals within the cell. To examine the role of tyrosine phosphorylation the authors have employed the following strategy. First, they have utilized antibodies to phosphotyrosine (anti-P.Tyr) to identify candidate substrates of various tyrosine kinases, such as pp60 c-src , the CSF- receptor, or the platelet-derived growth factor (PDGF) receptor. Second, they have attempted to characterize the biochemical properties of the putative substrates and to determine in what manner these properties are modified by phosphorylation on tyrosine residues. In this endeavor, they are recapitulating the classic biochemical analysis used to study the effect of kinases on metabolism. The final portion of our work consists of using modern molecular biological strategies to clone the genes or cDNAs for the substrates and overproduce the relevant proteins for studies in vitro in defined systems. This paper describes the first and second aspects of this strategy, the identification and characterization of novel substrate molecules

Modular Engineering of l-Tyrosine Production in Escherichia coli

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Juminaga, Darmawi; Baidoo, Edward E. K.; Redding-Johanson, Alyssa M.; Batth, Tanveer S.; Burd, Helcio; Mukhopadhyay, Aindrila; Petzold, Christopher J.

2012-01-01

Efficient biosynthesis of l-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for l-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to l-tyrosine on two plasmids. Rational engineering to improve l-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to l-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter l-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways. PMID:22020510

P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia

International Nuclear Information System (INIS)

Matteson, K.J.; Phillips, J.A. III; Miller, W.L.; Chung, B.C.; Orlando, P.J.; Frisch, H.; Ferrandez, A.; Burr, I.M.

1987-01-01

Congenital adrenal hyperplasia (CAH) is a common genetic disorder due to defective 21-hydroxylation of steroid hormones. The human P450XXIA2 gene encodes cytochrome P450c21 [steroid 21-monooxygenase (steroid 21-hydroxylase)], which mediates 21-hydroxylation. The P450XXIA2 gene may be distinguished from the duplicated P450XXIA1 pseudogene by cleavage with the restriction endonuclease Taq I, with the XXIA2 gene characterized by a 3.7-kilobase (kb) fragment and the XXIA1 pseudogene characterized by a 3.2-kb fragment. Restriction endonuclease mapping by several laboratories has suggested that deletion of the P450XXIA2 gene occurs in about 25% of patients with CAH, as their genomic DNA lacks detectable 3.7-kb Taq I fragments. The authors have cloned human P450c21 cDNA and used it to study genomic DNA prepared from 51 persons in 10 families, each of which includes 2 or more persons with CAH. After Taq I digestion, apparent deletions are seen in 7 of the 20 alleles of the probands; using EcoRI, apparent deletions are seen in 9 of the 20 alleles. However, the apparently deleted alleles seen with Taq I do not coincide with those seen with EcoRI. Furthermore, studies with Bgl II, EcoRI, Kpn I, and Xba I yield normal patterns with at least two enzymes in all cases. Since all probands yielded normal patterns with at least two of the five enzymes used, they conclude that the P450XXIA2 gene deletions widely reported in CAH patients probably represent gene conversions, unequal crossovers,or polymorphisms rather than simple gene deletions

Roles of the tyrosine isomers meta-tyrosine and ortho-tyrosine in oxidative stress.

Science.gov (United States)

Ipson, Brett R; Fisher, Alfred L

2016-05-01

The damage to cellular components by reactive oxygen species, termed oxidative stress, both increases with age and likely contributes to age-related diseases including Alzheimer's disease, atherosclerosis, diabetes, and cataract formation. In the setting of oxidative stress, hydroxyl radicals can oxidize the benzyl ring of the amino acid phenylalanine, which then produces the abnormal tyrosine isomers meta-tyrosine or ortho-tyrosine. While elevations in m-tyrosine and o-tyrosine concentrations have been used as a biological marker of oxidative stress, there is emerging evidence from bacterial, plant, and mammalian studies demonstrating that these isomers, particularly m-tyrosine, directly produce adverse effects to cells and tissues. These new findings suggest that the abnormal tyrosine isomers could in fact represent mediators of the effects of oxidative stress. Consequently the accumulation of m- and o-tyrosine may disrupt cellular homeostasis and contribute to disease pathogenesis, and as result, effective defenses against oxidative stress can encompass not only the elimination of reactive oxygen species but also the metabolism and ultimately the removal of the abnormal tyrosine isomers from the cellular amino acid pool. Future research in this area is needed to clarify the biologic mechanisms by which the tyrosine isomers damage cells and disrupt the function of tissues and organs and to identify the metabolic pathways involved in removing the accumulated isomers after exposure to oxidative stress. Published by Elsevier B.V.

RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua

OpenAIRE

Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

2016-01-01

Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of t...

[Cloning and bioinformatics analysis of abscisic acid 8'-hydroxylase from Pseudostellariae Radix].

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Li, Jun; Long, Deng-Kai; Zhou, Tao; Ding, Ling; Zheng, Wei; Jiang, Wei-Ke

2016-07-01

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress. Copyright© by the Chinese Pharmaceutical Association.

Differential in vivo regulation of TH and DBH mRNA in rat atria by maprotiline and fluoxetine

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Spasojević Nataša

2011-01-01

Full Text Available It is well known that antidepressants affect central monoaminergic neurotransmission and that they also modulate hormone release in peripheral tissues. Repeated maprotiline (a noradrenaline reuptake inhibitor and fluoxetine (a serotonin reuptake inhibitor treatment on gene expression of the catecholamine biosynthetic enzymes were examined in rat atria and ventricles in vivo. Maprotiline decreased the gene expression of tyrosine hydroxylase (TH and dopamineβ-hydroxylase (DBH in the rat atrium. Fluoxetine increased gene expression of TH and DBH, but not of phenylethanolamine N-methyltransferase (PNMT. Chronic application of antidepressants did not change the expression of these enzymes in the ventricles. We conclude that repeated administration of fluoxetine enhances gene transcription of TH and DBH and subsequently stimulates noradrenaline synthesis in rat atria in vivo.

Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

International Nuclear Information System (INIS)

Naranjo, J.R.; Mocchetti, I.; Schwartz, J.P.; Costa, E.

1986-01-01

In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 μM) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 μM veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 μM dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 μM) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated

Carotenoid β-Ring Hydroxylase and Ketolase from Marine Bacteria—Promiscuous Enzymes for Synthesizing Functional Xanthophylls

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Misawa, Norihiko

2011-01-01

Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C40-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′)-ketolase (4(4′)-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3′)-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′)-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2′)-hydroxylated carotenoids). PMID:21673887

Carotenoid β-Ring Hydroxylase and Ketolase from Marine Bacteria—Promiscuous Enzymes for Synthesizing Functional Xanthophylls

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Norihiko Misawa

2011-05-01

Full Text Available Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C40-type dicyclic carotenoids containing two β-end groups (β rings that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′-ketolase (4(4′-oxygenase; CrtW and hydroxylated by carotenoid β-ring 3(3′-hydroxylase (CrtZ. In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′-hydroxylase (CrtG. This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s-2(2′-hydroxylated carotenoids.

Carotenoid β-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls.

Science.gov (United States)

Misawa, Norihiko

2011-01-01

Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C₄₀-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4')-ketolase (4(4')-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3')-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2')-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2')-hydroxylated carotenoids).

Ethnic disparity in 21-hydroxylase gene mutations identified in Pakistani congenital adrenal hyperplasia patients

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Jabbar Abdul

2011-02-01

Full Text Available Abstract Background Congenital adrenal hyperplasia (CAH is a group of autosomal recessive disorders caused by defects in the steroid 21 hydroxylase gene (CYP21A2. We studied the spectrum of mutations in CYP21A2 gene in a multi-ethnic population in Pakistan to explore the genetics of CAH. Methods A cross sectional study was conducted for the identification of mutations CYP21A2 and their phenotypic associations in CAH using ARMS-PCR assay. Results Overall, 29 patients were analyzed for nine different mutations. The group consisted of two major forms of CAH including 17 salt wasters and 12 simple virilizers. There were 14 phenotypic males and 15 females representing all the major ethnic groups of Pakistan. Parental consanguinity was reported in 65% cases and was equally distributed in the major ethnic groups. Among 58 chromosomes analyzed, mutations were identified in 45 (78.6% chromosomes. The most frequent mutation was I2 splice (27% followed by Ile173Asn (26%, Arg 357 Trp (19%, Gln319stop, 16% and Leu308InsT (12%, whereas Val282Leu was not observed in this study. Homozygosity was seen in 44% and heterozygosity in 34% cases. I2 splice mutation was found to be associated with SW in the homozygous. The Ile173Asn mutation was identified in both SW and SV forms. Moreover, Arg357Trp manifested SW in compound heterozygous state. Conclusion Our study showed that CAH exists in our population with ethnic difference in the prevalence of mutations examined.

Genotyping of the 19-bp insertion/deletion polymorphism in the 5' flank of beta-hydroxylase gene by dissociation analysis of allele-specific PCR products

DEFF Research Database (Denmark)

Rasmussen, Henrik Berg; Werge, Thomas

2005-01-01

The 19-bp insertion/deletion polymorphism in the 5' flank of the dopamine beta-hydroxylase (DBH) gene has been associated with psychiatric disorders. We have developed a simple, reliable and inexpensive closed-tube assay for genotyping of this polymorphism based upon T(m) determination of amplified...... and a conventional approach based upon agarose gel electrophoresis of amplified fragments revealed complete concordance between the two procedures. The insights obtained in this study may be utilized to develop assays based upon dissociation analysis of PCR products for genotyping of other insertion...

24-Hydroxylase in Cancer: Impact on Vitamin D-based Anticancer Therapeutics

Science.gov (United States)

Luo, Wei; Hershberger, Pamela A.; Trump, Donald L.; Johnson, Candace S.

2013-01-01

The active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in regulating calcium homeostasis and bone mineralization. 1,25(OH)2D3 also modulates cellular proliferation and differentiation in a variety of cell types. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme which converts 1,25(OH)2D3 to less active calcitroic acid. Nearly all cell types express 24-hydroxylase, the highest activity being observed in the kidney. There is increasing evidence linking the incidence and prognosis of certain cancers to low serum 25 (OH)D3 levels and high expression of vitamin D 24-hydroxylase supporting the idea that elevated CYP24A1 expression may stimulate degradation of vitamin D metabolites including 25-(OH)D3 and 1,25(OH)2D3. The over expression of CYP24A1 in cancer cells may be a factor affecting 1,25(OH)2D3 bioavailability and anti-proliferative activity pre-clinically and clinically. The combination of 1,25(OH)2D3 with CYP24A1 inhibitors enhances 1,25(OH)2D3 mediated signaling and anti-proliferative effects and may be useful in overcoming effects of aberrant CYP24 expression. PMID:23059474

Dectin-1-mediated signaling leads to characteristic gene expressions and cytokine secretion via spleen tyrosine kinase (Syk) in rat mast cells.

Science.gov (United States)

Kimura, Yukihiro; Chihara, Kazuyasu; Honjoh, Chisato; Takeuchi, Kenji; Yamauchi, Shota; Yoshiki, Hatsumi; Fujieda, Shigeharu; Sada, Kiyonao

2014-11-07

Dectin-1 recognizes β-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 domain of Syk. These responses depended on tyrosine phosphorylation of the hemi-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of Dectin-1, whereas they were independent of the γ-subunit of high-affinity IgE receptor. DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1, IL-3, IL-4, IL-13, and tumor necrosis factor (TNF)-α. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by FcϵRI activation. In addition, these gene expressions induced by curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the antifungal immunity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Presence of corticotrophin-releasing factor and/or tyrosine hydroxylase in cells of a neural brain-testicular pathway that are labelled by a transganglionic tracer.

Science.gov (United States)

James, P; Rivier, C; Lee, S

2008-02-01

Our laboratory has shown that male testosterone levels are not solely controlled by the release of hypothalamic gonadotrophin-releasing hormone and pituitary luteinising hormone, but are also regulated by a multisynaptic pathway connecting the brain and the testis that interferes with the testosterone response to gonadotrophins. This pathway, which is independent of the pituitary gland, is activated by an i.c.v. injection of either the stress-related peptide corticotrophin-releasing factor (CRF) or of beta-adrenoceptor agonists, both of which alter androgen release and decrease levels of the peripheral-type benzodiazepine receptor and the steroidogenic acute regulatory protein within Leydig cells. Our original studies used the retrograde transganglionic tracer pseudorabies virus (PRV) to map progression of the virus from the testes to upper brain levels. The present study aimed to extend this work by identifying the regions where CRF and catecholamine neurones represented components of the stress-activated, brain-testicular pathway that prevents testosterone increases. To this end, anaesthetised adult male rats received an intra-testicular injection of PRV. Using immunofluorescence, we identified co-labelling of PRV and either CRF or tyrosine hydroxylase (TH), the enzyme responsible for biogenic amine synthesis. Co-labelling of PRV and CRF was found in the bed nucleus of the stria terminalis, the paraventricular nucleus of the hypothalamus (PVN) and the central amygdala. Co-labelling of PRV and TH was found in the PVN, substantia nigra, A7/Kölliker-Fuse area, area of A5, locus coeruleus, nucleus of solitary tract, area of C3, area of C2 and the area of C1/A1. These results indicate that most cell groups of the ventral noradrenergic pathway have neurones that are a part of the brain-testicular pathway. This suggests that the stress hormones CRF and catecholamines may act as neurotransmitters that signal the pathway to inhibit increases in plasma testosterone levels.

Hypoxic-ischemic injury decreases anxiety-like behavior in rats when associated with loss of tyrosine-hydroxylase immunoreactive neurons of the substantia nigra

International Nuclear Information System (INIS)

Hei, Ming-Yan; Luo, Ya-Li; Zhang, Xiao-Chun; Liu, Hong; Gao, Ru; Wu, Jing-Jiang

2011-01-01

Neonatal Sprague-Dawley rats were randomly divided into normal control, mild hypoxia-ischemia (HI), and severe HI groups (N = 10 in each group at each time) on postnatal day 7 (P7) to study the effect of mild and severe HI on anxiety-like behavior and the expression of tyrosine hydroxylase (TH) in the substantia nigra (SN). The mild and severe HI groups were exposed to hypoxia (8% O 2 /92% N 2 ) for 90 and 150 min, respectively. The elevated plus-maze (EPM) test was performed to assess anxiety-like behavior by measuring time spent in the open arms (OAT) and OAT%, and immunohistochemistry was used to determine the expression of TH in the SN at P14, P21, and P28. OAT and OAT% in the EPM were significantly increased in both the mild (1.88-, 1.99-, and 2.04-fold, and 1.94-, 1.51-, and 1.46-fold) and severe HI groups (1.69-, 1.68-, and 1.87-fold, and 1.83-, 1.43-, and 1.39-fold, respectively; P < 0.05). The percent of TH-positive cells occupying the SN area was significantly and similarly decreased in both the mild (17.7, 40.2, and 47.2%) and severe HI groups (16.3, 32.2, and 43.8%, respectively; P < 0.05). The decrease in the number of TH-positive cells in the SN and the level of protein expression were closely associated (Pearson correlation analysis: r = 0.991, P = 0.000 in the mild HI group and r = 0.974, P = 0.000 in the severe HI group) with the impaired anxiety-like behaviors. We conclude that neonatal HI results in decreased anxiety-like behavior during the juvenile period of Sprague-Dawley rats, which is associated with the decreased activity of TH in the SN. The impairment of anxiety and the expression of TH are not likely to be dependent on the severity of HI

Hypoxic-ischemic injury decreases anxiety-like behavior in rats when associated with loss of tyrosine-hydroxylase immunoreactive neurons of the substantia nigra

Directory of Open Access Journals (Sweden)

Hei Ming-Yan

2012-01-01

Full Text Available Neonatal Sprague-Dawley rats were randomly divided into normal control, mild hypoxia-ischemia (HI, and severe HI groups (N = 10 in each group at each time on postnatal day 7 (P7 to study the effect of mild and severe HI on anxiety-like behavior and the expression of tyrosine hydroxylase (TH in the substantia nigra (SN. The mild and severe HI groups were exposed to hypoxia (8% O2/92% N2 for 90 and 150 min, respectively. The elevated plus-maze (EPM test was performed to assess anxiety-like behavior by measuring time spent in the open arms (OAT and OAT%, and immunohistochemistry was used to determine the expression of TH in the SN at P14, P21, and P28. OAT and OAT% in the EPM were significantly increased in both the mild (1.88-, 1.99-, and 2.04-fold, and 1.94-, 1.51-, and 1.46-fold and severe HI groups (1.69-, 1.68-, and 1.87-fold, and 1.83-, 1.43-, and 1.39-fold, respectively; P < 0.05. The percent of TH-positive cells occupying the SN area was significantly and similarly decreased in both the mild (17.7, 40.2, and 47.2% and severe HI groups (16.3, 32.2, and 43.8%, respectively; P < 0.05. The decrease in the number of TH-positive cells in the SN and the level of protein expression were closely associated (Pearson correlation analysis: r = 0.991, P = 0.000 in the mild HI group and r = 0.974, P = 0.000 in the severe HI group with the impaired anxiety-like behaviors. We conclude that neonatal HI results in decreased anxiety-like behavior during the juvenile period of Sprague-Dawley rats, which is associated with the decreased activity of TH in the SN. The impairment of anxiety and the expression of TH are not likely to be dependent on the severity of HI.

Molecular Characterization of Ferulate 5-Hydroxylase Gene from Kenaf (Hibiscus cannabinus L.

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Jonggeun Kim

2013-01-01

Full Text Available The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.. Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84, is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF encoding 518 amino acids (GenBank Accession number JX524278. The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78% with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h, wounding (24 h, cold (24 h, abscisic acid (24 h, and methyl jasmonate (24 h.

Muscle-directed gene therapy for phenylketonuria (PKU): Development of transgenic mice with muscle-specific phenylalanine hydroxylase expression

Energy Technology Data Exchange (ETDEWEB)

Harding, C.O.; Messing, A.; Wolff, J.A. [Univ. of Wisconsin, Madison, WI (United States)

1994-09-01

Phenylketonuria (PKU) is an attractive target for gene therapy because of shortcomings in current therapy including lifelong commitment to a difficult and expensive diet, persistent mild cognitive deficits in some children despite adequate dietary therapy, and maternal PKU syndrome. Phenylalanine hydroxylase (PAH) is normally expressed only in liver, but we propose to treat PKU by introducing the gene for PAH into muscle. In order to evaluate both the safety and efficacy of this approach, we have a developed a trangenic mouse which expresses PAH in both cardiac and skeletal muscle. The transgene includes promoter and enhancer sequences from the mouse muscle creatine kinase (MCK) gene fused to the mouse liver PAH cDNA. Mice which have inherited the transgene are healthy, active, and do not exhibit any signs of muscle weakness or wasting. Ectopic PAH expression in muscle is not detrimental to the health, neurologic function, or reproduction of the mice. Pah{sup enu2} hyperphenylalaninemic mice, a model of human PAH deficiency, bred to carry the transgene have substantial PAH expression in cardiac and skeletal muscle but none in liver. Muscle PAH expression alone does not complement the hyperphenylalaninemic phenotype of Pah{sup enu2} mice. However, administration of reduced tetrahydrobiopterin to transgenic Pah{sup enu2} mice is associated with a 25% mean decrease in serum phenylalanine levels. We predict that ectopic expression of PAH in muscle along with adequate muscle supplies of reduced biopterin cofactor will decrease hyperphenylalaninemia in PKU.

Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

DEFF Research Database (Denmark)

Mijakovic, Ivan; Petranovic, Dina; Macek, B

2006-01-01

for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (Sc......SSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically...... by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis

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Marta Perez

2017-11-01

Full Text Available Enterococci are considered mainly responsible for the undesirable accumulation of the biogenic amines tyramine and putrescine in cheeses. The biosynthesis of tyramine and putrescine has been described as a species trait in Enterococcus faecalis. Tyramine is formed by the decarboxylation of the amino acid tyrosine, by the tyrosine decarboxylase (TDC route encoded in the tdc cluster. Putrescine is formed from agmatine by the agmatine deiminase (AGDI pathway encoded in the agdi cluster. These biosynthesis routes have been independently studied, tyrosine and agmatine transcriptionally regulate the tdc and agdi clusters. The objective of the present work is to study the possible co-regulation among TDC and AGDI pathways in E. faecalis. In the presence of agmatine, a positive correlation between putrescine biosynthesis and the tyrosine concentration was found. Transcriptome studies showed that tyrosine induces the transcription of putrescine biosynthesis genes and up-regulates pathways involved in cell growth. The tyrosine modulation over AGDI route was not observed in the mutant Δtdc strain. Fluorescence analyses using gfp as reporter protein revealed PaguB (the promoter of agdi catabolic genes was induced by tyrosine in the wild-type but not in the mutant strain, confirming that tdc cluster was involved in the tyrosine induction of putrescine biosynthesis. This study also suggests that AguR (the transcriptional regulator of agdi was implicated in interaction among the two clusters.

Cloning and expression analysis of tyrosine hydroxylase and changes in catecholamine levels in brain during ontogeny and after sex steroid analogues exposure in the catfish, Clarias batrachus.

Science.gov (United States)

Mamta, Sajwan Khatri; Raghuveer, Kavarthapu; Sudhakumari, Cheni-Chery; Rajakumar, Anbazhagan; Basavaraju, Yaraguntappa; Senthilkumaran, Balasubramanian

2014-02-01

Tyrosine hydroxylase (Th) is the rate-limiting enzyme for catecholamine (CA) biosynthesis and is considered to be a marker for CA-ergic neurons, which regulate the levels of gonadotropin-releasing hormone in brain and gonadotropins in the pituitary. In the present study, we cloned full-length cDNA of Th from the catfish brain and evaluated its expression pattern in the male and female brain during early development and after sex-steroid analogues treatment using quantitative real-time PCR. We measured the CA levels to compare our results on Th. Cloned Th from catfish brain is 1.591 kb, which encodes a putative protein of 458 amino acid residues and showed high homology with other teleosts. The tissue distribution of Th revealed ubiquitous expression in all the tissues analyzed with maximum expression in male and female brain. Copy number analysis showed two-fold more transcript abundance in the female brain when compared with the male brain. A differential expression pattern of Th was observed in which the mRNA levels were significantly higher in females compared with males, during early brain development. CAs, l-3,4-dihydroxyphenylalanine, dopamine, and norepinephrine levels measured using high-performance liquid chromatography with electrochemical detection in the developing male and female brain confirmed the prominence of the CA-ergic system in the female brain. Sex-steroid analogue treatment using methyltestosterone and ethinylestradiol confirmed our findings of the differential expression of Th related to CA levels. Copyright © 2013 Elsevier Inc. All rights reserved.

Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

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Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

1992-01-01

The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

Comparative transcriptomic analyses of differentially expressed genes in transgenic melatonin biosynthesis ovine HIOMT gene in switchgrass

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Shan Yuan

2016-11-01

Full Text Available Melatonin serves pleiotropic functions in prompting plant growth and resistance to various stresses. The accurate biosynthetic pathway of melatonin remains elusive in plant species, while the N-acetyltransferase and O-methyltransferase were considered to be the last two key enzymes during its biosynthesis. To investigate the biosynthesis and metabolic pathway of melatonin in plants, the RNA-seq profile of overexpression of the ovine HIOMT was analyzed and compared with the previous transcriptome of transgenic oAANAT gene in switchgrass, a model plant for cellulosic ethanol production. A total of 946, 405 and 807 differentially expressed unigenes were observed in AANAT vs. control, HIOMT vs. control, and AANAT vs. HIOMT, respectively. The significantly upregulated (F-box/kelch-repeat protein, zinc finger BED domain-containing protein-3 genes were consistent with enhanced phenotypes of shoot, stem and root growth in transgenic oHIOMT switchgrass. Early flowering in overexpression of oHIOMT switchgrass involved in the regulation of flowering-time genes (APETALA2. Several stress resistant related genes (SPX domain-containing membrane protein, copper transporter 1, late blight resistance protein homolog R1A-6 OS etc. were specifically and significantly upregulated in transgenic oHIOMT only, while metabolism-related genes (phenylalanine-4-hydroxylase, tyrosine decarboxylase 1, protein disulfide-isomerase and galactinol synthase 2 etc. were significantly upregulated in transgenic oAANAT only. These results provide new sights into the biosynthetic and physiological functional networks of melatonin in plants.

Dopamine and Mushroom Bodies in Drosophila: Experience-Dependent and -Independent Aspects of Sexual Behavior

Science.gov (United States)

Neckameyer, Wendi S.

1998-01-01

Depletion of dopamine in Drosophila melanogaster adult males, accomplished through systemic introduction of the tyrosine hydroxylase inhibitor 3-iodo-tyrosine, severely impaired the ability of these flies to modify their courtship responses to immature males. Mature males, when first exposed to immature males, will perform courtship rituals; the intensity and duration of this behavior rapidly diminshes with time. Dopamine is also required for normal female sexual receptivity; dopamine-depleted females show increased latency to copulation. One kilobase of 5′ upstream information from the Drosophila tyrosine hydroxylase (DTH) gene, when fused to the Escherichia coli β-galactosidase reporter and transduced into the genome of Drosophila melanogaster, is capable of directing expression of the reporter gene in the mushroom bodies, which are believed to mediate learning acquisition and memory retention in flies. Ablation of mushroom bodies by treatment of newly hatched larva with hydroxyurea resulted in the inability of treated mature adult males to cease courtship when placed with untreated immature males. However, functional mushroom bodies were not required for the dopaminergic modulation of an innate behavior, female sexual receptivity. These data suggest that dopamine acts as a signaling molecule within the mushroom bodies to mediate a simple form of learning. PMID:10454380

Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice.

Science.gov (United States)

Lewandowski, M; Chui, Y C; Levi, P; Hodgson, E

1991-02-01

A simple and sensitive method for the separation of 14C-labelled acetanilide, 4-hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBA2/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [14C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.

Involvement of tryptophan hydroxylase 2 gene polymorphisms in susceptibility to tic disorder in Chinese Han population

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Zheng Ping

2013-01-01

Full Text Available Abstract Background Tryptophan hydroxylase-2 (TPH2 is a potential candidate gene for screening tic disorder (TD. Methods A case–control study was performed to examine the association between the TPH2 gene and TD. The Sequenom® Mass ARRAY iPLEX GOLD System was used to genotype two single nucleotide polymorphisms (SNPs of the TPH2 gene in 149 TD children and in 125 normal controls. Results For rs4565946, individuals with the TT genotype showed a significantly higher risk of TD than those with TC plus CC genotypes [odds ratio (OR =3.077, 95% confidence interval (CI: 1.273–7.437; P = 0.009], as did male TD children with the TT genotype (OR = 3.228, 95% CI: 1.153–9.040; P = 0.020. The G allele of rs4570625 was significantly more frequent in TD children with higher levels of tic symptoms (Yale Global Tic Severity Scale, YGTSS than those in controls among the male children (OR = 1.684, 95%: 1.097–2.583; P = 0.017]. TD children with severe tic symptoms had significantly higher frequencies of rs4546946 TT genotype than did normal controls in boys (OR = 3.292, 95% CI: 1.139–9.513; P = 0.022. We also found that genotype distributions of both SNPs were different between the Asian and European populations. Conclusions Our results indicated that the TT genotype of rs4565946 is a potential genetic risk factor for TD, and the allele G of rs4570625 might be associated with the severity of tic symptoms in boys. These polymorphisms might be susceptibility loci for TD in the Chinese Han population. Because of the confounding of co-existing attention deficit hyperactivity disorder (ADHD,these findings need to be confirmed by studies in much larger samples.

Involvement of tryptophan hydroxylase 2 gene polymorphisms in susceptibility to tic disorder in Chinese Han population.

Science.gov (United States)

Zheng, Ping; Li, Erzhen; Wang, Jianhua; Cui, Xiaodai; Wang, Liwen

2013-01-29

Tryptophan hydroxylase-2 (TPH2) is a potential candidate gene for screening tic disorder (TD). A case-control study was performed to examine the association between the TPH2 gene and TD. The Sequenom® Mass ARRAY iPLEX GOLD System was used to genotype two single nucleotide polymorphisms (SNPs) of the TPH2 gene in 149 TD children and in 125 normal controls. For rs4565946, individuals with the TT genotype showed a significantly higher risk of TD than those with TC plus CC genotypes [odds ratio (OR) =3.077, 95% confidence interval (CI): 1.273-7.437; P = 0.009], as did male TD children with the TT genotype (OR = 3.228, 95% CI: 1.153-9.040; P = 0.020). The G allele of rs4570625 was significantly more frequent in TD children with higher levels of tic symptoms (Yale Global Tic Severity Scale, YGTSS) than those in controls among the male children (OR = 1.684, 95%: 1.097-2.583; P = 0.017]. TD children with severe tic symptoms had significantly higher frequencies of rs4546946 TT genotype than did normal controls in boys (OR = 3.292, 95% CI: 1.139-9.513; P = 0.022). We also found that genotype distributions of both SNPs were different between the Asian and European populations. Our results indicated that the TT genotype of rs4565946 is a potential genetic risk factor for TD, and the allele G of rs4570625 might be associated with the severity of tic symptoms in boys. These polymorphisms might be susceptibility loci for TD in the Chinese Han population. Because of the confounding of co-existing attention deficit hyperactivity disorder (ADHD),these findings need to be confirmed by studies in much larger samples.

Electrode Potentials of l-Tryptophan, l-Tyrosine, 3-Nitro-l-tyrosine, 2,3-Difluoro-l-tyrosine, and 2,3,5-Trifluoro-l-tyrosine.

Science.gov (United States)

Mahmoudi, Leila; Kissner, Reinhard; Nauser, Thomas; Koppenol, Willem H

2016-05-24

Electrode potentials for aromatic amino acid radical/amino acid couples were deduced from cyclic voltammograms and pulse radiolysis experiments. The amino acids investigated were l-tryptophan, l-tyrosine, N-acetyl-l-tyrosine methyl ester, N-acetyl-3-nitro-l-tyrosine ethyl ester, N-acetyl-2,3-difluoro-l-tyrosine methyl ester, and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester. Conditional potentials were determined at pH 7.4 for all compounds listed; furthermore, Pourbaix diagrams for l-tryptophan, l-tyrosine, and N-acetyl-3-nitro-l-tyrosine ethyl ester were obtained. Electron transfer accompanied by proton transfer is reversible, as confirmed by detailed analysis of the current waves, and because the slopes of the Pourbaix diagrams obey Nernst's law. E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) at pH 7 are 0.99 ± 0.01 and 0.97 ± 0.01 V, respectively. Pulse radiolysis studies of two dipeptides that contain both amino acids indicate a difference in E°' of approximately 0.06 V. Thus, in small peptides, we recommend values of 1.00 and 0.96 V for E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH), respectively. The electrode potential of N-acetyl-3-nitro-l-tyrosine ethyl ester is higher, while because of mesomeric stabilization of the radical, those of N-acetyl-2,3-difluoro-l-tyrosine methyl ester and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester are lower than that of tyrosine. Given that the electrode potentials at pH 7 of E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) are nearly equal, they would be, in principle, interchangeable. Proton-coupled electron transfer pathways in proteins that use TrpH and TyrOH are thus nearly thermoneutral.

The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver.

OpenAIRE

Taylor, D S; Dahl, H H; Mercer, J F; Green, A K; Fisher, M J

1989-01-01

The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabe...

From the Cover: Prenatal Nicotinic Exposure Attenuates Respiratory Chemoreflexes Associated With Downregulation of Tyrosine Hydroxylase and Neurokinin 1 Receptor in Rat Pup Carotid Body.

Science.gov (United States)

Zhao, Lei; Zhuang, Jianguo; Gao, Xiuping; Ye, Chunyan; Lee, Lu-Yuan; Xu, Fadi

2016-09-01

Maternal cigarette smoke is the major risk of sudden infant death syndrome (SIDS). A depressed ventilatory response to hypoxia (HVR) and hypercapnia (HCVR) is thought to be responsible for the pathogenesis of SIDS and the carotid body is critically involved in these responses. We have recently reported that prenatal nicotinic exposure (PNE) over the full gestation induces depressed HVR in rat pups. Here, we asked whether PNE (1) depressed not only HVR but also HCVR that were dependent on the carotid body, (2) affected some important receptors and neurochemicals expressed in the carotid body, such as tyrosine hydroxylase (TH), neurokinin-1 receptor (NK1R), and α7 nicotinic acetylcholine receptor (α7nAChR), and (3) blunted the ventilatory responses to activation of these receptors. To this end, HVR and HCVR in Ctrl and PNE pups were measured with plethysmography before and after carotid body ablation (Series I), mRNA expression and/or immunoreactivity (IR) of TH, NK1R, and α7nAChR in the carotid body were examined by RT-PCR and immunohistochemistry (Series II), and the ventilatory responses were tested before and after intracarotid injection of substance P (NK1R agonist) and AR-R17779 (α7nAChR agonist) (Series III). Our results showed that PNE (1) significantly depressed both HVR and HCVR and these depressions were abolished by carotid body ablation, (2) reduced the relative population of glomus cells, mRNA NK1R, and α7nAChR and IR of NK1R and TH in the carotid body, and (3) decreased ventilatory responses to intracarotid injection of substance P or AR-R17779. These results suggest that PNE acting via the carotid body could strikingly blunt HVR and HCVR, likely through downregulating TH and NK1R. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Species differences in the immunoreactive expression of oxytocin, vasopressin, tyrosine hydroxylase and estrogen receptor alpha in the brain of Mongolian gerbils (Meriones unguiculatus and Chinese striped hamsters (Cricetulus barabensis.

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Yu Wang

Full Text Available Species differences in neurochemical expression and activity in the brain may play an important role in species-specific patterns of social behavior. In the present study, we used immunoreactive (ir labeling to compare the regional density of cells containing oxytocin (OT, vasopressin (AVP, tyrosine hydroxylase (TH, or estrogen receptor alpha (ERα staining in the brains of social Mongolian gerbils (Meriones unguiculatus and solitary Chinese striped hamsters (Cricetulus barabensis. Multiple region- and neurochemical-specific species differences were found. In the anterior hypothalamus (AH, Mongolian gerbils had higher densities of AVP-ir and ERα-ir cells than Chinese striped hamsters. In the lateral hypothalamus (LH, Mongolian gerbils also had higher densities of AVP-ir and TH-ir cells, but a lower density of OT-ir cells, than Chinese striped hamsters. Furthermore, in the anterior nucleus of the medial preoptic area (MPOAa, Mongolian gerbils had higher densities of OT-ir and AVP-ir cells than Chinese striped hamsters, and an opposite pattern was found in the posterior nucleus of the MPOA (MPOAp. Some sex differences were also observed. Females of both species had higher densities of TH-ir cells in the MPOAa and of OT-ir cells in the intermediate nucleus of the MPOA (MPOAi than males. Given the role of these neurochemicals in social behaviors, our data provide additional evidence to support the notion that species-specific patterns of neurochemical expression in the brain may be involved in species differences in social behaviors associated with different life strategies.

Tyrosine content, influx and accumulation rate, and catecholamine biosynthesis measured in vivo, in the central nervous system and in peripheral organs of the young rat. Influence of neonatal hypo- and hyperthyroidism.

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Diarra, A; Lefauconnier, J M; Valens, M; Georges, P; Gripois, D

1989-10-01

The influence of neonatal hypo- and hyperthyroidism on different aspects of tyrosine metabolism in the hypothalamus, striatum, brainstem, adrenal glands, heart and brown adipose tissue (BAT) were studied in 14-day old rats. The synthesis rate of catecholamines (CA) was also determined in vivo after the injection of labelled tyrosine. Hypothyroidism increases tyrosinaemia and endogenous tyrosine concentration in the hypothalamus and BAT. Hyperthyroidism decreases tyrosinaemia and endogenous tyrosine levels in the striatum, adrenals and heart. The accumulation rate of tyrosine determined 30 min after an intravenous injection of the labelled amino acid has been determined in the organs, together with the influx of the amino acid, determined within 20s. Hypothyroidism increases tyrosine accumulation rate in all the organs studied, and tyrosine clearance is decreased in the striatum and brainstem; together with an increased tyrosinaemia, this leads to a normal influx. The influx of tyrosine is increased in the hypothalamus. Hyperthyroidism decreases tyrosine accumulation rate in all the organs except the adrenals. These results indicate that the thyroid status of the young rat can influence tyrosine uptake mechanisms, without modifying an organ's tyrosine content. The fact that hypothyroidism increases tyrosine influx in the hypothalamus without modifying it in the brainstem and striatum reflects an heterogeneous reactivity to the lack of thyroid hormones in different brain structures. Neonatal hypothyroidism decreases the CA synthesis rate in the striatum, the heart and the interscapular brown adipose tissue, while synthesis was enhanced in the brainstem and the adrenals. It is likely that these variations in CA synthesis are due to thyroid hormone modulation of tyrosine hydroxylase activity, the enzyme which catalyses the rate limiting step in CA biosynthesis.

Recent advances in biochemical and molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

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Jin-Ho Choi

2016-03-01

Full Text Available The term congenital adrenal hyperplasia (CAH covers a group of autosomal recessive disorders caused by defects in one of the steroidogenic enzymes involved in the synthesis of cortisol or aldosterone from cholesterol in the adrenal glands. Approximately 95% of all CAH cases are caused by 21-hydroxylase deficiency encoded by the CYP21A2 gene. The disorder is categorized into classical forms, including the salt-wasting and the simple virilizing types, and nonclassical forms based on the severity of the disease. The severity of the clinical features varies according to the level of residual 21-hydroxylase activity. Newborn screening for CAH is performed in many countries to prevent salt-wasting crises in the neonatal period, to prevent male sex assignment in affected females, and to reduce long-term morbidities, such as short stature, gender confusion, and psychosexual disturbances. 17α-hydroxyprogesterone is a marker for 21-hydroxylase deficiency and is measured using a radioimmunoassay, an enzyme-linked immunosorbent assay, or a fluoroimmunoassay. Recently, liquid chromatography linked with tandem mass spectrometry was developed for rapid, highly specific, and sensitive analysis of multiple analytes. Urinary steroid analysis by gas chromatography mass spectrometry also provides qualitative and quantitative data on the excretion of steroid hormone metabolites. Molecular analysis of CYP21A2 is useful for genetic counseling, confirming diagnosis, and predicting prognoses. In conclusion, early detection using neonatal screening tests and treatment can prevent the worst outcomes of 21-hydroxylase deficiency.

An artificial TCA cycle selects for efficient α-ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli.

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Theodosiou, Eleni; Breisch, Marina; Julsing, Mattijs K; Falcioni, Francesco; Bühler, Bruno; Schmid, Andreas

2017-07-01

Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate α-ketoglutarate (α-KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase α-KG flux through proline-4-hydroxylase (P4H). The genes sucA (α-KG dehydrogenase E1 subunit) and sucC (succinyl-CoA synthetase β subunit) were alternately deleted together with aceA (isocitrate lyase) in proline degradation-deficient Escherichia coli strains (ΔputA) expressing the p4h gene. Whereas, the ΔsucCΔaceAΔputA strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the ΔsucAΔaceAΔputA strictly depended on P4H activity, thus coupling growth to proline hydroxylation. P4H restored growth, even when proline was not externally added. However, the reduced succinyl-CoA pool caused a 27% decrease of the average cell size compared to the wildtype strain. Medium supplementation partially restored the morphology and, in some cases, enhanced proline hydroxylation activity. The specific proline hydroxylation rate doubled when putP, encoding the Na + /l-proline transporter, was overexpressed in the ΔsucAΔaceAΔputA strain. This is in contrast to wildtype and ΔputA single-knock out strains, in which α-KG availability obviously limited proline hydroxylation. Such α-KG limitation was relieved in the ΔsucAΔaceAΔputA strain. Furthermore, the ΔsucAΔaceAΔputA strain was used to demonstrate an agar plate-based method for the identification and selection of active α-KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome α-KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of α-KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511-1520. © 2017 Wiley Periodicals, Inc. �

Structural and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase

NARCIS (Netherlands)

Montersino, S.

2012-01-01

The thesis deals with the characterization of a new flavoprotein hydroxylase 3 hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1. 3HB6H is able to insert exclusively oxygen in para-position and the enzyme has been chosen to study the structural basis of such regioselectivity. As

Studies on estradiol-2/4-hydroxylase activity in rat brain and liver

International Nuclear Information System (INIS)

Theron, C.N.

1985-03-01

A sensitive and specific radio-enzymatic assay was used to study estradiol-2/4-hydroxylase activity in rat liver microsomes and in microsomes obtained from 6 discrete brain areas of the rat. Kinetic parameters were determined for these enzyme activities. The effects of different P-450 inhibitors on estradiol-2/4-hydroxylase activity in brain and liver microsomes were also studied. In both organs these enzyme activities were found to be located mainly in the microsomal fraction and were inhibited by the 3 P-450 inhibitors tested. The hepatic estradiol-2/4-hydroxylase activity in adult male rats was significantly higher than that of females, but the enzyme activity in the brain did not exhibit a similar sex difference. Furthermore, estradiol-2/4-hydroxylase activity in rat liver was strongly induced by phenobarbitone treatment, but not in the brain. The phenobarbitone-induced activity in male and female rats exhibited significant kinetic differences. In female rats sexual maturation was associated with significant changes in the apparent Km of estradiol-2/4-hydroxylases in the liver and hypothalamus. Evidence was found that the in vitro estradiol-2/4-hydroxylase activity in rat brain and liver is due to more than one form of microsomal P-450. Kinetic studies showed important differences between the estradiol-2/4-hydroxylase activities in the hippocampus and hypothalamus. Significant differences in estradiol-2/4-hydroxylase activities were observed in the 6 brain areas studied, with the hippocampus showing the highest, and the hypothalamus the lowest activity at all developmental stages in both male and female rats

Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

International Nuclear Information System (INIS)

Rawluszko, Agnieszka A; Bujnicka, Katarzyna E; Horbacka, Karolina; Krokowicz, Piotr; Jagodziński, Paweł P

2013-01-01

Colorectal cancer (CRC) is one of the most common and comprehensively studied malignancies. Hypoxic conditions during formation of CRC may support the development of more aggressive cancers. Hypoxia inducible factor (HIF), a major player in cancerous tissue adaptation to hypoxia, is negatively regulated by the family of prolyl hydroxylase enzymes (PHD1, PHD2, PHD3) and asparaginyl hydroxylase, called factor inhibiting HIF (FIH). PHD1, PHD2, PHD3 and FIH gene expression was evaluated using quantitative RT-PCR and western blotting in primary colonic adenocarcinoma and adjacent histopathologically unchanged colonic mucosa from patients who underwent radical surgical resection of the colon (n = 90), and the same methods were used for assessment of PHD3 gene expression in HCT116 and DLD-1 CRC cell lines. DNA methylation levels of the CpG island in the promoter regulatory region of PHD1, PHD2, PHD3 and FIH were assessed using bisulfite DNA sequencing and high resolution melting analysis (HRM) for patients and HRM analysis for CRC cell lines. We found significantly lower levels of PHD1, PHD2 and PHD3 transcripts (p = 0.00026; p < 0.00001; p < 0.00001) and proteins (p = 0.004164; p = 0.0071; p < 0.00001) in primary cancerous than in histopathologically unchanged tissues. Despite this, we did not observe statistically significant differences in FIH transcript levels between cancerous and histopathologically unchanged colorectal tissue, but we found a significantly increased level of FIH protein in CRC (p = 0.0169). The reduced PHD3 expression was correlated with significantly increased DNA methylation in the CpG island of the PHD3 promoter regulatory region (p < 0.0001). We did not observe DNA methylation in the CpG island of the PHD1, PHD2 or FIH promoter in cancerous and histopathologically unchanged colorectal tissue. We also showed that 5-Aza-2’-deoxycytidine induced DNA demethylation leading to increased PHD3 transcript and protein level in HCT116 cells. We

Tyrosine Residues Regulate Multiple Nuclear Functions of P54nrb.

Science.gov (United States)

Lee, Ahn R; Hung, Wayne; Xie, Ning; Liu, Liangliang; He, Leye; Dong, Xuesen

2017-04-01

The non-POU-domain-containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi-functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site-directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co-immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein-protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non-specific binding affinity to anti-phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co-repression and RNA splicing in gene context-dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co-regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. J. Cell. Physiol. 232: 852-861, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

Science.gov (United States)

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

International Nuclear Information System (INIS)

Teutschbein, Janka; Haydn, Johannes M; Samans, Birgit; Krause, Michael; Eilers, Martin; Schartl, Manfred; Meierjohann, Svenja

2010-01-01

Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development

Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

Directory of Open Access Journals (Sweden)

Krause Michael

2010-07-01

Full Text Available Abstract Background Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1, early growth response 1 (Egr1, osteopontin (Opn, insulin-like growth factor binding protein 3 (Igfbp3, dual-specificity phosphatase 4 (Dusp4, and tumor-associated antigen L6 (Taal6. Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute

Neural control of left ventricular contractility in the dog heart: synaptic interactions of negative inotropic vagal preganglionic neurons in the nucleus ambiguus with tyrosine hydroxylase immunoreactive terminals.

Science.gov (United States)

Massari, V J; Dickerson, L W; Gray, A L; Lauenstein, J M; Blinder, K J; Newsome, J T; Rodak, D J; Fleming, T J; Gatti, P J; Gillis, R A

1998-08-17

Recent physiological evidence indicates that vagal postganglionic control of left ventricular contractility is mediated by neurons found in a ventricular epicardial fat pad ganglion. In the dog this region has been referred to as the cranial medial ventricular (CMV) ganglion [J.L. Ardell, Structure and function of mammalian intrinsic cardiac neurons, in: J.A. Armour, J.L. Ardell (Eds.). Neurocardiology, Oxford Univ. Press, New York, 1994, pp. 95-114; B.X. Yuan, J.L. Ardell, D.A. Hopkins, A.M. Losier, J.A. Armour, Gross and microscopic anatomy of the canine intrinsic cardiac nervous system, Anat. Rec., 239 (1994) 75-87]. Since activation of the vagal neuronal input to the CMV ganglion reduces left ventricular contractility without influencing cardiac rate or AV conduction, this ganglion contains a functionally selective pool of negative inotropic parasympathetic postganglionic neurons. In the present report we have defined the light microscopic distribution of preganglionic negative inotropic neurons in the CNS which are retrogradely labeled from the CMV ganglion. Some tissues were also processed for the simultaneous immunocytochemical visualization of tyrosine hydroxylase (TH: a marker for catecholaminergic neurons) and examined with both light microscopic and electron microscopic methods. Histochemically visualized neurons were observed in a long slender column in the ventrolateral nucleus ambiguus (NA-VL). The greatest number of retrogradely labeled neurons were observed just rostral to the level of the area postrema. TH perikarya and dendrites were commonly observed interspersed with vagal motoneurons in the NA-VL. TH nerve terminals formed axo-dendritic synapses upon negative inotropic vagal motoneurons, however the origin of these terminals remains to be determined. We conclude that synaptic interactions exist which would permit the parasympathetic preganglionic vagal control of left ventricular contractility to be modulated monosynaptically by

The tryptophan hydroxylase 1 (TPH1) gene, schizophrenia susceptibility, and suicidal behavior: a multi-centre case-control study and meta-analysis

DEFF Research Database (Denmark)

Saetre, Peter; Lundmark, Per; Wang, August

2010-01-01

Serotonin (5-hydroxytryptamin; 5-HT) alternations has since long been suspected in the pathophysiology of schizophrenia. Tryptophan hydroxylase (tryptophan 5-monooxygenase; TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT, and sequence variation in intron 6 of the TPH1 gene has been...... associated with schizophrenia. The minor allele (A) of this polymorphism (A218C) is also more frequent in patients who have attempted suicide and individuals who died by suicide, than in healthy control individuals. In an attempt to replicate previous findings, five single nucleotide polymorphisms (SNPs......) were genotyped in 837 Scandinavian schizophrenia patients and 1,473 controls. Three SNPs spanning intron 6 and 7, including the A218C and A779C polymorphisms, were associated with schizophrenia susceptibility (P = 0.019). However there were no differences in allele frequencies of these loci between...

RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua.

Science.gov (United States)

Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

2016-05-25

Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H.

Phenylalanine hydroxylase gene mutations in the United States: Report from the maternal PKU collaborative study

Energy Technology Data Exchange (ETDEWEB)

Guldberg, P.; Henriksen, K.F.; Guettler, F. [John F. Kennedy Inst., Glostrup (Denmark)] [and others

1996-07-01

The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g{r_arrow}a, and Y414C, accounting for 18.7%, 7.8% and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies {le}1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. 47 refs., 1 fig., 5 tabs.

A potential benefit of albinism in Astyanax cavefish: downregulation of the oca2 gene increases tyrosine and catecholamine levels as an alternative to melanin synthesis.

Science.gov (United States)

Bilandžija, Helena; Ma, Li; Parkhurst, Amy; Jeffery, William R

2013-01-01

Albinism, the loss of melanin pigmentation, has evolved in a diverse variety of cave animals but the responsible evolutionary mechanisms are unknown. In Astyanax mexicanus, which has a pigmented surface dwelling form (surface fish) and several albino cave-dwelling forms (cavefish), albinism is caused by loss of function mutations in the oca2 gene, which operates during the first step of the melanin synthesis pathway. In addition to albinism, cavefish have evolved differences in behavior, including feeding and sleep, which are under the control of the catecholamine system. The catecholamine and melanin synthesis pathways diverge after beginning with the same substrate, L-tyrosine. Here we describe a novel relationship between the catecholamine and melanin synthesis pathways in Astyanax. Our results show significant increases in L-tyrosine, dopamine, and norepinephrine in pre-feeding larvae and adult brains of Pachón cavefish relative to surface fish. In addition, norepinephrine is elevated in cavefish adult kidneys, which contain the teleost homologs of catecholamine synthesizing adrenal cells. We further show that the oca2 gene is expressed during surface fish development but is downregulated in cavefish embryos. A key finding is that knockdown of oca2 expression in surface fish embryos delays the development of pigmented melanophores and simultaneously increases L-tyrosine and dopamine. We conclude that a potential evolutionary benefit of albinism in Astyanax cavefish may be to provide surplus L-tyrosine as a precursor for the elevated catecholamine synthesis pathway, which could be important for adaptation to the challenging cave environment.

A potential benefit of albinism in Astyanax cavefish: downregulation of the oca2 gene increases tyrosine and catecholamine levels as an alternative to melanin synthesis.

Directory of Open Access Journals (Sweden)

Helena Bilandžija

Full Text Available Albinism, the loss of melanin pigmentation, has evolved in a diverse variety of cave animals but the responsible evolutionary mechanisms are unknown. In Astyanax mexicanus, which has a pigmented surface dwelling form (surface fish and several albino cave-dwelling forms (cavefish, albinism is caused by loss of function mutations in the oca2 gene, which operates during the first step of the melanin synthesis pathway. In addition to albinism, cavefish have evolved differences in behavior, including feeding and sleep, which are under the control of the catecholamine system. The catecholamine and melanin synthesis pathways diverge after beginning with the same substrate, L-tyrosine. Here we describe a novel relationship between the catecholamine and melanin synthesis pathways in Astyanax. Our results show significant increases in L-tyrosine, dopamine, and norepinephrine in pre-feeding larvae and adult brains of Pachón cavefish relative to surface fish. In addition, norepinephrine is elevated in cavefish adult kidneys, which contain the teleost homologs of catecholamine synthesizing adrenal cells. We further show that the oca2 gene is expressed during surface fish development but is downregulated in cavefish embryos. A key finding is that knockdown of oca2 expression in surface fish embryos delays the development of pigmented melanophores and simultaneously increases L-tyrosine and dopamine. We conclude that a potential evolutionary benefit of albinism in Astyanax cavefish may be to provide surplus L-tyrosine as a precursor for the elevated catecholamine synthesis pathway, which could be important for adaptation to the challenging cave environment.

Tyrosine kinase inhibitors: Multi-targeted or single-targeted?

Science.gov (United States)

Broekman, Fleur; Giovannetti, Elisa; Peters, Godefridus J

2011-02-10

Since in most tumors multiple signaling pathways are involved, many of the inhibitors in clinical development are designed to affect a wide range of targeted kinases. The most important tyrosine kinase families in the development of tyrosine kinase inhibitors are the ABL, SCR, platelet derived growth factor, vascular endothelial growth factor receptor and epidermal growth factor receptor families. Both multi-kinase inhibitors and single-kinase inhibitors have advantages and disadvantages, which are related to potential resistance mechanisms, pharmacokinetics, selectivity and tumor environment. In different malignancies various tyrosine kinases are mutated or overexpressed and several resistance mechanisms exist. Pharmacokinetics is influenced by interindividual differences and differs for two single targeted inhibitors or between patients treated by the same tyrosine kinase inhibitor. Different tyrosine kinase inhibitors have various mechanisms to achieve selectivity, while differences in gene expression exist between tumor and stromal cells. Considering these aspects, one type of inhibitor can generally not be preferred above the other, but will depend on the specific genetic constitution of the patient and the tumor, allowing personalized therapy. The most effective way of cancer treatment by using tyrosine kinase inhibitors is to consider each patient/tumor individually and to determine the strategy that specifically targets the consequences of altered (epi)genetics of the tumor. This strategy might result in treatment by a single multi kinase inhibitor for one patient, but in treatment by a couple of single kinase inhibitors for other patients.

Suppression of bcr-abl synthesis by siRNAs or tyrosine kinase activity by Glivec alters different oncogenes, apoptotic/antiapoptotic genes and cell proliferation factors (microarray study).

Science.gov (United States)

Zhelev, Zhivko; Bakalova, Rumiana; Ohba, Hideki; Ewis, Ashraf; Ishikawa, Mitsuru; Shinohara, Yasuo; Baba, Yoshinobu

2004-07-16

Short 21-mer double-stranded/small-interfering RNAs (ds/siRNAs) were designed to target bcr-abl mRNA in chronic myelogenous leukemia. The ds/siRNAs were transfected into bcr-abl-positive K-562 (derived from blast crisis chronic myelogenous leukemia), using lipofectamine. Penetrating of ds/siRNAs into the cells was detected by fluorescent confocal microscopy, using fluorescein-labeled ds/siRNAs. The cells were treated with mix of three siRNA sequences (3 x 60 nM) during 6 days with three repetitive transfections. The siRNA-treatment was accompanied with significant reduction of bcr-abl mRNA, p210, protein tyrosine kinase activity and cell proliferation index. Treatment of cells with Glivec (during 8 days with four repetitive doses, 180 nM single dose) resulted in analogous reduction of cell proliferation activity, stronger suppression of protein tyrosine kinase activity, and very low reduction of p210. siRNA-mix and Glivec did not affect significantly the viability of normal lymphocytes. Microarray analysis of siRNA- and Glivec-treated K-562 cells demonstrated that both pathways of bcr-abl suppression were accompanied with overexpression and suppression of many different oncogenes, apoptotic/antiapoptotic and cell proliferation factors. The following genes of interest were found to decrease in relatively equal degree in both siRNA- and Glivec-treated cells: Bcd orf1 and orf2 proto-oncogene, chromatin-specific transcription elongation factor FACT 140-kDa subunit mRNA, gene encoding splicing factor SF1, and mRNA for Tec protein tyrosine kinase. siRNA-mix and Glivec provoked overexpression of the following common genes: c-jun proto-oncogene, protein kinase C-alpha, pvt-1 oncogene homologue (myc activator), interleukin-6, 1-8D gene from interferon-inducible gene family, tumor necrosis factor receptor superfamily (10b), and STAT-induced STAT inhibitor.

Regulation and Functional Expression of Cinnamate 4-Hydroxylase from Parsley

Science.gov (United States)

Koopmann, Edda; Logemann, Elke; Hahlbrock, Klaus

1999-01-01

A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10). Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley. The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues. These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit. PMID:9880345

Expression of tyrosine kinase gene in mouse thymic stromal cells

NARCIS (Netherlands)

Rinke de Wit, T. F.; Izon, D. J.; Revilla, C.; Oosterwegel, M.; Bakker, A. Q.; van Ewijk, W.; Kruisbeek, A. M.

1996-01-01

Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both

Effect of tryptophan hydroxylase gene polymorphism on aggression in major depressive disorder and undifferentiated somatoform disorder.

Science.gov (United States)

Koh, Kyung Bong; Kim, Chan Hyung; Choi, Eun Hee; Lee, Young-joon; Seo, Won Youl

2012-05-01

Aggression and anger have been linked with depression, and anger suppression has been linked with somatic symptoms of somatoform disorders. However, the relationship between aggression or anger and genes in patients with depression and somatoform disorders has not been clearly elucidated. The objective of this study was to examine the effect of serotonin-related gene polymorphism on aggression in depressive disorders and somatoform disorders. A serotonin-related polymorphic marker was assessed by using single nucleotide polymorphism (SNP) genotyping. 106 outpatients with major depressive disorder (MDD), 102 outpatients with undifferentiated somatoform disorder, and 133 healthy subjects were enrolled between October 2005 and May 2008. Diagnoses were made according to the Korean version of the Structured Clinical Interview Schedule for DSM-IV. The allele and genotype frequencies of tryptophan hydroxylase-1 (TPH1) A218C were compared between groups. The Hamilton Depression Rating Scale and the Aggression Questionnaire were used for psychological assessment. Each of the 2 disorder groups scored significantly higher on all the Aggression Questionnaire subscales and on the total Aggression Questionnaire score than the healthy subjects (P sex and age. However, no significant differences were found in TPH1 C allele and CC homozygote frequencies between the undifferentiated somatoform disorder patients and the healthy subjects. TPH1 CC homozygote in the MDD group scored significantly higher in terms of verbal aggression (P = .03) and total Aggression Questionnaire score (P = .04) than A-carrier genotypes, regardless of sex and age. However, no significant differences were found in the scores of all the Aggression Questionnaire subscales and the total Aggression Questionnaire score between TPH1 CC homozygote and A-carrier genotypes in the undifferentiated somatoform disorder group and the control group, respectively. Aggression in MDD patients is more susceptible to an

[Congenital adrenal hyperplasia due to lack of 17α-hydroxylase: a report of a new mutation in the gene CYP17A1].

Science.gov (United States)

Perales Martínez, J I; Pina Marqués, B; de Arriba Muñoz, A; Mayayo Dehesa, E; Labarta Aizpún, J I; Loidi Fernández, L

2015-01-01

P450c17 enzyme catalyses two different reactions: the 17α-hydroxylation of progesterone and pregnenolone, and segmenting the carbon 17-20 binding from the 17,20lyase producing adrenal androgens. This enzyme is coded by the CYP17A1 gene. The case is presented of a 14 year old patient with delayed pubertal development and a high blood pressure for height and age. 46,XX karyotype. Hormonal studies highlighted hypergonadotropic hypogonadism, adrenal insufficiency and mineralocorticoid excess. Subsequent genetic studies showed a homozygous mutation in the CYP17A1 gene (c.753+G>A), not previously described, which is responsible for the pathophysiology of 17α-hydroxylase deficiency. This entity is a rare form of congenital adrenal hyperplasia. The disease often goes unnoticed until adolescence or early adult life, and should be suspected in 46,XY individuals with ambiguous genitalia or 46,XX with delayed puberty associated with hypertension and/or hypokalaemia. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

Characterization of mutations at the mouse phenylalanine hydroxylase locus

Energy Technology Data Exchange (ETDEWEB)

McDonald, J.D.; Charlton, C.K. [Wichita State Univ., KS (United States)

1997-02-01

Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. In PAH{sup ENU1}, the phenotype is mild. The Pah{sup enu1} mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. In PAH{sup ENU2} the phenotype is severe. The Pah{sup enu2} mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. In PAH{sup ENU2}, the sequence information was used to devise a direct genotyping system based on the creation of a new Alw26I restriction endonuclease site. 26 refs., 2 figs., 1 tab.

Treatment of Nonclassic 11-Hydroxylase Deficiency with Ashwagandha Root

Directory of Open Access Journals (Sweden)

Daniel Powell

2017-01-01

Full Text Available An elderly woman presented with acne and male pattern alopecia, which upon diagnostic evaluation was found to be due to nonclassic 11-hydroxylase deficiency. We previously reported that Ashwagandha root ameliorates nonclassic 3-β-ol dehydrogenase and aldosterone synthase deficiencies. This is the first report of its use being associated with amelioration of nonclassic 11-hydroxylase deficiency, where its apparent effects appear to be dose-related.

The tryptophan hydroxylase 1 (TPH1) gene, schizophrenia susceptibility, and suicidal behavior: a multi-centre case-control study and meta-analysis

DEFF Research Database (Denmark)

Saetre, Peter; Lundmark, Per; Wang, August

2010-01-01

Serotonin (5-hydroxytryptamin; 5-HT) alternations has since long been suspected in the pathophysiology of schizophrenia. Tryptophan hydroxylase (tryptophan 5-monooxygenase; TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT, and sequence variation in intron 6 of the TPH1 gene has been...... affected individuals having attempted suicide at least once and patients with no history of suicide attempts (P = 0.84). A systematic literature review and meta-analysis support the A218C polymorphism as a susceptibility locus for schizophrenia (odds ratio 1.17, 95% confidence interval 1.......07-1.29). Association studies on suicide attempts are however conflicting (heterogeneity index I(2) = 0.54) and do not support the A218C/A779C polymorphisms being a susceptibility locus for suicidal behavior among individuals diagnosed with a psychiatric disorder (OR = 0.96 [0.80-1.16]). We conclude that the TPH1 A218...

Radiolytic dimerization of tyrosine in alkaline solutions of poly-L-tyrosine, glycyl-L-tyrosine and tyrosine

International Nuclear Information System (INIS)

Boguta, G.; Dancewicz, A.M.

1982-01-01

Blue fluorescence characteristic of dityrosine appeared in γ-irradiated solutions of tyrosine, glycyl-L-tyrosine or polytyrosine (MW 110,000). The intensity of fluorescence was used for the determination of the dityrosine concentration in hydrolysed samples. The radiation-induced formation of dityrosine depended on pH and on the presence of oxygen during radiolysis carried out with a total dose of the order of 1000 Gy. The presence of oxygen in the system suppressed the formation of dityrosine in solution at low or neutral pH but had no effect on this process in alkaline solutions. Except for the radiolysis of air-saturated poly-L-tyrosine solutions, where G(Dityrosine) = 0.35, the yields of dityrosine at high pH were lower than the yields obtained during radiolysis at low pH and in the absence of oxygen. (author)

Loss of Function Studies in Mice and Genetic Association Link Receptor Protein Tyrosine Phosphatase a to Schizophrenia

DEFF Research Database (Denmark)

Takahashi, Nagahide; Nielsen, Karin Sandager; Aleksic, Branko

2011-01-01

Solid evidence links schizophrenia (SZ) susceptibility to neurodevelopmental processes involving tyrosine phosphorylation-mediated signaling. Mouse studies implicate the Ptpra gene, encoding protein tyrosine phosphatase RPTPa, in the control of radial neuronal migration, cortical cytoarchitecture...

Investigations into potential extrasynaptic communication between the dopaminergic and nitrergic systems

Directory of Open Access Journals (Sweden)

Miso eMitkovski

2012-09-01

Full Text Available Nitric oxide is unconstrained by cell membranes and can therefore act along a broad distance as a volume transmitter. Spillover of nitric oxide between neurons may have a major impact on central nervous system diseases and particularly on neurodegeneration. There is evidence whereby communication between nitrergic and dopaminergic systems plays an essential role in the control of the nigrostriatal pathway. However, there is sparse information for either the coexistence or overlap of nitric oxide and dopaminergic structures. The present study used double-labeling immunofluorescent microscopy to investigate the degree of cellular co-localization between nitric oxide synthase and tyrosine hydroxylase, enzymes responsible for the synthesis of nitric oxide and dopamine, respectively, was examined in neurons of the nigrostriatal pathway regions in the rat brain. After perfusional fixation, the brains were cut and double immunostained. A proximity analysis of tyrosine hydroxylase and nitric oxide synthase structures was made using confocal laser scanning microscopy, in nigrostriatal regions of the rat brain. We used image acquired at the optical limit and generated binary masks at 2µm-wide margin from the respective maximum projections. Co-localization between the two antigens was infrequent (<10% in most areas examined. However, tyrosine hydroxylase labeling was particularly concentrated close to nitric oxide synthase dendrites/axons and the cell bodies. These results further substantiate an extrasynaptic substrate for interaction between nitrergic and dopaminergic systems, thereby modulating sensitivity to neural inputs and its gene expression.

Nigral dopaminergic neuron replenishment in adult mice through VE-cadherin-expressing neural progenitor cells

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Abir A Rahman

2017-01-01

Full Text Available The function of dopaminergic neurons in the substantia nigra is of central importance to the coordination of movement by the brain's basal ganglia circuitry. This is evidenced by the loss of these neurons, resulting in the cardinal motor deficits associated with Parkinson's disease. In order to fully understand the physiology of these key neurons and develop potential therapies for their loss, it is essential to determine if and how dopaminergic neurons are replenished in the adult brain. Recent work has presented evidence for adult neurogenesis of these neurons by Nestin+/Sox2– neural progenitor cells. We sought to further validate this finding and explore a potential atypical origin for these progenitor cells. Since neural progenitor cells have a proximal association with the vasculature of the brain and subsets of endothelial cells are Nestin+, we hypothesized that dopaminergic neural progenitors might share a common cell lineage. Therefore, we employed a VE-cadherin promoter-driven CREERT2:THlox/THlox transgenic mouse line to ablate the tyrosine hydroxylase gene from endothelial cells in adult animals. After 26 weeks, but not 13 weeks, following the genetic blockade of tyrosine hydroxylase expression in VE-cadherin+ cells, we observed a significant reduction in tyrosine hydroxylase+ neurons in the substantia nigra. The results from this genetic lineage tracing study suggest that dopaminergic neurons are replenished in adult mice by a VE-cadherin+ progenitor cell population potentially arising from an endothelial lineage.

Dopamine beta-hydroxylase deficiency

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Senard Jean-Michel

2006-03-01

Full Text Available Abstract Dopamine beta-hydroxylase (DβH deficiency is a very rare form of primary autonomic failure characterized by a complete absence of noradrenaline and adrenaline in plasma together with increased dopamine plasma levels. The prevalence of DβH deficiency is unknown. Only a limited number of cases with this disease have been reported. DβH deficiency is mainly characterized by cardiovascular disorders and severe orthostatic hypotension. First symptoms often start during a complicated perinatal period with hypotension, muscle hypotonia, hypothermia and hypoglycemia. Children with DβH deficiency exhibit reduced ability to exercise because of blood pressure inadaptation with exertion and syncope. Symptoms usually worsen progressively during late adolescence and early adulthood with severe orthostatic hypotension, eyelid ptosis, nasal stuffiness and sexual disorders. Limitation in standing tolerance, limited ability to exercise and traumatic morbidity related to falls and syncope may represent later evolution. The syndrome is caused by heterogeneous molecular alterations of the DBH gene and is inherited in an autosomal recessive manner. Restoration of plasma noradrenaline to the normal range can be achieved by therapy with the synthetic precursor of noradrenaline, L-threo-dihydroxyphenylserine (DOPS. Oral administration of 100 to 500 mg DOPS, twice or three times daily, increases blood pressure and reverses the orthostatic intolerance.

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

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L. Gavrilovic

2009-12-01

Full Text Available Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH, dopamine-β-hydroxylase (DBH and phenylethanolamine N-methyltransferase (PNMT and protein levels in the right and left heart auricles of naive control and long-term (12 weeks socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70% compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62% and left (about 81% auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%, DBH (about 37% and PNMT (about 60% only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

No evidence for a major gene effect of the dopamine D{sub 4} receptor gene in the susceptibility to Gilles de la Tourette Syndrome in five Canadian families

Energy Technology Data Exchange (ETDEWEB)

Barr, C.L.; Wigg, K.G.; Tsui, Lap-Chee [Univ. of Toronto, Ontario (Canada)] [and others

1996-05-31

Gilles de la Tourette Syndrome (TS) is a neuropsychiatric disorder characterized by both motor and vocal tics affecting approximately 1/10,000 females and 1/2000 males. Because of the success of neuroleptics and other agents interacting with the dopaminergic system in the suppression of tics, a defect in the dopamine system has been hypothesized in the etiology of TS. In this paper we test the hypothesis that the dopamine D{sub 4} receptor (DRD44) is linked to the genetic susceptibility to TS in five families. We tested three polymorphisms in the DRD4 gene and a polymorphism in the closely linked locus, tyrosine hydroxylase (TH). We found no evidence for linkage of DRD4 or TH to TS using an autosomal dominant model with reduced penetrance or using non-parametric methods. The presence of a mutation that results in a truncated non-functional D{sub 4} receptor protein was also tested for, but was not observed in these families. 36 refs., 1 tab.

Oxidative stress and regulation of Pink1 in zebrafish (Danio rerio.

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Madhusmita Priyadarshini

Full Text Available Oxidative stress-mediated neuronal dysfunction is characteristic of several neurodegenerative disorders, including Parkinson's disease (PD. The enzyme tyrosine hydroxylase (TH catalyzes the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine. A lack of dopamine in the striatum is the most characteristic feature of PD, and the cause of the most dominant symptoms. Loss of function mutations in the PTEN-induced putative kinase (PINK1 gene cause autosomal recessive PD. This study explored the basic mechanisms underlying the involvement of pink1 in oxidative stress-mediated PD pathology using zebrafish as a tool. We generated a transgenic line, Tg(pink1:EGFP, and used it to study the effect of oxidative stress (exposure to H2O2 on pink1 expression. GFP expression was enhanced throughout the brain of zebrafish larvae subjected to oxidative stress. In addition to a widespread increase in pink1 mRNA expression, mild oxidative stress induced a clear decline in tyrosine hydroxylase 2 (th2, but not tyrosine hydroxylase 1 (th1 expression, in the brain of wild-type larvae. The drug L-Glutathione Reduced (LGR has been associated with anti-oxidative and possible neuroprotective properties. Administration of LGR normalized the increased fluorescence intensity indicating pink1 transgene expression and endogenous pink1 mRNA expression in larvae subjected to oxidative stress by H2O2. In the pink1 morpholino oliogonucleotide-injected larvae, the reduction in the expression of th1 and th2 was partially rescued by LGR. The pink1 gene is a sensitive marker of oxidative stress in zebrafish, and LGR effectively normalizes the consequences of mild oxidative stress, suggesting that the neuroprotective effects of pink1 and LGR may be significant and useful in drug development.

RNAi inhibition of feruloyl CoA 6'-hydroxylase reduces scopoletin biosynthesis and post-harvest physiological deterioration in cassava (Manihot esculenta Crantz) storage roots.

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Liu, Shi; Zainuddin, Ima M; Vanderschuren, Herve; Doughty, James; Beeching, John R

2017-05-01

Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.

Effects of transgenic expression of dopamine beta hydroxylase (Dbh) gene on blood pressure in spontaneously hypertensive rats

Czech Academy of Sciences Publication Activity Database

Pravenec, Michal; Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šilhavý, Jan; Mir, S.A.; Vaingankar, S. M.; Wang, J.; Kurtz, T. W.

2016-01-01

Roč. 65, č. 6 (2016), s. 1039-1044 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP301/12/0696; GA TA ČR(CZ) TA02010013 Institutional support: RVO:67985823 Keywords : spontaneously hypertensive rat * transgenic * dopamine beta hydroxylase * catecholamines * blood pressure * left ventricular mass Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.461, year: 2016

Tracking neuronal marker expression inside living differentiating cells using molecular beacons

DEFF Research Database (Denmark)

Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole

2013-01-01

and tyrosine hydroxylase mRNAs were expressed 2 and 3 days post induction of differentiation, respectively. Oct 4 was not detected with MB in these cells and signal was not increased over time suggesting that MB are generally stable inside the cells. The gene expression changes measured using MBs were...... confirmed using qRT-PCR. These results suggest that MBs are simple to use sensors inside living cell, and particularly useful for studying dynamic gene expression in heterogeneous cell populations....

Environmental Exposures, Genetic Polymorphisms and p53 Mutational Spectra in a Case-Control Study of Breast Cancer

Science.gov (United States)

1999-01-01

activities in alimentary tract. Gastroenterology 1997;112:766-75. 38. Moreno A, Pares A, Ortiz J, Enriquez J, Pares X. Alcohol dehydrogenase from human...preliminary data analysis, Dr. Terri Lehman and Bioserve Biotechnologies (Laurel, MD) for their technical expertise, Drs. Joel Gelernter, David Comings...genes with the tyrosine hydroxylase was performed at Bioserve cigarette smoking. In a recent study of the polymorphic Biotechnologies (Laurel, MD, USA

Tyrosine kinome sequencing of pediatric acute lymphoblastic leukemia: a report from the Children's Oncology Group TARGET Project | Office of Cancer Genomics

Science.gov (United States)

TARGET researchers sequenced the tyrosine kinome and downstream signaling genes in 45 high-risk pediatric ALL cases with activated kinase signaling, including Ph-like ALL, to establish the incidence of tyrosine kinase mutations in this cohort. The study confirmed previously identified somatic mutations in JAK and FLT3, but did not find novel alterations in any additional tyrosine kinases or downstream genes. The mechanism of kinase signaling activation in this high-risk subgroup of pediatric ALL remains largely unknown.

Lignification in transgenics deficient in 4-coumarate 3-hydroxylase (C3H)or the associated hydroxycinnamoyl transferase (HCT)

Science.gov (United States)

John Ralph; Takuya Akiyama; Hoon Kim; Fachuang Lu; Sally A. Ralph; Clint Chapple; Ramesh B. Nair; Armin Wagner; Fang Chen; M.S. Srinivasa Reddy; Richard A Dixon; Heather D. Coleman; Shawn D. Mansfield

2006-01-01

Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in angiosperms massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to the normally dominant syringyl (S) and guaiacyl (G) units. Alfalfa stem levels of up to ~65% P (from wild-type (WT) levels of ~1%) resulting from down-regulation of C3H were measured by...

Ror receptor tyrosine kinases: orphans no more.

Science.gov (United States)

Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

2008-11-01

Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

Comparison of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase induction by polycyclic aromatic compounds in human and mouse cell lines.

Science.gov (United States)

Jaiswal, A K; Nebert, D W; Eisen, H W

1985-08-01

The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus. The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells. On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM). P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line. P1-450 mRNA was induced markedly by TCDD and benzo[a] anthracene, whereas P3-450 mRNA was induced negligibly. A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line. Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line. Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells. Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures. The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility. This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism.

Polymorphism screening and haplotype analysis of the tryptophan hydroxylase gene (TPH1 and association with bipolar affective disorder in Taiwan

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Lin Yi-Mei J

2005-03-01

Full Text Available Abstract Background Disturbances in serotonin neurotransmission are implicated in the etiology of many psychiatric disorders, including bipolar affective disorder (BPD. The tryptophan hydroxylase gene (TPH, which codes for the enzyme catalyzing the rate-limiting step in serotonin biosynthetic pathway, is one of the leading candidate genes for psychiatric and behavioral disorders. In a preliminary study, we found that TPH1 intron7 A218C polymorphism was associated with BPD. This study was designed to investigate sequence variants of the TPH1 gene in Taiwanese and to test whether the TPH1 gene is a susceptibility factor for the BPD. Methods Using a systematic approach, we have searched the exons and promoter region of the TPH1 gene for sequence variants in Taiwanese Han and have identified five variants, A-1067G, G-347T, T3804A, C27224T, and A27237G. These five variants plus another five taken from the literature and a public database were examined for an association in 108 BPD patients and 103 controls; no association was detected for any of the 10 variants. Results Haplotype constructions using these 10 SNPs showed that the 3 most common haplotypes in both patients and controls were identical. One of the fourth common haplotype in the patient group (i.e. GGGAGACCCA was unique and showed a trend of significance with the disease (P = 0.028. However, the significance was abolished after Bonferroni correction thus suggesting the association is weak. In addition, three haplotype-tagged SNPs (htSNPs were selected to represent all haplotypes with frequencies larger than 2% in the Taiwanese Han population. The defined TPH1 htSNPs significantly reduce the marker number for haplotype analysis thus provides useful information for future association studies in our population. Conclusion Results of this study did not support the role of TPH1 gene in BPD etiology. As the current studies found the TPH1 gene under investigation belongs to the peripheral

Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

Science.gov (United States)

Rabatel, Andréane; Febvay, Gérard; Gaget, Karen; Duport, Gabrielle; Baa-Puyoulet, Patrice; Sapountzis, Panagiotis; Bendridi, Nadia; Rey, Marjolaine; Rahbé, Yvan; Charles, Hubert; Calevro, Federica; Colella, Stefano

2013-04-10

Nutritional symbioses play a central role in insects' adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology. We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch

Spectrum of Phenylalanine Hydroxylase Gene Mutations in Hamadan and Lorestan Provinces of Iran and Their Associations with Variable Number of Tandem Repeat Alleles.

Science.gov (United States)

Alibakhshi, Reza; Moradi, Keivan; Biglari, Mostafa; Shafieenia, Samaneh

2018-05-01

Phenylketonuria (PKU) is one of the most common known inherited metabolic diseases. The present study aimed to investigate the status of molecular defects in phenylalanine hydroxylase ( PAH ) gene in western Iranian PKU patients (predominantly from Kermanshah, Hamadan, and Lorestan provinces) during 2014-2016. Additionally, the results were compared with similar studies in Iran. Nucleotide sequence analysis of all 13 exons and their flanking intronic regions of the PAH gene was performed in 18 western Iranian PKU patients. Moreover, a variable number of tandem repeat (VNTR) located in the PAH gene was studied. The results revealed a mutational spectrum encompassing 11 distinct mutations distributed along the PAH gene sequence on 34 of the 36 mutant alleles (diagnostic efficiency of 94.4%). Also, four PAH VNTR alleles (with repeats of 3, 7, 8 and 9) were detected. The three most frequent mutations were IVS9+5G>A, IVS7-5T>C, and p.P281L with the frequency of 27.8%, 11%, and 11%, respectively. The results showed that there is not only a consanguineous relation, but also a difference in PAH characters of mutations between Kermanshah and the other two parts of western Iran (Hamadan and Lorestan). Also, it seems that the spectrum of mutations in western Iran is relatively distinct from other parts of the country, suggesting that this region might be a special PAH gene distribution region. Moreover, our findings can be useful in the identification of genotype to phenotype relationship in patients, and provide future abilities for confirmatory diagnostic testing, prognosis, and predict the severity of PKU patients.

Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon.

Science.gov (United States)

Kang, Hong-Gyu; Jun, Sung-Hoon; Kim, Joonyul; Kawaide, Hiroshi; Kamiya, Yuji; An, Gynheung

2002-02-01

We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.

Evolutionary mechanisms driving the evolution of a large polydnavirus gene family coding for protein tyrosine phosphatases

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Serbielle Céline

2012-12-01

Full Text Available Abstract Background Gene duplications have been proposed to be the main mechanism involved in genome evolution and in acquisition of new functions. Polydnaviruses (PDVs, symbiotic viruses associated with parasitoid wasps, are ideal model systems to study mechanisms of gene duplications given that PDV genomes consist of virulence genes organized into multigene families. In these systems the viral genome is integrated in a wasp chromosome as a provirus and virus particles containing circular double-stranded DNA are injected into the parasitoids’ hosts and are essential for parasitism success. The viral virulence factors, organized in gene families, are required collectively to induce host immune suppression and developmental arrest. The gene family which encodes protein tyrosine phosphatases (PTPs has undergone spectacular expansion in several PDV genomes with up to 42 genes. Results Here, we present strong indications that PTP gene family expansion occurred via classical mechanisms: by duplication of large segments of the chromosomally integrated form of the virus sequences (segmental duplication, by tandem duplications within this form and by dispersed duplications. We also propose a novel duplication mechanism specific to PDVs that involves viral circle reintegration into the wasp genome. The PTP copies produced were shown to undergo conservative evolution along with episodes of adaptive evolution. In particular recently produced copies have undergone positive selection in sites most likely involved in defining substrate selectivity. Conclusion The results provide evidence about the dynamic nature of polydnavirus proviral genomes. Classical and PDV-specific duplication mechanisms have been involved in the production of new gene copies. Selection pressures associated with antagonistic interactions with parasitized hosts have shaped these genes used to manipulate lepidopteran physiology with evidence for positive selection involved in

Silencing of flavanone-3-hydroxylase in apple (Malus × domestica Borkh.) leads to accumulation of flavanones, but not to reduced fire blight susceptibility.

Science.gov (United States)

Flachowsky, Henryk; Halbwirth, Heidi; Treutter, Dieter; Richter, Klaus; Hanke, Magda-Viola; Szankowski, Iris; Gosch, Christian; Stich, Karl; Fischer, Thilo C

2012-02-01

Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora. Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Carotenoid β-Ring Hydroxylase and Ketolase from Marine Bacteria—Promiscuous Enzymes for Synthesizing Functional Xanthophylls

OpenAIRE

Misawa, Norihiko

2011-01-01

Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C40-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′)-ketolase (4(4′)-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3′)-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene fo...

Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461.

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Liang Zhu

Full Text Available Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB and a β-carotene hydroxylase gene (crtZ located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.

Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461.

Science.gov (United States)

Zhu, Liang; Wu, Xuechang; Li, Ou; Qian, Chaodong; Gao, Haichun

2012-01-01

Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.

Exploring oxidative modifications of tyrosine

DEFF Research Database (Denmark)

Houée-Lévin, C; Bobrowski, K; Horakova, L

2015-01-01

residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different...... effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor...... residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation...

Expression of the vitamin D receptor, 25-hydroxylases, 1alpha-hydroxylase and 24-hydroxylase in the human kidney and renal clear cell cancer

DEFF Research Database (Denmark)

Blomberg Jensen, Martin; Andersen, Claus B.; Nielsen, John E

2010-01-01

The vitamin D receptor (VDR), CYP27B1 and CYP24A1 are expressed in the human kidney, but the segmental expression of the 25-hydroxylases is unknown. A comprehensive analysis of CYP2R1, CYP27A1, CYP27B1, VDR and CYP24A1 expression in normal kidney and renal clear cell cancer (CCc) would reveal...

HFE Genotype Restricts the Response to Paraquat in a Mouse Model of Neurotoxicity.

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Nixon, Anne M; Meadowcroft, Mark D; Neely, Elizabeth B; Snyder, Amanda M; Purnell, Carson J; Wright, Justin; Lamendella, Regina; Nandar, Wint; Huang, Xuemei; Connor, James R

2018-05-01

Parkinson's disease is marked clinically by motor dysfunction and pathologically by dopaminergic cell loss in the substantia nigra and iron accumulation in the substantia nigra. The driver underlying iron accumulation remains unknown and could be genetic or environmental. The HFE protein is critical for the regulation of cellular iron uptake. Mutations within this protein are associated with increased iron accumulation including in the brain. We have focused on the commonly occurring H63D variant of the HFE gene as a disease modifier in a number of neurodegenerative diseases. To investigate the role of H63D HFE genotype, we generated a mouse model in which the wild-type (WT) HFE gene is replaced by the H67D gene variant (mouse homolog of the human H63D gene variant). Using paraquat toxicity as the model for Parkinson's disease, we found that WT mice responded as expected with significantly greater motor function, loss of tyrosine hydroxylase staining and increase microglial staining in the substantia nigra, and an increase in R 2 relaxation rate within the substantia nigra of the paraquat-treated mice compared to their saline-treated counterparts. In contrast, the H67D mice showed a remarkable resistance to paraquat treatment; specifically differing from the WT mice with no changes in motor function or changes in R 2 relaxation rates following paraquat exposure. At baseline, there were differences between the H67D HFE mice and WT mice in gut microbiome profile and increased L-ferritin staining in the substantia nigra that could account for the resistance to paraquat. Of particular note, the H67D HFE mice regardless of whether or not they were treated with paraquat had significantly less tyrosine hydroxylase immunostaining than WT. Our results clearly demonstrate that the HFE genotype impacts the expression of tyrosine hydroxylase in the substantia nigra, the gut microbiome and the response to paraquat providing additional support that the HFE genotype is a disease

Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear.

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Gumucio, J P; Flood, M D; Bedi, A; Kramer, H F; Russell, A J; Mendias, C L

2017-01-01

Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics. Chronic supraspinatus tears were induced in adult rats, and repaired 28 days later. Rats received 0 mg/kg, 3 mg/kg, or 10 mg/kg GSK1120360A daily. Collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis were then assessed two weeks following surgical repair. At two weeks following repair, treatment groups showed increased muscle mass but there was a 15% decrease in force production in the 10 mg/kg group from controls, and no difference between the 0 mg/kg and the 3 mg/kg groups. There was a decrease in the expression of several gene transcripts related to matrix accumulation and fibrosis, and a 50% decrease in collagen content in both treated groups compared with controls. Additionally, the expression of inflammatory genes was reduced in the treated groups compared with controls. Finally, PHD inhibition improved the maximum stress and displacement to failure in repaired tendons. GSK1120360A resulted in improved enthesis mechanics with variable effects on muscle function. PHD inhibition may be beneficial for connective tissue injuries in which muscle atrophy has not occurred.Cite this article: J. P. Gumucio, M. D. Flood, A. Bedi, H. F. Kramer, A. J

Genetics Home Reference: dopamine beta-hydroxylase deficiency

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... common features include an unusually large range of joint movement (hypermobility) and muscle weakness. Related Information What ... Dopamine beta-hydroxylase deficiency Washington Univeristy, St. Louis: Neuromuscular Disease Center Patient Support and Advocacy Resources (1 ...

Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.. Localizations in the enteric nervous and endocrine systems

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A Veggetti

2009-12-01

Full Text Available The gut of silver eels (Anguilla anguilla L. was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-diaphorase and acetylcholinesterese (AChEase were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP, bombesin, vasoactive intestinal peptide (VIP, neuropeptide Y (NPY, somatostatin, cholecystokinin-octapeptide (CCK-8, serotonin, cholineacetyltransferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin. Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations to those of similar NADPHdiaphorase- reactivity, and in the same nerve bundles in which substance P- and CGRP-likeimmunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.

Finasteride inhibited brain dopaminergic system and open-field behaviors in adolescent male rats.

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Li, Li; Kang, Yun-Xiao; Ji, Xiao-Ming; Li, Ying-Kun; Li, Shuang-Cheng; Zhang, Xiang-Jian; Cui, Hui-Xian; Shi, Ge-Ming

2018-02-01

Finasteride inhibits the conversion of testosterone to dihydrotestosterone. Because androgen regulates dopaminergic system in the brain, it could be hypothesized that finasteride may inhibit dopaminergic system. The present study therefore investigates the effects of finasteride in adolescent and early developmental rats on dopaminergic system, including contents of dopamine and its metabolites (dihydroxy phenyl acetic acid and homovanillic acid) and tyrosine hydroxylase expressions both at gene and protein levels. Meanwhile, open-field behaviors of the rats are examined because of the regulatory effect of dopaminergic system on the behaviors. Open-field behaviors were evaluated by exploratory and motor behaviors. Dopamine and its metabolites were assayed by liquid chromatography-mass spectrometry. Tyrosine hydroxylase mRNA and protein expressions were determined by real-time qRT-PCR and western blot, respectively. It was found that in adolescent male rats, administration of finasteride at doses of 25 and 50 mg/kg for 14 days dose dependently inhibited open-field behaviors, reduced contents of dopamine and its metabolites in frontal cortex, hippocampus, caudate putamen, nucleus accumbens, and down-regulated tyrosine hydroxylase mRNA and protein expressions in substantia nigra and ventral tegmental area. However, there was no significant change of these parameters in early developmental rats after finasteride treatment. These results suggest that finasteride inhibits dopaminergic system and open-field behaviors in adolescent male rats by inhibiting the conversion of testosterone to dihydrotestosterone, and imply finasteride as a potential therapeutic option for neuropsychiatric disorders associated with hyperactivities of dopaminergic system and androgen. © 2017 John Wiley & Sons Ltd.

Screening non-classical 21-hydroxylase gene deficiency from patients diagnosed as polycystic ovary syndrome by gene assay

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Jie HU

2016-04-01

Full Text Available Objective 　To screen non-classical 21-hydroxylase deficiency (NC-21OHD from patients diagnosed as polycystic ovary syndrome (PCOS by gene assay. Methods 　Ninety-eight patients with PCOS were enrolled according to 2003 Rotterdam criteria from Department of Endocrinology, Tangdu Hospital of Fourth Military Medical University, and they were divided into three groups according to the modified Ferriman-Gallway (mF-G score as follows: group A with score 0-2; group B with score 3-5, and group C with score ≥6. Meanwhile, 30 healthy subjects from the Medical Center of the Hospital were recruited as control group. Peripheral blood of all subjects were collected for extracting DNA, the CYP21A2 gene were amplified by 5 pairs of specific primers, and then the PCR products were sequenced by Shanghai Sangon Co. The subjects would accept test for serum cortisol and adrenocorticotropic hormone (ACTH at 8:00am if their CYP21A2 was proved to be abnormal. Results 　Thirty subjects of control group had no any defects in CYP21A2, but 5 of 98 patients with PCOS were proved to be deficient in CYP21A2, and the genotypes were V281L/920-921insT (P1, V281L/I230M (P2, V281L/Normal (P3, P4, P5, respectively, and all of them were heterozygous mutations. The incidences of NC-21OHD in group C and B were 28.6% and 3.3%, respectively. Genotype P1 had been identified to belong to NC-21OHD, which was consistent with its clinical phenotype. All genotypes P3, P4 and P5 belonged to carriers. But for P2, since I230M hadn't been reported in literature, the patient with V281L/I230M couldn't be classified now. Serum biochemical results showed that only in P1 the cortisol was close to the normal lower level, and ACTH was close to the normal upper limit of the reported level in the literature, and the remainders were all normal. Conclusions 　Although PCOS and NC-21OHD are very similar in clinical manifestations, they are different completely in the pathogenesis and treatment. So it

Spectrum of Phenylalanine Hydroxylase Gene Mutations in Hamadan and Lorestan Provinces of Iran and Their Associations with Variable Number of Tandem Repeat Alleles

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Reza Alibakhshi

2018-05-01

Full Text Available Phenylketonuria (PKU is one of the most common known inherited metabolic diseases. The present study aimed to investigate the status of molecular defects in phenylalanine hydroxylase (PAH gene in western Iranian PKU patients (predominantly from Kermanshah, Hamadan, and Lorestan provinces during 2014-2016. Additionally, the results were compared with similar studies in Iran. Nucleotide sequence analysis of all 13 exons and their flanking intronic regions of the PAH gene was performed in 18 western Iranian PKU patients. Moreover, a variable number of tandem repeat (VNTR located in the PAH gene was studied. The results revealed a mutational spectrum encompassing 11 distinct mutations distributed along the PAH gene sequence on 34 of the 36 mutant alleles (diagnostic efficiency of 94.4%. Also, four PAH VNTR alleles (with repeats of 3, 7, 8 and 9 were detected. The three most frequent mutations were IVS9+5G>A, IVS7-5T>C, and p.P281L with the frequency of 27.8%, 11%, and 11%, respectively. The results showed that there is not only a consanguineous relation, but also a difference in PAH characters of mutations between Kermanshah and the other two parts of western Iran (Hamadan and Lorestan. Also, it seems that the spectrum of mutations in western Iran is relatively distinct from other parts of the country, suggesting that this region might be a special PAH gene distribution region. Moreover, our findings can be useful in the identification of genotype to phenotype relationship in patients, and provide future abilities for confirmatory diagnostic testing, prognosis, and predict the severity of PKU patients.

Dmp53, basket and drICE gene knockdown and polyphenol gallic acid increase life span and locomotor activity in a Drosophila Parkinson's disease model

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Hector Flavio Ortega-Arellano

2013-01-01

Full Text Available Understanding the mechanism(s by which dopaminergic (DAergic neurons are eroded in Parkinson's disease (PD is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH-Gal4/UAS-X RNAi Drosophila melanogaster system, we report that Dmp53, basket and drICE gene knockdown in dopaminergic neurons prolong life span (p < 0.05; log-rank test and locomotor activity (p < 0.05; χ² test in D. melanogaster lines chronically exposed to (1 mM paraquat (PQ, oxidative stress (OS generator compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. We propose that a therapy with antioxidants and selectively "switching off" death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition.

Development of vitamin D3 25-hydroxylase activity in rat liver microsomes

International Nuclear Information System (INIS)

Thierry-Palmer, M.; Cullins, S.; Rashada, S.; Gray, T.K.; Free, A.

1986-01-01

The authors have determined the ontogeny of vitamin D 3 25-hydroxylase activity in rat liver microsomes. Microsomes from fetuses, neonates, and their mothers were incubated with 44 nM 3 H-vitamin D 3 in the presence of an NADPH generating system, oxygen, KCl, and MgCl 2 . Lipid extracts of the incubation samples were partially purified by thin-layer chromatography. Tritiated 25-hydroxy vitamin D 3 (250HD 3 ) was analyzed by high-pressure liquid chromatography using 94/6 hexane/isopropanol. Production rate for 250HD 3 in the mothers ranged from 0.22 to 0.30 pmol/mg protein/hr. Activities in the fetuses and neonates were 2.1, 12.9, 32.0, 35.8, and 71.0% of that of their mothers at -3, 0, 2, 7, and 15 days of age. The cytosolic fraction protected the substrate from degradation, stimulated the vitamin D 3 25-hydroxylase reaction in neonates and mothers (1.4 to 1.7 fold increase), and was absolutely required for 25-hydroxylase activity in fetuses. These data suggest that microsomal vitamin D 3 25-hydroxylase activity develops slowly and approaches full activity near the weaning stage. A cytosolic factor present as early as -3 days of age stimulates the activity of the microsomal vitamin D 3 25-hydroxylase

Tyrosine kinase receptor status in endometrial stromal sarcoma: an immunohistochemical and genetic-molecular analysis.

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Cossu-Rocca, Paolo; Contini, Marcella; Uras, Maria Gabriela; Muroni, Maria Rosaria; Pili, Francesca; Carru, Ciriaco; Bosincu, Luisanna; Massarelli, Giovannino; Nogales, Francisco F; De Miglio, Maria Rosaria

2012-11-01

Endometrial stromal sarcomas (ESS) are rare uterine malignant mesenchymal neoplasms, which are currently treated by surgery, as effective adjuvant therapies have not yet been established. Tyrosine kinase inhibitors have rarely been applied in ESS therapy, with few reports describing imatinib responsivity. The aim of this study was to analyze the status of different tyrosine kinase receptors in an ESS series, in order to evaluate their potential role as molecular targets. Immunohistochemistry was performed for EGFR, c-KIT, PDGFR-α, PDGFR-β, and ABL on 28 ESS. EGFR, PDGFR-α, and PDGFR-β gene expression was investigated by real-time polymerase chain reaction (qRT-PCR) on selected cases. "Hot-spot" mutations were screened for on EGFR, c-KIT, PDGFR-α, and PDGFR-β genes, by sequencing. All analysis was executed from formalin-fixed, paraffin-embedded specimens. Immunohistochemical overexpression of 2 or more tyrosine kinase receptors was observed in 18 of 28 tumors (64%), whereas only 5 tumors were consistently negative. Gene expression profiles were concordant with immunohistochemical overexpression in only 1 tumor, which displayed both high mRNA levels and specific immunoreactivity for PDGFR-α, and PDGFR-β. No activating mutations were found on the tumors included in the study. This study confirms that TKRs expression is frequently observed in ESS. Considering that the responsiveness to tyrosine kinase inhibitors is known to be related to the presence of specific activating mutations or gene over-expression, which are not detectable in ESS, TKRs immunohistochemical over-expression alone should not be considered as a reliable marker for targeted therapies in ESS. Specific post-translational abnormalities, responsible for activation of TKRs, should be further investigated.

Functionalization of protected tyrosine via Sonogashira reaction: synthesis of 3-(1,2,3-triazolyl)-tyrosine.

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Vasconcelos, Stanley N S; Shamim, Anwar; Ali, Bakhat; de Oliveira, Isadora M; Stefani, Hélio A

2016-05-01

1,2,3-Triazol tyrosines were synthesized from tyrosine alkynes that were in turn prepared via Sonogashira cross-coupling reaction. The tyrosine alkynes were subjected to click-chemistry reaction conditions leading to the corresponding 3-(1,2,3-triazolyl)-tyrosines in yields ranging from moderate to good.

The crystal structure of tryptophan hydroxylase with bound amino acid substrate

DEFF Research Database (Denmark)

Windahl, Michael Skovbo; Petersen, Charlotte Rode; Christensen, Hans Erik Mølager

2008-01-01

Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O2, and tetrahydrobiopterin (BH4) to produce 5-hydroxytryptophan and 4a-hydroxytetrahydrobiopterin. This is the first and rate-limiting step in the biosynthesis of the neurotransmi......Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O2, and tetrahydrobiopterin (BH4) to produce 5-hydroxytryptophan and 4a-hydroxytetrahydrobiopterin. This is the first and rate-limiting step in the biosynthesis...... acid hydroxylase with bound natural amino acid substrate. The iron coordination can be described as distorted trigonal bipyramidal coordination with His273, His278, and Glu318 (partially bidentate) and one imidazole as ligands. The tryptophan stacks against Pro269 with a distance of 3.9 Å between...

Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

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Oates, A C; Wollberg, P; Achen, M G; Wilks, A F

1998-08-28

The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

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McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.

2006-01-01

Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus.

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Jae Hoon Jeong

Full Text Available The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC, plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat.

Elucidation of a carotenoid biosynthesis gene cluster encoding a novel enzyme, 2,2'-beta-hydroxylase, from Brevundimonas sp. strain SD212 and combinatorial biosynthesis of new or rare xanthophylls.

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Nishida, Yasuhiro; Adachi, Kyoko; Kasai, Hiroaki; Shizuri, Yoshikazu; Shindo, Kazutoshi; Sawabe, Akiyoshi; Komemushi, Sadao; Miki, Wataru; Misawa, Norihiko

2005-08-01

A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium Brevundimonas sp. strain SD212 by using a common crtI sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes, crtW, crtY, crtI, crtB, crtE, idi, and crtZ. The individual ORFs were functionally analyzed by complementation studies using Escherichia coli that accumulated various carotenoid precursors due to the presence of other bacterial crt genes. In addition to functionally identifying the known crt genes, we found that one (ORF11, named crtG) coded for a novel enzyme, carotenoid 2,2'-beta-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this crtG gene was introduced into E. coli accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2'-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation.

Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines.

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Baptista, Melisa J; O'Farrell, Casey; Daya, Sneha; Ahmad, Rili; Miller, David W; Hardy, John; Farrer, Matthew J; Cookson, Mark R

2003-05-01

Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.

Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

LENUS (Irish Health Repository)

Ward, Joseph B J

2012-02-01

Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 +\\/- 2.6 and 38.8 +\\/- 6.7% (n=16; P<\\/=0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

LENUS (Irish Health Repository)

Ward, Joseph B J

2011-02-01

Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 ± 2.6 and 38.8 ± 6.7% (n=16; P≤0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

A novel missense mutation pattern of the GCH1 gene in dopa-responsive dystonia Novo padrão de mutação missense no gene GCH1 na distonia dopa-responsiva

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Rosana H. Scola

2007-12-01

Full Text Available Dopa-responsive dystonia (DRD is an inherited metabolic disorder now classified as DYT5 with two different biochemical defects: autosomal dominant GTP cyclohydrolase 1 (GCH1 deficiency or autosomal recessive tyrosine hydroxylase deficiency. We report the case of a 10-years-old girl with progressive generalized dystonia and gait disorder who presented dramatic response to levodopa. The phenylalanine to tyrosine ratio was significantly higher after phenylalanine loading test. This condition had two different heterozygous mutations in the GCH1 gene: the previously reported P23L mutation and a new Q182E mutation. The characteristics of the DRD and the molecular genetic findings are discussed.Distonia dopa-responsiva (DRD, classificada como DYT5, é um erro inato do metabolismo que pode ser causado por dois diferentes tipos de defeito bioquímico: deficiência de GTP ciclo-hidrolase 1 (GCH1 (autossômica dominante ou de tirosina hidroxilase (autossômica recessiva. Descrevemos o caso de menina de 10 anos com distonia generalizada progressiva e alteração da marcha com importante melhora após uso de levodopa. A relação fenilalanina/tirosina estava aumentada após teste de sobrecarga com fenilalanina. O estudo molecular mostrou que o paciente apresenta uma combinação hererozigótica de mutação no gene GCH1: a já conhecida mutação P23L e uma nova mutação Q182E. Discutem-se as características da DRD e as alterações genéticas possíveis.

Role of the pathotype-specific ACRTS1 gene encoding a hydroxylase involved in the biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata.

Science.gov (United States)

Izumi, Yuriko; Kamei, Eri; Miyamoto, Yoko; Ohtani, Kouhei; Masunaka, Akira; Fukumoto, Takeshi; Gomi, Kenji; Tada, Yasuomi; Ichimura, Kazuya; Peever, Tobin L; Akimitsu, Kazuya

2012-08-01

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of the rootstock species rough lemon (Citrus jambhiri) and Rangpur lime (C. limonia). Genes controlling toxin production were localized to a 1.5-Mb chromosome carrying the ACR-toxin biosynthesis gene cluster (ACRT) in the genome of the rough lemon pathotype. A genomic BAC clone containing a portion of the ACRT cluster was sequenced which allowed identification of three open reading frames present only in the genomes of ACR-toxin producing isolates. We studied the functional role of one of these open reading frames, ACRTS1 encoding a putative hydroxylase, in ACR-toxin production by homologous recombination-mediated gene disruption. There are at least three copies of ACRTS1 gene in the genome and disruption of two copies of this gene significantly reduced ACR-toxin production as well as pathogenicity; however, transcription of ACRTS1 and production of ACR-toxin were not completely eliminated due to remaining functional copies of the gene. RNA-silencing was used to knock down the remaining ACRTS1 transcripts to levels undetectable by reverse transcription-polymerase chain reaction. The silenced transformants did not produce detectable ACR-toxin and were not pathogenic. These results indicate that ACRTS1 is an essential gene in ACR-toxin biosynthesis in the rough lemon pathotype of A. alternata and is required for full virulence of this fungus.

High frequency of cytolytic 21-hydroxylase-specific CD8+ T cells in autoimmune Addison's disease patients.

Science.gov (United States)

Dawoodji, Amina; Chen, Ji-Li; Shepherd, Dawn; Dalin, Frida; Tarlton, Andrea; Alimohammadi, Mohammad; Penna-Martinez, Marissa; Meyer, Gesine; Mitchell, Anna L; Gan, Earn H; Bratland, Eirik; Bensing, Sophie; Husebye, Eystein S; Pearce, Simon H; Badenhoop, Klaus; Kämpe, Olle; Cerundolo, Vincenzo

2014-09-01

The mechanisms behind destruction of the adrenal glands in autoimmune Addison's disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in >90% of patients, but these autoantibodies are not thought to mediate the disease. In this article, we demonstrate highly frequent 21-hydroxylase-specific T cells detectable in 20 patients with Addison's disease. Using overlapping 18-aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8(+) and CD4(+) T cell responses in a large proportion of Addison's patients both ex vivo and after in vitro culture of PBLs ≤20 y after diagnosis. In a large proportion of patients, CD8(+) and CD4(+) 21-hydroxylase-specific T cells were very abundant and detectable in ex vivo assays. HLA class I tetramer-guided isolation of 21-hydroxylase-specific CD8(+) T cells showed their ability to lyse 21-hydroxylase-positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate that strong CTL responses to 21-hydroxylase often occur in vivo, and that reactive CTLs have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer. Copyright © 2014 by The American Association of Immunologists, Inc.

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Suicidal inactivation by acetylenic fatty acids.

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Shak, S; Reich, N O; Goldstein, I M; Ortiz de Montellano, P R

1985-10-25

Human polymorphonuclear leukocytes (PMN) not only generate and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation (probably due to the action of a cytochrome P-450 enzyme). To develop pharmacologically useful inhibitors of the LTB4 omega-hydroxylase in human PMN, we devised a general scheme for synthesizing terminal acetylenic fatty acids based on the "acetylenic zipper" reaction. We found that the LTB4 omega-hydroxylase in intact PMN and in PMN sonicates is inactivated in a concentration-dependent fashion by terminal acetylenic analogues of lauric, palmitic, and stearic acids (i.e. 11-dodecynoic, 15-hexadecynoic, and 17-octadecynoic acids). Consistent with a suicidal process, inactivation of the LTB4 omega-hydroxylase requires molecular oxygen and NADPH, is time-dependent, and follows pseudo-first-order kinetics. Inactivation of the omega-hydroxylase by acetylenic fatty acids also is dependent on the terminal acetylenic moiety and the carbon chain length. Saturated fatty acids lacking a terminal acetylenic moiety do not inactivate the omega-hydroxylase. In addition, the two long-chain (C16, C18) acetylenic fatty acids inactivate the omega-hydroxylase at much lower concentrations (less than 5.0 microM) than those required for inactivation by the short-chain (C12) terminal acetylenic fatty acid (100 microM). Potent suicidal inhibitors of the LTB4 omega-hydroxylase in human PMN will help elucidate the roles played by LTB4 and its omega-oxidation products in regulating PMN function and in mediating inflammation.

Epidermal growth factor receptor tyrosine kinase defines critical prognostic genes of stage I lung adenocarcinoma.

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Mai Yamauchi

Full Text Available PURPOSE: To identify stage I lung adenocarcinoma patients with a poor prognosis who will benefit from adjuvant therapy. PATIENTS AND METHODS: Whole gene expression profiles were obtained at 19 time points over a 48-hour time course from human primary lung epithelial cells that were stimulated with epidermal growth factor (EGF in the presence or absence of a clinically used EGF receptor tyrosine kinase (RTK-specific inhibitor, gefitinib. The data were subjected to a mathematical simulation using the State Space Model (SSM. "Gefitinib-sensitive" genes, the expressional dynamics of which were altered by addition of gefitinib, were identified. A risk scoring model was constructed to classify high- or low-risk patients based on expression signatures of 139 gefitinib-sensitive genes in lung cancer using a training data set of 253 lung adenocarcinomas of North American cohort. The predictive ability of the risk scoring model was examined in independent cohorts of surgical specimens of lung cancer. RESULTS: The risk scoring model enabled the identification of high-risk stage IA and IB cases in another North American cohort for overall survival (OS with a hazard ratio (HR of 7.16 (P = 0.029 and 3.26 (P = 0.0072, respectively. It also enabled the identification of high-risk stage I cases without bronchioalveolar carcinoma (BAC histology in a Japanese cohort for OS and recurrence-free survival (RFS with HRs of 8.79 (P = 0.001 and 3.72 (P = 0.0049, respectively. CONCLUSION: The set of 139 gefitinib-sensitive genes includes many genes known to be involved in biological aspects of cancer phenotypes, but not known to be involved in EGF signaling. The present result strongly re-emphasizes that EGF signaling status in cancer cells underlies an aggressive phenotype of cancer cells, which is useful for the selection of early-stage lung adenocarcinoma patients with a poor prognosis. TRIAL REGISTRATION: The Gene Expression Omnibus (GEO GSE31210.

Characterization of the beta-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice.

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Du, Hao; Wang, Nili; Cui, Fei; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

2010-11-01

Drought is a major limiting factor for crop production. To identify critical genes for drought resistance in rice (Oryza sativa), we screened T-DNA mutants and identified a drought-hypersensitive mutant, dsm2. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative β-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2 mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem II and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2 in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress-related ABA-responsive genes were up-regulated in the overexpression line. DSM2 is a chloroplast protein, and the response of DSM2 to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2 gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice.

Inhibition of biofilm formation by D-tyrosine: Effect of bacterial type and D-tyrosine concentration.

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Yu, Cong; Li, Xuening; Zhang, Nan; Wen, Donghui; Liu, Charles; Li, Qilin

2016-04-01

D-Tyrosine inhibits formation and triggers disassembly of bacterial biofilm and has been proposed for biofouling control applications. This study probes the impact of D-tyrosine in different biofilm formation stages in both G+ and G- bacteria, and reveals a non-monotonic correlation between D-tyrosine concentration and biofilm inhibition effect. In the attachment stage, cell adhesion was studied in a flow chamber, where D-tyrosine caused significant reduction in cell attachment. Biofilms formed by Pseudomonas aeruginosa and Bacillus subtilis were characterized by confocal laser scanning microscopy as well as quantitative analysis of cellular biomass and extracellular polymeric substances. D-Tyrosine exhibited strong inhibitive effects on both biofilms with an effective concentration as low as 5 nM; the biofilms responded to D-tyrosine concentration change in a non-monotonic, bi-modal pattern. In addition, D-tyrosine showed notable and different impact on EPS production by G+ and G- bacteria. Extracellular protein was decreased in P. aeruginosa biofilms, but increased in those of B. subtilis. Exopolysaccharides production by P. aeruginosa was increased at low concentrations and reduced at high concentrations while no impact was found in B. subtilis. These results suggest that distinct mechanisms are at play at different D-tyrosine concentrations and they may be species specific. Dosage of D-tyrosine must be carefully controlled for biofouling control applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Imidacloprid, a neonicotinoid insecticide, facilitates tyrosine hydroxylase transcription and phenylethanolamine N-methyltransferase mRNA expression to enhance catecholamine synthesis and its nicotine-evoked elevation in PC12D cells.

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Kawahata, Ichiro; Yamakuni, Tohru

2018-02-01

Imidacloprid is a neonicotinoid insecticide acting as an agonist of nicotinic acetylcholine receptors (nAChRs) in the target insects. However, questions about the safety to mammals, including human have emerged. Overactivation of mammalian peripheral catecholaminergic systems leads to onset of tachycardia, hypertension, vomiting, etc., which have been observed in acutely imidacloprid-poisoned patients as well. Physiological activation of the nAChRs is known to drive catecholamine biosynthesis and secretion in mammalian adrenal chromaffin cells. Yet, the impacts of imidacloprid on the catecholaminergic function of the chromaffin cells remain to be evaluated. In this study using PC12D cells, a catecholaminergic cell line derived from the medulla chromaffin-cell tumors of rat adrenal gland, we examined whether imidacloprid itself could impact the catecholamine-synthesizing ability. Imidacloprid alone did facilitate tyrosine hydroxylase (TH) transcription via activation of α3β4 nAChR and the α7 subunit-comprising receptor. The insecticide showed the TH transcription-facilitating ability at the concentrations of 3 and 30 μM, at which acetylcholine is known to produce physiological responses, including catecholamine secretion through the nAChRs in adrenal chromaffin cells. The insecticide-facilitated TH transcription was also dependent on PKA- and RhoA-mediated signaling pathways. The insecticide coincidentally raised levels of TH and phenylethanolamine N-methyltransferase (PNMT) mRNA, and as a consequence, increased catecholamine production, although the efficacy of the neonicotinoid was lesser than that of nicotine, indicating its partial agonist-like action. Intriguingly, in cultured rat adrenal chromaffin cells, imidacloprid did increase levels of TH and PNMT protein. When the chromaffin cells were treated with nicotine in the presence of the insecticide, nicotine-elevated adrenaline production was enhanced due to facilitation of nicotine-increased TH and PNMT

Leptin Intake at Physiological Doses Throughout Lactation in Male Wistar Rats Normalizes the Decreased Density of Tyrosine Hydroxylase-Immunoreactive Fibers in the Stomach Caused by Mild Gestational Calorie Restriction

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Nara Szostaczuk

2018-03-01

Full Text Available Introduction: Gestational under nutrition in rats has been shown to decrease expression of sympathetic innervation markers in peripheral tissues of offspring, including the stomach. This has been linked to lower gastric secretion and decreased circulating levels of ghrelin. Considering the critical role of leptin intake during lactation in preventing obesity and reversing adverse developmental programming effects, we aimed to find out whether leptin supplementation may reverse the above mentioned alterations caused by mild gestational calorie restriction.Methods: Three groups of male rats were studied at a juvenile age (25 days old and during adulthood (3 and 6 months old: the offspring of ad libitum fed dams (controls, the offspring of dams that were diet restricted (20% from days 1 to 12 of gestation (CR, and CR rats supplemented with a daily oral dose of leptin (equivalent to 5 times the average amount they could receive each day from maternal milk throughout lactation (CR-Leptin. The density of TyrOH-immunoreactive (TyrOH+ fibers and the levels of Tyrosine hydroxylase (TyrOH—used as potential markers of functional sympathetic innervation—were measured in stomach. Plasma leptin and ghrelin levels were also determined.Results: Twenty five-day-old CR rats, but not CR-Leptin rats, displayed lower density of TyrOH+ fibers (−46% and TyrOH levels (−47% in stomach compared to controls. Alterations in CR animals were mitigated at 6 months of age, and differences were not significant. Adult CR-Leptin animals showed higher plasma ghrelin levels than CR animals, particularly at 3 months (+16%, and a lower leptin/ghrelin ratio (−28 and −37% at 3 and 6 months, respectively.Conclusion: Leptin intake during lactation is able to reverse the alterations in the density of TyrOH+ fibers in the stomach and normalize the increased leptin/ghrelin ratio linked to a mild gestational calorie restriction in rats, supporting the relevance of leptin as an

Elucidation of a Carotenoid Biosynthesis Gene Cluster Encoding a Novel Enzyme, 2,2′-β-Hydroxylase, from Brevundimonas sp. Strain SD212 and Combinatorial Biosynthesis of New or Rare Xanthophylls

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Nishida, Yasuhiro; Adachi, Kyoko; Kasai, Hiroaki; Shizuri, Yoshikazu; Shindo, Kazutoshi; Sawabe, Akiyoshi; Komemushi, Sadao; Miki, Wataru; Misawa, Norihiko

2005-01-01

A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium Brevundimonas sp. strain SD212 by using a common crtI sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes, crtW, crtY, crtI, crtB, crtE, idi, and crtZ. The individual ORFs were functionally analyzed by complementation studies using Escherichia coli that accumulated various carotenoid precursors due to the presence of other bacterial crt genes. In addition to functionally identifying the known crt genes, we found that one (ORF11, named crtG) coded for a novel enzyme, carotenoid 2,2′-β-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this crtG gene was introduced into E. coli accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2′-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation. PMID:16085816

High frequency of cytolytic 21-Hydroxylase specific CD8+ T cells in autoimmune Addison’s disease patients1

Science.gov (United States)

Dawoodji, Amina; Chen, Ji-Li; Shepherd, Dawn; Dalin, Frida; Tarlton, Andrea; Alimohammadi, Mohammad; Penna-Martinez, Marissa; Meyer, Gesine; Mitchell, Anna L; Gan, Earn H; Bratland, Eirik; Bensing, Sophie; Husebye, Eystein; Pearce, Simon H.; Badenhoop, Klaus; Kämpe, Olle; Cerundolo, Vincenzo

2016-01-01

The mechanisms behind the destruction of the adrenal glands in autoimmune Addison’s disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in over 90% of patients, but these autoantibodies are not thought to mediate the disease. Here we demonstrate highly frequent 21-hydroxylase specific T cells detectable in 20 patients with Addison’s disease. Using overlapping 18aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8+ and CD4+ T cell responses in a large proportion of Addison’s patients both ex-vivo and after in-vitro culture of peripheral blood lymphocytes up to 20 years after diagnosis. In a large proportion of patients, CD8+ 21-hydroxylase specific T cells and CD4+ 21-hydroxylase specific T cells were very abundant and detectable in ex-vivo assays. HLA class-I tetramer-guided isolation of 21-hydroxylase specific CD8+ T cells showed their ability to lyse 21-hydroxylase positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate strong cytotoxic T lymphocyte responses to 21-hydroxylase often occur in-vivo, and that reactive cytotoxic T lymphocytes have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer. PMID:25063864

Identification and Partial Characterization of an L-Tyrosine Aminotransferase (TAT from Arabidopsis thaliana

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Pranav R. Prabhu

2010-01-01

Full Text Available The aminotransferase gene family in the model plant Arabidopsis thaliana consists of 44 genes. Twenty six of these enzymes are classified as characterized meaning that the reaction(s that the enzyme catalyzes are documented using experimental means. The remaining 18 enzymes are uncharacterized and are therefore deemed putative. Our laboratory is interested in elucidating the function(s of the remaining putative aminotransferase enzymes. To this end, we have identified and partially characterized an aminotransferase (TAT enzyme from Arabidopsis annotated by the locus tag At5g36160. The full-length cDNA was cloned and the purified recombinant enzyme was characterized using in vitro and in vivo experiments. In vitro analysis showed that the enzyme is capable of interconverting L-Tyrosine and 4-hydroxyphenylpyruvate, and L-Phenylalanine and phenylpyruvate. In vivo analysis by functional complementation showed that the gene was able to complement an E. coli with a background of aminotransferase mutations that confers auxotrophy for L-Tyrosine and L-Phenylalanine.

The Role of Oxygen Sensors, Hydroxylases, and HIF in Cardiac Function and Disease

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W. H. Davin Townley-Tilson

2015-01-01

Full Text Available Ischemic heart disease is the leading cause of death worldwide. Oxygen-sensing proteins are critical components of the physiological response to hypoxia and reperfusion injury, but the role of oxygen and oxygen-mediated effects is complex in that they can be cardioprotective or deleterious to the cardiac tissue. Over 200 oxygen-sensing proteins mediate the effects of oxygen tension and use oxygen as a substrate for posttranslational modification of other proteins. Hydroxylases are an essential component of these oxygen-sensing proteins. While a major role of hydroxylases is regulating the transcription factor HIF, we investigate the increasing scope of hydroxylase substrates. This review discusses the importance of oxygen-mediated effects in the heart as well as how the field of oxygen-sensing proteins is expanding, providing a more complete picture into how these enzymes play a multifaceted role in cardiac function and disease. We also review how oxygen-sensing proteins and hydroxylase function could prove to be invaluable in drug design and therapeutic targets for heart disease.

Gene expression in the lignin biosynthesis pathway during soybean seed development.

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Baldoni, A; Von Pinho, E V R; Fernandes, J S; Abreu, V M; Carvalho, M L M

2013-02-28

The study of gene expression in plants is fundamental, and understanding the molecular mechanisms involved in important biological processes, such as biochemical pathways or signaling that are used or manipulated in improvement programs, are key for the production of high-quality soybean seeds. Reports related to gene expression of lignin in seeds are scarce in the literature. We studied the expression of the phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase, 4-hydroxycinnamate 3-hydroxylase, and cinnamyl alcohol dehydrogenase genes involved in lignin biosynthesis during the development of soybean (Glycine max L. Merrill) seeds. As the endogenous control, the eukaryotic elongation factor 1-beta gene was used in two biological replicates performed in triplicate. Relative quantitative expression of these genes during the R4, R5, R6, and R7 development stages was analyzed. Real-time polymerase chain reaction was used for the gene expression study. The analyses were carried out in an ABI PRISM 7500 thermocycler using the comparative Ct method and SYBR Green to detect amplification. The seed samples at the R4 stage were chosen as calibrators. Increased expression of the cinnamate-4-hydroxylase and PAL genes occurred in soybean seeds at the R5 and R6 development stages. The cinnamyl alcohol dehydrogenase gene was expressed during the final development phases of soybean seeds. In low-lignin soybean cultivars, the higher expression of the PAL gene occurs at development stages R6 and R7. Activation of the genes involved in the lignin biosynthesis pathway occurs at the beginning of soybean seed development.

Receptor Tyrosine Kinases in Drosophila Development

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Sopko, Richelle; Perrimon, Norbert

2013-01-01

Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

Dose-dependent effects of oral tyrosine administration on plasma tyrosine levels and cognition in aging

NARCIS (Netherlands)

Rest, van de Ondine; Bloemendaal, Mirjam; Heus, De Rianne; Aarts, Esther

2017-01-01

The effects of tyrosine on plasma response and cognition in aging are unknown. We assessed the dose-dependent response to tyrosine administration in older adults in both plasma tyrosine concentrations and working memory performance. In this double blind randomized cross-over trial 17 older adults

Dose-Dependent Effects of Oral Tyrosine Administration on Plasma Tyrosine Levels and Cognition in Aging

NARCIS (Netherlands)

Rest, O. van de; Bloemendaal, M.; Heus, R.A.A. de; Aarts, E.

2017-01-01

The effects of tyrosine on plasma response and cognition in aging are unknown. We assessed the dose-dependent response to tyrosine administration in older adults in both plasma tyrosine concentrations and working memory performance. In this double blind randomized cross-over trial 17 older adults

MAP kinase-independent signaling in angiotensin II regulation of neuromodulation in SHR neurons.

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Yang, H; Raizada, M K

1998-09-01

Angiotensin II (Ang II), via its interaction with the angiotensin type 1 (AT1) receptor subtype, causes enhanced stimulation of norepinephrine (NE) neuromodulation. This involves increased transcription of NE transporter, tyrosine hydroxylase, and dopamine ss-hydroxylase genes in Wistar-Kyoto rat (WKY) brain neurons. AT1 receptor-mediated regulation of certain signaling events (such as activation of the Ras-Raf-1-mitogen activated protein (MAP) kinase signaling pathway, nuclear translocation of transcription factors such as Fos and Jun, and the interactions of these factors with AP-1 binding sites) is involved in this NE neuromodulation (Lu et al. J Cell Biol. 1996;135:1609-1617). The aim of this study was to compare the signal transduction mechanism of Ang II regulation of NE neuromodulation in WKY and spontaneously hypertensive rat (SHR) brain neurons, in view of the fact that AT1 receptor expression and Ang II stimulation of NE neuromodulation are higher in SHR neurons compared with WKY neurons. Despite this hyperactivity, Ang II stimulation of Ras, Raf-1, and MAP kinase activities was comparable between the neurons from WKY and SHR. Similarly, central injections of Ang II caused a comparable stimulation of MAP kinase in the hypothalamic and brain stem areas of adult WKY and SHR. Inhibition of MAP kinase by either an MAP kinase kinase inhibitor (PD98059) or an MAP kinase antisense oligonucleotide completely attenuated the stimulatory effects of Ang II on [3H]-NE uptake, NE transporter mRNA, and tyrosine hydroxylase mRNA levels in WKY neurons. These treatments resulted in only 43% to 50% inhibition of [3H]-NE uptake and NE transporter and tyrosine hydroxylase mRNAs in SHR neurons. Thus, Ang II stimulation of NE neuromodulation was completely blocked by MAP kinase inhibition in WKY neurons and only partially blocked in the SHR neurons. These observations suggest the presence of an additional signal transduction pathway involved in NE neuromodulation in SHR neurons

Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA.

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Fox, Rebecca M; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J

2013-05-01

FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously.

Developmental role of phenylalanine-ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) genes during adventitious rooting of Juglans regia L. microshoots.

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Cheniany, Monireh; Ganjeali, Ali

2016-12-01

Phenylalanine-ammonia-lyase and cinnamate-4-hydroxylase play important role in the phenylpropanoid pathway, which produces many biologically important secondary metabolites participating in normal plant development. Flavonol quercetin is the main representant of these compounds that has been identified in numerous Juglans spp. In this survey, the developmental expression patterns of PAL and C4H genes during in vitro rooting of two walnut cultivars 'Sunland' and 'Howard' was examined by RT-PCR. To understand the potential role in rooting, the changing pattern of endogenous content of quercetin was also analyzed by HPLC. The 'Sunland' with better capacity to root had more quercetin content during the "inductive phase" of rooting than 'Howard'. In each cultivar, the level of PAL transcripts showed the same behavior with the changing patterns of quercetin during root formation of microshoots. The positive correlation between the changes of quercetin and PAL-mRNA indicated that PAL gene may have an immediate effect on flavonoid pathway metabolites including quercetin. Although the behavioral change of C4H expression was similar in both cultivars during root formation (with significantly more level for 'Howard'), it was not coincide with the changes of quercerin concentrations. Our results showed that C4H function is important for the normal development, but its transcriptional regulation does not correlate with quercetin as an efficient phenolic compound for walnut rhizogenesis.

Aversive odorant causing appetite decrease downregulates tyrosine decarboxylase gene expression in the olfactory receptor neuron of the blowfly, Phormia regina

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Ishida, Yuko; Ozaki, Mamiko

2012-01-01

In the blowfly Phormia regina, exposure to d-limonene for 5 days during feeding inhibits proboscis extension reflex behavior due to decreasing tyramine (TA) titer in the brain. TA is synthesized by tyrosine decarboxylase (Tdc) and catalyzed into octopamine (OA) by TA ß-hydroxylase (Tbh). To address the mechanisms of TA titer regulation in the blowfly, we cloned Tdc and Tbh cDNAs from P. regina (PregTdc and PregTbh). The deduced amino acid sequences of both proteins showed high identity to those of the corresponding proteins from Drosophila melanogaster at the amino acid level. PregTdc was expressed in the antenna, labellum, and tarsus whereas PregTbh was expressed in the head, indicating that TA is mainly synthesized in the sensory organs whereas OA is primarily synthesized in the brain. d-Limonene exposure significantly decreased PregTdc expression in the antenna but not in the labellum and the tarsus, indicating that PregTdc expressed in the antenna is responsible for decreasing TA titer. PregTdc-like immunoreactive material was localized in the thin-walled sensillum. In contrast, the OA/TA receptor (PregOAR/TAR) was localized to the thick-walled sensillum. The results indicated that d-limonene inhibits PregTdc expression in the olfactory receptor neurons in the thin-walled sensilla, likely resulting in reduced TA levels in the receptor neurons in the antenna. TA may be transferred from the receptor neuron to the specific synaptic junction in the antennal lobe of the brain through the projection neurons and play a role in conveying the aversive odorant information to the projection and local neurons.

21 CFR 582.5920 - Tyrosine.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Tyrosine. 582.5920 Section 582.5920 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS... § 582.5920 Tyrosine. (a) Product. Tyrosine (L- and DL-forms). (b) Conditions of use. This substance is...

Phenylalanine metabolism in isolated rat liver cells. Effects of glucagon and diabetes.

OpenAIRE

Carr, F P; Pogson, C I

1981-01-01

1. Methods are described for monitoring the metabolic flux through phenylalanine hydroxylase, the tyrosine catabolic pathway and phenylalanine: pyruvate transaminase in isolated liver cell incubations. 2. The relationship between hydroxylase flux and phenylalanine concentration is sigmoidal. 3. Glucagon increases hydroxylase activity at low, near-physiological, substrate concentrations only. The hormone does not affect the rate of formation of phenylpyruvate. 4. Experimental diabetes (for 10 ...

Transcriptome analysis and anthocyanin-related genes in red leaf lettuce.

Science.gov (United States)

Zhang, Y Z; Xu, S Z; Cheng, Y W; Ya, H Y; Han, J M

2016-01-29

This study aimed to analyze the transcriptome profile of red lettuce and identify the genes involved in anthocyanin accumulation. Red leaf lettuce is a popular vegetable and popular due to its high anthocyanin content. However, there is limited information available about the genes involved in anthocyanin biosynthesis in this species. In this study, transcriptomes of 15-day-old seedlings and 40-day-old red lettuce leaves were analyzed using an Illuminia HiseqTM 2500 platform. A total of 10.6 GB clean data were obtained and de novo assembled into 83,333 unigenes with an N50 of 1067. After annotation against public databases, 51,850 unigene sequences were identified, among which 46,087 were annotated in the NCBI non-redundant protein database, and 41,752 were annotated in the Swiss-Prot database. A total of 9125 unigenes were mapped into 163 pathways using the Kyoto Encyclopedia of Genes and Genomes database. Thirty-four structural genes were found to cover the main steps of the anthocyanin pathway, including chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase. Seven MYB, three bHLH, and two WD40 genes, considered anthocyanin regulatory genes, were also identified. In addition, 3607 simple sequence repeat (SSR) markers were identified from 2916 unigenes. This research uncovered the transcriptomic characteristics of red leaf lettuce seedlings and mature plants. The identified candidate genes related to anthocyanin biosynthesis and the detected SSRs provide useful tools for future molecular breeding studies.

Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

Science.gov (United States)

Torrens-Spence, Michael P; Gillaspy, Glenda; Zhao, Bingyu; Harich, Kim; White, Robert H; Li, Jianyong

2012-02-10

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine. Copyright © 2012 Elsevier Inc. All rights reserved.

Assays for mammalian tyrosinase: a comparative study

International Nuclear Information System (INIS)

Jara, J.R.; Solano, F.; Lozano, J.A.

1988-01-01

This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed

The ducky2J mutation in Cacna2d2 results in reduced spontaneous Purkinje cell activity and altered gene expression

Science.gov (United States)

Donato, Roberta; Page, Karen M.; Koch, Dietlind; Nieto-Rostro, Manuela; Foucault, Isabelle; Davies, Anthony; Wilkinson, Tonia; Rees, Michele; Edwards, Frances A.; Dolphin, Annette C.

2006-01-01

The mouse mutant ducky and its allele ducky2J represent a model for absence epilepsy characterized by spike-wave seizures, and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the α2δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α2δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2du2J mutation results in a two base-pair deletion in the coding region and a complete loss of α2δ-2 protein. Here we show that du2J/du2J mice have a 30% reduction in somatic calcium current, and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C du2J/du2J PCs show no spontaneous intrinsic activity. Du2J/du2J mice also have alterations in the cerebellar expression of several genes related to PC function. At P21 there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du2J/+ mice have a marked reduction in α2δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However, du2J/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α2δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α2δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma. PMID:17135419

[Characterisation of three polymorphisms of the tryptophan hydroxylase 2 gene in a sample of Colombian population with major depressive disorder].

Science.gov (United States)

Martínez-Idárraga, Adriana; Riveros-Barrera, Irene; Sánchez, Ricardo; Jaramillo, Luis Eduardo; Calvo-Gómez, José Manuel; Yunis-Londoño, Juan José

Identify whether rs11179000, rs136494 and rs4570625 polymorphisms of the tryptophan hydroxylase 2 gene, are associated with a major depressive disorder in a sample of the Colombian population. Case-control study was conducted in which a comparison was made between subjects diagnosed with major depressive disorder at some point in adulthood or active symptoms at the time of evaluation, and subjects with no psychiatric disease. Subjects were studied in the Department of Psychiatry, Faculty of Medicine and the Institute of Genetics at the National University of Colombia. Polymorphisms were genotyped using Taqman probes in real time PCR. As well as studying the association between major depressive disorder and these (single nucleotide polymorphisms (SNPs), the association with other factors previously associated with depression were also analysed. No statistically significant association between genotypic and allelic frequencies of each polymorphism and major depressive disorder was found. Association between sex and complication during pregnancy / childbirth and major depressive disorder was observed. Association between sex and complication during pregnancy / childbirth and major depressive disorder was observed. There was no association between any polymorphism and major depressive disorder. Copyright © 2016 Asociación Colombiana de Psiquiatría. Publicado por Elsevier España. All rights reserved.

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosine-26 and -64

International Nuclear Information System (INIS)

Roepe, P.; Scherrer, P.; Ahl, P.L.; Gupta, S.K.D.; Bogomolni, R.A.; Herzfeld, J.; Rothschild, K.J.

1987-01-01

Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR 570 → K 630 or BR 570 → M 412 transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K 630 and the deprotonation of a tyrosine by M 412 . Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M 412 as concluded previously. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm -1 in the BR 570 → M 412 FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group

Src protein-tyrosine kinase structure and regulation

International Nuclear Information System (INIS)

Roskoski, Robert

2004-01-01

Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPα displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the αD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu 4 Tyr)

Mutation analysis of the phenylalanine hydroxylase gene in Azerbaijani population, a report from West Azerbaijan province of Iran

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Morteza Bagheri

2015-07-01

Full Text Available Objective(s:Phenylketonuria (PKU is a genetic inborn error of phenylalanine (Phe metabolism resulting from insufficiency in the hepatic enzyme, phenylalanine hydroxylase (PAH, which leads to elevated levels of Phe in the blood. The present study was carried out for mutation analysis of the PAH gene in West Azerbaijan province of Iran. Materials and Methods:A total of 218 alleles from 40 PKU families were studied using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR method. Results:The frequencies of IVS10-11, S67P, R261Q, R252W, IVS11nt-1 g>c, R408Q, and Q232Q mutations were 28(35, 17(21.25, 15(18.75, 3(3.75, 3(3.75, 2(2.5, and 1(1.25, in cases group, and 51(23.4, 31(14.2, 27(12.4, 6(2.75, 6(2.75, 4(1.83, and 2(0.92 in total group, respectively. The mutations of R243Q, 364delG, L333F, 261X, I65T, and R408W were not detected in our samples. Conclusion: It can be concluded that the IVS10-11 mutation has the highest frequency in the tested population. To our knowledge, this report is the first in its own kind and provides better understanding of the genetic heterogeneity, the origin and distributions of PAH mutations in West Azerbaijan province of Iran.

Selective WGA uptake in the hippocampus from the locus coeruleus of DBH-WGA transgenic mice

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Susan G eWalling

2012-05-01

Full Text Available We generated transgenic mice in which a transsynaptic tracer, wheat germ agglutinin (WGA, was specifically expressed in the locus coeruleus neurons under the control of the dopamine-β-hydroxylase gene promoter. WGA protein was produced in more than 95% of the tyrosine hydroxylase-positive locus coeruleus neurons sampled. Transynaptic transfer of WGA was most evident in CA3 neurons of the hippocampus, but appeared absent in CA1 neurons. Faint but significant WGA immunoreactivity was observed surrounding the nuclei of dentate granule cells. Putative hilar mossy cells, identified by the presence of calretinin in the ventral hippocampus, appeared uniformly positive for transynaptically transferred WGA protein. GAD67-positive interneurons in the hilar and CA3 regions tended to be WGA-positive, although a subset of them did not show WGA co-localization. The same mixed WGA uptake profile was apparent when examining co-localization with parvalbumin. The selective uptake of WGA by dentate granule cells, mossy cells and CA3 pyramidal neurons is consistent with evidence for a large proportion of conventional synapses adjacent to locus coeruleus axonal varicosities in these regions. The lack of WGA uptake in the CA1 region and its relatively sparse innervation by dopamine-β-hydroxylase-positive fibers suggest that a majority of the tyrosine hydroxylase-positive classical synapses revealed by electron microscopy in that region may be producing dopamine. The overall pattern of WGA uptake in these transgenic mice suggests a selective role for the granule cell-mossy cell-CA3 network in processing novelty or the salient environmental contingency changes signaled by locus coeruleus activity.

Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.

Science.gov (United States)

Herbst, R; Munemitsu, S; Ullrich, A

1995-01-19

The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.

Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

LENUS (Irish Health Repository)

Meehan, Maria

2012-02-05

Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

Structural Insights into Diversity and n-Alkane Biodegradation Mechanisms of Alkane Hydroxylases

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Yurui eJi

2013-03-01

Full Text Available Environmental microbes utilize four degradation pathways for the oxidation of n-alkanes. Although the enzymes degrading n-alkanes in different microbes may vary, enzymes functioning in the first step in the aerobic degradation of alkanes all belong to the alkane hydroxylases. Alkane hydroxylases are a class of enzymes that insert oxygen atoms derived from molecular oxygen into different sites of the alkane terminus (or termini depending on the type of enzymes. In this review, we summarize the different types of alkane hydroxylases, their degrading steps and compare typical enzymes from various classes with regard to their three dimensional structures, in order to provide insights into how the enzymes mediate their different roles in the degradation of n-alkanes and what determines their different substrate ranges. Through the above analyses, the degrading mechanisms of enzymes can be elucidated and molecular biological methods can be utilized to expand their catalytic roles in the petrochemical industry or in bioremediation of oil-contaminated environments.

Enhancement of Naringenin Biosynthesis from Tyrosine by Metabolic Engineering of Saccharomyces cerevisiae.

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Lyu, Xiaomei; Ng, Kuan Rei; Lee, Jie Lin; Mark, Rita; Chen, Wei Ning

2017-08-09

Flavonoids are an important class of plant polyphenols that possess a variety of health benefits. In this work, S. cerevisiae was metabolically engineered to produce the flavonoid naringenin, using tyrosine as the precursor. Our strategy to improve naringenin production comprised three modules. In module 1, we employed a modified GAL system to overexpress the genes of the naringenin biosynthesis pathway and investigated their synergistic action. In module 2, we simultaneously up-regulated acetyl-CoA production and down-regulated fatty acid biosynthesis in order to increase the precursor supply, malonyl-CoA. In module 3, we engineered the tyrosine biosynthetic pathway to eliminate the feedback inhibition of tyrosine and also down-regulated competing pathways. It was found that modules 1 and 3 played important roles in improving naringenin production. We succeeded in producing up to ∼90 mg/L of naringenin in our final strain, which is a 20-fold increase as compared to the parental strain.

Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD.

Science.gov (United States)

Kazi, Julhash U; Chougule, Rohit A; Li, Tianfeng; Su, Xianwei; Moharram, Sausan A; Rupar, Kaja; Marhäll, Alissa; Gazi, Mohiuddin; Sun, Jianmin; Zhao, Hui; Rönnstrand, Lars

2017-07-01

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.

Antisense-induced suppression of taxoid 14β- hydroxylase gene ...

African Journals Online (AJOL)

Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the 14OH mRNA level in transgenic cells dropped dramatically, suggesting that the expression of endogenous14OH gene was significantly suppressed by the exogenous as14OH gene. Correspondingly, the total yield of three major C-14 ...

To Cheat or Not To Cheat: Tryptophan Hydroxylase 2 SNP Variants Contribute to Dishonest Behavior.

Science.gov (United States)

Shen, Qiang; Teo, Meijun; Winter, Eyal; Hart, Einav; Chew, Soo H; Ebstein, Richard P

2016-01-01

Although, lying (bear false witness) is explicitly prohibited in the Decalogue and a focus of interest in philosophy and theology, more recently the behavioral and neural mechanisms of deception are gaining increasing attention from diverse fields especially economics, psychology, and neuroscience. Despite the considerable role of heredity in explaining individual differences in deceptive behavior, few studies have investigated which specific genes contribute to the heterogeneity of lying behavior across individuals. Also, little is known concerning which specific neurotransmitter pathways underlie deception. Toward addressing these two key questions, we implemented a neurogenetic strategy and modeled deception by an incentivized die-under-cup task in a laboratory setting. The results of this exploratory study provide provisional evidence that SNP variants across the tryptophan hydroxylase 2 (TPH2) gene, that encodes the rate-limiting enzyme in the biosynthesis of brain serotonin, contribute to individual differences in deceptive behavior.

To cheat or not to cheat: Tryptophan hydroxylase 2 SNP variants contribute to dishonest behavior

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Qiang eShen

2016-05-01

Full Text Available Although lying (bear false witness is explicitly prohibited in the Decalogue and a focus of interest in philosophy and theology, more recently the behavioral and neural mechanisms of deception are gaining increasing attention from diverse fields especially economics, psychology and neuroscience. Despite the considerable role of heredity in explaining individual differences in deceptive behavior, few studies have investigated which specific genes contribute to the heterogeneity of lying behavior across individuals. Also, little is known concerning which specific neurotransmitter pathways underlie deception. Towards addressing these two key questions, we implemented a neurogenetic strategy and modeled deception by an incentivized die-under-cup task in a laboratory setting. The results of this exploratory study provide provisional evidence that SNP variants across the tryptophan hydroxylase 2 (TPH2 gene, that encodes the rate-limiting enzyme in the biosynthesis of brain serotonin, contribute to individual differences in deceptive behavior.

Three novel variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) of the phenylalanine hydroxylase (PAH) gene impair protein expression and function in vitro.

Science.gov (United States)

Zong, Yanan; Liu, Ning; Ma, Shanshan; Bai, Ying; Guan, Fangxia; Kong, Xiangdong

2018-08-20

Phenylketonuria (PKU) is the most common inherited metabolic disease, an autosomal recessive disorder affecting >10,000 newborns each year globally. It can be caused by over 1000 different naturally occurring mutations in the phenylalanine hydroxylase (PAH) gene. We analyzed three novel naturally occurring PAH gene variants: p.Glu178Lys (c.532G>A), p.Val245Met (c.733G>A) and p.Ser250Phe (c.749C>T). The mutant effect on the PAH enzyme structure and function was predicted by bioinformatics software. Vectors expressing the corresponding PAH variants were generated for expression in E. coli and in HEK293T cells. The RNA expression of the three PAH variants was measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The mutant PAH protein levels were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot and enzyme-linked immunosorbent assay (ELISA). All three variants were predicted to be pathogenic by bioinformatics analysis. The transcription of the three PAH variants was similar to the wild type PAH gene in HEK293T cells. In contrast, the levels of mutant PAH proteins decreased significantly compared to the wild type control, in both E. coli and HEK293T cells. Our results indicate that the three novel PAH gene variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) impair PAH protein expression and function in prokaryotic and eukaryotic cells. Copyright © 2018. Published by Elsevier B.V.

Polycystic ovary syndrome: association of a C/T single nucleotide polymorphism at tyrosine kinase domain of insulin receptor gene with pathogenesis among lean Japanese women.

Science.gov (United States)

Kashima, Katsunori; Yahata, Tetsuro; Fujita, Kazuyuki; Tanaka, Kenichi

2013-01-01

To assess whether the insulin receptor (INSR) gene contributes to genetic susceptibility to polycystic ovary syndrome (PCOS) in a Japanese population. We ex-amined the frequency of the His 1058 C/T single nucleotide polymorphism (SNP) found in exon 17 of the INSR gene in 61 Japanese PCOS patients and 99 Japanese healthy controls. In addition, we analyzed the association between the genotype of this SNP and the clinical phenotypes. The frequency of the C/C genotype was not significantly different between all PCOS patients (47.5%) and controls (35.4%). However, among the lean cases (body mass index PCOS patients (65.0%) as compared with controls (36.6%). We concluded that the His 1058 C/T polymorphism at the tyrosine kinase domain of the INSR gene had a relationship to the pathogenesis of lean PCOS patients in a Japanese population.

Tyrosine kinase domain mutations of EGFR gene in head and neck squamous cell carcinoma

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Vatte C

2017-03-01

Full Text Available Chittibabu Vatte,1 Ali M Al Amri,2 Cyril Cyrus,1 Shahanas Chathoth,1 Sadananda Acharya,3 Tariq Mohammad Hashim,4 Zhara Al Ali,2 Saleh Tawfeeq Alshreadah,2 Ahmed Alsayyah,4 Amein K Al-Ali5 1Department of Genetic Research, Institute for Research and Medical Consultation, University of Dammam, Dammam, 2Department of Internal Medicine, King Fahd Hospital of the University, University of Dammam, Al-Khobar, 3Department of Stemcell Research, Institute for Research and Medical Consultation, 4Department of Pathology, King Fahd Hospital of the University, University of Dammam, Al-Khobar, 5Department of Biochemistry, College of Medicine, University of Dammam, Dammam, Kingdom of Saudi Arabia Background: Epidermal growth factor receptor (EGFR is a commonly altered gene that is identified in various cancers, including head and neck squamous cell carcinoma (HNSCC. Therefore, EGFR is a promising molecular marker targeted by monoclonal antibodies and small molecule inhibitors targeting the tyrosine kinase (TK domain. Objective: The objective of this study was to investigate the spectrum of mutations in exons 18, 19, 20, and 21 of the EGFR gene in HNSCC patients. Materials and methods: This retrospective study included 47 confirmed HNSCC cases. Mutations in the TK domain, exons 18, 19, 20, and 21 of the EGFR gene, were detected by Scorpion® chemistry and ARMS® technologies on Rotor-Gene Q real-time polymerase chain reaction.Results: The tumors exhibited EGFR-TK domain mutations in 57% of cases. Four cases of T790M mutations were reported for the first time among HNSCC patients. Out of the total mutations, L861Q (exon 21, exon 20 insertions and deletions of exon 19 accounted for the majority of mutations (21%, 19%, and 17%, respectively. EGFR mutation status was correlated with the higher grade (P=0.026 and advanced stage (P=0.034 of HNSCC tumors.Conclusion: Higher frequency of EGFR-TK domain mutations together with the presence of the T790M mutation suggests

PPAR/RXR Regulation of Fatty Acid Metabolism and Fatty Acid -Hydroxylase (CYP4 Isozymes: Implications for Prevention of Lipotoxicity in Fatty Liver Disease

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James P. Hardwick

2009-01-01

Full Text Available Fatty liver disease is a common lipid metabolism disorder influenced by the combination of individual genetic makeup, drug exposure, and life-style choices that are frequently associated with metabolic syndrome, which encompasses obesity, dyslipidemia, hypertension, hypertriglyceridemia, and insulin resistant diabetes. Common to obesity related dyslipidemia is the excessive storage of hepatic fatty acids (steatosis, due to a decrease in mitochondria -oxidation with an increase in both peroxisomal -oxidation, and microsomal -oxidation of fatty acids through peroxisome proliferator activated receptors (PPARs. How steatosis increases PPAR activated gene expression of fatty acid transport proteins, peroxisomal and mitochondrial fatty acid -oxidation and -oxidation of fatty acids genes regardless of whether dietary fatty acids are polyunsaturated (PUFA, monounsaturated (MUFA, or saturated (SFA may be determined by the interplay of PPARs and HNF4 with the fatty acid transport proteins L-FABP and ACBP. In hepatic steatosis and steatohepatitis, the -oxidation cytochrome P450 CYP4A gene expression is increased even with reduced hepatic levels of PPAR. Although numerous studies have suggested the role ethanol-inducible CYP2E1 in contributing to increased oxidative stress, Cyp2e1-null mice still develop steatohepatitis with a dramatic increase in CYP4A gene expression. This strongly implies that CYP4A fatty acid -hydroxylase P450s may play an important role in the development of steatohepatitis. In this review and tutorial, we briefly describe how fatty acids are partitioned by fatty acid transport proteins to either anabolic or catabolic pathways regulated by PPARs, and we explore how medium-chain fatty acid (MCFA CYP4A and long-chain fatty acid (LCFA CYP4F -hydroxylase genes are regulated in fatty liver. We finally propose a hypothesis that increased CYP4A expression with a decrease in CYP4F genes may promote the progression of steatosis to

TATN-1 mutations reveal a novel role for tyrosine as a metabolic signal that influences developmental decisions and longevity in Caenorhabditis elegans.

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Annabel A Ferguson

Full Text Available Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people.

TATN-1 Mutations Reveal a Novel Role for Tyrosine as a Metabolic Signal That Influences Developmental Decisions and Longevity in Caenorhabditis elegans

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Ferguson, Annabel A.; Dumas, Kathleen J.; Ritov, Vladimir B.; Matern, Dietrich; Hu, Patrick J.; Fisher, Alfred L.

2013-01-01

Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR) signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people. PMID:24385923

Tyrosine supplementation for phenylketonuria.

Science.gov (United States)

Webster, Diana; Wildgoose, Joanne

2013-06-05

Phenylketonuria is an inherited disease for which the main treatment is the dietary restriction of the amino acid phenylalanine. The diet has to be initiated in the neonatal period to prevent or reduce mental handicap. However, the diet is very restrictive and unpalatable and can be difficult to follow. A deficiency of the amino acid tyrosine has been suggested as a cause of some of the neuropsychological problems exhibited in phenylketonuria. Therefore, this review aims to assess the efficacy of tyrosine supplementation for phenylketonuria. To assess the effects of tyrosine supplementation alongside or instead of a phenylalanine-restricted diet for people with phenylketonuria, who commenced on diet at diagnosis and either continued on the diet or relaxed the diet later in life. To assess the evidence that tyrosine supplementation alongside, or instead of a phenylalanine-restricted diet improves intelligence, neuropsychological performance, growth and nutritional status, mortality rate and quality of life. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Register which is comprised of references identified from comprehensive electronic database searches, handsearches of relevant journals and abstract books of conference proceedings. Additional studies were identified from handsearches of the Journal of Inherited Metabolic Disease (from inception in 1978 to 1998). The manufacturers of prescribable dietary products used in the treatment of phenylketonuria were also contacted for further references.Date of the most recent search of the Group's Inborn Errors of Metabolism Trials Register: 28 June 2012. All randomised or quasi-randomised trials investigating the use of tyrosine supplementation versus placebo in people with phenylketonuria in addition to, or instead of, a phenylalanine-restricted diet. People treated for maternal phenylketonuria were excluded. Two authors independently assessed the trial eligibility, methodological quality

Genome-Wide Identification and Characterization of Tyrosine Kinases in the Silkworm, Bombyx mori

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Songzhen He

2018-03-01

Full Text Available The tyrosine kinases (TKs are important parts of metazoan signaling pathways and play significant roles in cell growth, development, apoptosis and disease. Genome-wide characterization of TKs has been conducted in many metazoans, however, systematic information about this family in Lepidoptera is still lacking. We retrieved 33 TK-encoding genes in silkworm and classified them into 25 subfamilies by sequence analysis, without members in AXL, FRK, PDGFR, STYK1 and TIE subfamilies. Although domain sequences in each subfamily are conserved, TKs in vertebrates tend to be remarkably conserved and stable. Our results of phylogenetic analysis supported the previous conclusion for the second major expansion of TK family. Gene-Ontology (GO analysis revealed that a higher proportion of BmTKs played roles in binding, catalysis, signal transduction, metabolism, biological regulation and response to stimulus, compared to all silkworm genes annotated in GO. Moreover, the expression profile analysis of BmTKs among multiple tissues and developmental stages demonstrated that many genes exhibited stage-specific and/or sex-related expression during embryogenesis, molting and metamorphosis, and that 8 BmTKs presented tissue-specific high expression. Our study provides systematic description of silkworm tyrosine kinases, and may also provide further insights into metazoan TKs and assist future studies addressing their functions.

Prolyl hydroxylase domain enzymes: important regulators of cancer metabolism

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Yang M

2014-08-01

Full Text Available Ming Yang,1 Huizhong Su,1 Tomoyoshi Soga,2 Kamil R Kranc,3 Patrick J Pollard1 1Cancer Biology and Metabolism Group, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK; 2Institute for Advanced Biosciences, Keio University, Mizukami, Tsuruoka, Yamagata, Japan; 3MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Abstract: The hypoxia-inducible factor (HIF prolyl hydroxylase domain enzymes (PHDs regulate the stability of HIF protein by post-translational hydroxylation of two conserved prolyl residues in its α subunit in an oxygen-dependent manner. Trans-4-prolyl hydroxylation of HIFα under normal oxygen (O2 availability enables its association with the von Hippel-Lindau (VHL tumor suppressor pVHL E3 ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway. Due to the obligatory requirement of molecular O2 as a co-substrate, the activity of PHDs is inhibited under hypoxic conditions, resulting in stabilized HIFα, which dimerizes with HIFβ and, together with transcriptional co-activators CBP/p300, activates the transcription of its target genes. As a key molecular regulator of adaptive response to hypoxia, HIF plays important roles in multiple cellular processes and its overexpression has been detected in various cancers. The HIF1α isoform in particular has a strong impact on cellular metabolism, most notably by promoting anaerobic, whilst inhibiting O2-dependent, metabolism of glucose. The PHD enzymes also seem to have HIF-independent functions and are subject to regulation by factors other than O2, such as by metabolic status, oxidative stress, and abnormal levels of endogenous metabolites (oncometabolites that have been observed in some types of cancers. In this review, we aim to summarize current understandings of the function and regulation of PHDs in cancer with an emphasis on their roles in metabolism. Keywords: prolyl hydroxylase domain (PHD

Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

DEFF Research Database (Denmark)

Su, J; Batzer, A; Sap, J

1994-01-01

Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine......-phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation...... links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function....

1α-hydroxylase and innate immune responses to 25-hydroxyvitamin D in colonic cell lines

Science.gov (United States)

Lagishetty, Venu; Chun, Rene F.; Liu, Nancy Q.; Lisse, Thomas S.; Adams, John S.; Hewison, Martin

2010-01-01

Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase). In cells such as macrophages, expression of 1α-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and β-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1α-hydroxylase. Alternative activators of 1α-hydroxylase such as transforming growth factor β were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D. PMID:20152900

Role of tyrosine phosphatase inhibitors in cancer treatment with emphasis on SH2 domain-containing tyrosine phosphatases (SHPs)

NARCIS (Netherlands)

Irandoust, Mahban; van den Berg, Timo K.; Kaspers, Gertjan J. L.; Cloos, Jacqueline

2009-01-01

Protein tyrosine phosphorylation is one of the key mechanisms involved in signal transduction pathways. This modification is regulated by concerted action of protein tyrosine phosphatases and protein tyrosine kinases. Deregulation of either of these key regulators lead to abnormal cellular

Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

Energy Technology Data Exchange (ETDEWEB)

Venkitachalam, Srividya; Chueh, Fu-Yu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States); Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States)

2012-01-20

Highlights: Black-Right-Pointing-Pointer Lmo2 expression is elevated in Lck-transformed cells. Black-Right-Pointing-Pointer Both endogenous and exogenous Lck localize in the nucleus. Black-Right-Pointing-Pointer Nuclear Lck is active in Lck-transformed cells. Black-Right-Pointing-Pointer Lck binds to the promoter region of Lmo2 gene in vivo. Black-Right-Pointing-Pointer In contrast to JAK2, Lck does not increase histone H3 phosphorylation on Tyr 41. -- Abstract: LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle

DEFF Research Database (Denmark)

Lassiter, David G; Nylén, Carolina; Sjögren, Rasmus J O

2018-01-01

the FAK gene, PTK2. RESULTS: AMPK activation reduced tyrosine phosphorylation of FAK in skeletal muscle. AICAR reduced p-FAKY397in isolated human skeletal muscle and cultured myotubes. Insulin stimulation did not alter FAK phosphorylation. Serum starvation increased AMPK activation, as demonstrated...

The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

DEFF Research Database (Denmark)

Voena, Claudia; Conte, Chiara; Ambrogio, Chiara

2007-01-01

Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades......, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine...... phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542...

Effect of Tryptophan Hydroxylase-2 rs7305115 SNP on suicide attempts risk in major depression

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Zhang Yuqi

2010-08-01

Full Text Available Abstract Background Suicide and major depressive disorders (MDD are strongly associated, and genetic factors are responsible for at least part of the variability in suicide risk. We investigated whether variation at the tryptophan hydroxylase-2 (TPH2 gene rs7305115 SNP may predispose to suicide attempts in MDD. Methods We genotyped TPH2 gene rs7305115 SNP in 215 MDD patients with suicide and matched MDD patients without suicide. Differences in behavioral and personality traits according to genotypic variation were investigated by logistic regression analysis. Results There were no significant differences between MDD patients with suicide and controls in genotypic (AG and GG frequencies for rs7305115 SNP, but the distribution of AA genotype differed significantly (14.4% vs. 29.3%, p p p Conclusions The study suggested that hopelessness, negative life events and family history of suicide were risk factors of attempted suicide in MDD while the TPH2 rs7305115A remained a significant protective predictor of suicide attempts.

No effect of C1473G polymorphism in the tryptophan hydroxylase 2 gene on the response of the brain serotonin system to chronic fluoxetine treatment in mice.

Science.gov (United States)

Bazhenova, Ekaterina Y; Sinyakova, Nadezhda A; Kulikova, Elizabeth A; Kazarinova, Irina A; Bazovkina, Daria V; Gainetdinov, Raul R; Kulikov, Alexander V

2017-07-13

Selective serotonin reuptake inhibitors (SSRIs) are antidepressants that block serotonin transporter (SERT) and increase serotonin (5-HT) level in the synaptic cleft. The interaction between SERT and the key enzyme of 5-HT synthesis in the brain, tryptophan hydroxylase 2 (TPH2), is essential to maintain the brain 5-HT level. The G allele of C1473G polymorphism in Tph2 gene decreases enzyme activity by half in mouse brain. Here we studied effect of C1473G polymorphism on the reaction of brain 5-HT system to chronic fluoxetine treatment (120mg/l in drinking water, for 3 weeks) in adult males of the congenic B6-1473C and B6-1473G mouse lines with high and low enzyme activity, respectively. The polymorphism did not affect the levels of 5-HT, its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) and Tph2 gene mRNA in the brain. Fluoxetine significantly attenuated 5-HT levels in the cortex and striatum, 5-HIAA concentrations in the cortex, hippocampus, striatum and midbrain, and Tph2 gene expression in the midbrain. However, we did not observed any effect of the genotype x treatment interaction on these neurochemical characteristics. Therefore, C1473G polymorphism does not seem to play an essential role in the reaction of the brain 5-HT system to chronic fluoxetine treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

A sandwich immunoassay for human prolyl 4-hydroxylase using monoclonal antibody

International Nuclear Information System (INIS)

Yoshida, Shinichi

1986-01-01

Monoclonal antibody was used in a sandwich enzyme immunoassay and in a radioimmunoassay for human serum immunoreactive prolyl 4-hydroxylase. The enzyme immunoassay utilized a monoclonal antibody as a solid phase and horseradish peroxidase-labeled rabbit antibody to human prolyl 4-hydroxylase as a conjugate. Sensitivity was 0.1 ng of enzyme per tube. With a conjugate purified by an enzyme-bound affinity column, sensitivity was increased to 0.01 ng per tube, and linearity was obtained between 0.01 to 30 ng per tube. The radioimmunoassay used a 125 I-labeled rabbit antibody (IgG) as the conjugate. Sensitivity of this technique was 0.4 ng of enzyme per tube. (Auth.)

Tyrosine kinases in rheumatoid arthritis

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Kobayashi Akiko

2011-08-01

Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

Synthesis of deuterium and tritium labelled tyrosine

International Nuclear Information System (INIS)

Kanska, M.; Drabarek, S.

1980-01-01

A new method of synthesis of tyrosine labelled with deuterium and tritium in the aromatic ring has been developed. Deuterated and tritiated tyrosine was obtained by isotope exchange between tyrosine and deuterated or tritiated water at elevated temperature in hydrochloric acid medium using K 2 PtCl 4 as a catalyst. For synthesis of tritiated tyrosine 1 Ci HTO was used; the specific activity of the product was 5 mCi/mMol. (author)

Tyrosine-sensitized photodimerization of thymine in aqueous solution

International Nuclear Information System (INIS)

Kaneko, M.; Matsuyama, A.; Nagata, C.

1978-01-01

Photodimerization of thymine in aqueous solution in the presence of tyrosine was studied with monochromatic UV irradiation. The total dimer formation was sensitized in the presence of tyrosine. The action spectrum of sensitized total dimer formation has a peak near 280 nm corresponding to the absorption maximum of tyrosine. Triplet quenchers reduced the sensitization substantially. It seems probable that tyrosine-sensitized photodimerization of thymine occurred via triplet-triplet energy transfer from tyrosine to thymine. (author)

Multiplicity of 3-Ketosteroid-9 alpha-Hydroxylase Enzymes in Rhodococcus rhodochrous DSM43269 for Specific Degradation of Different Classes of Steroids

OpenAIRE

Petrusma, Mirjan; Hessels, Gerda; Dijkhuizen, Lubbert; van der Geize, Robert

2011-01-01

The well-known large catabolic potential of rhodococci is greatly facilitated by an impressive gene multiplicity. This study reports on the multiplicity of kshA, encoding the oxygenase component of 3-ketosteroid 9 alpha-hydroxylase, a key enzyme in steroid catabolism. Five kshA homologues (kshA1 to kshA5) were previously identified in Rhodococcus rhodochrous DSM43269. These KshA(DSM43269) homologues are distributed over several phylogenetic groups. The involvement of these KshA homologues in ...

Homeostatic mechanisms in dopamine synthesis and release: a mathematical model

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Nijhout H Frederik

2009-09-01

Full Text Available Abstract Background Dopamine is a catecholamine that is used as a neurotransmitter both in the periphery and in the central nervous system. Dysfunction in various dopaminergic systems is known to be associated with various disorders, including schizophrenia, Parkinson's disease, and Tourette's syndrome. Furthermore, microdialysis studies have shown that addictive drugs increase extracellular dopamine and brain imaging has shown a correlation between euphoria and psycho-stimulant-induced increases in extracellular dopamine 1. These consequences of dopamine dysfunction indicate the importance of maintaining dopamine functionality through homeostatic mechanisms that have been attributed to the delicate balance between synthesis, storage, release, metabolism, and reuptake. Methods We construct a mathematical model of dopamine synthesis, release, and reuptake and use it to study homeostasis in single dopaminergic neuron terminals. We investigate the substrate inhibition of tyrosine hydroxylase by tyrosine, the consequences of the rapid uptake of extracellular dopamine by the dopamine transporters, and the effects of the autoreceoptors on dopaminergic function. The main focus is to understand the regulation and control of synthesis and release and to explicate and interpret experimental findings. Results We show that the substrate inhibition of tyrosine hydroxylase by tyrosine stabilizes cytosolic and vesicular dopamine against changes in tyrosine availability due to meals. We find that the autoreceptors dampen the fluctuations in extracellular dopamine caused by changes in tyrosine hydroxylase expression and changes in the rate of firing. We show that short bursts of action potentials create significant dopamine signals against the background of tonic firing. We explain the observed time courses of extracellular dopamine responses to stimulation in wild type mice and mice that have genetically altered dopamine transporter densities and the observed

Bacterial Protein-Tyrosine Kinases

DEFF Research Database (Denmark)

Shi, Lei; Kobir, Ahasanul; Jers, Carsten

2010-01-01

in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

1alpha-hydroxylase and innate immune responses to 25-hydroxyvitamin D in colonic cell lines.

Science.gov (United States)

Lagishetty, Venu; Chun, Rene F; Liu, Nancy Q; Lisse, Thomas S; Adams, John S; Hewison, Martin

2010-07-01

Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). In cells such as macrophages, expression of 1alpha-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and beta-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1alpha-hydroxylase. Alternative activators of 1alpha-hydroxylase such as transforming growth factor beta were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Mapping the functional landscape of frequent phenylalanine hydroxylase (PAH) genotypes promotes personalised medicine in phenylketonuria.

Science.gov (United States)

Danecka, Marta K; Woidy, Mathias; Zschocke, Johannes; Feillet, François; Muntau, Ania C; Gersting, Søren W

2015-03-01

In phenylketonuria, genetic heterogeneity, frequent compound heterozygosity, and the lack of functional data for phenylalanine hydroxylase genotypes hamper reliable phenotype prediction and individualised treatment. A literature search revealed 690 different phenylalanine hydroxylase genotypes in 3066 phenylketonuria patients from Europe and the Middle East. We determined phenylalanine hydroxylase function of 30 frequent homozygous and compound heterozygous genotypes covering 55% of the study population, generated activity landscapes, and assessed the phenylalanine hydroxylase working range in the metabolic (phenylalanine) and therapeutic (tetrahydrobiopterin) space. Shared patterns in genotype-specific functional landscapes were linked to biochemical and pharmacological phenotypes, where (1) residual activity below 3.5% was associated with classical phenylketonuria unresponsive to pharmacological treatment; (2) lack of defined peak activity induced loss of response to tetrahydrobiopterin; (3) a higher cofactor need was linked to inconsistent clinical phenotypes and low rates of tetrahydrobiopterin response; and (4) residual activity above 5%, a defined peak of activity, and a normal cofactor need were associated with pharmacologically treatable mild phenotypes. In addition, we provide a web application for retrieving country-specific information on genotypes and genotype-specific phenylalanine hydroxylase function that warrants continuous extension, updates, and research on demand. The combination of genotype-specific functional analyses with biochemical, clinical, and therapeutic data of individual patients may serve as a powerful tool to enable phenotype prediction and to establish personalised medicine strategies for dietary regimens and pharmacological treatment in phenylketonuria. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

The crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of beta-carotene into astaxanthin and other xanthophylls.

Science.gov (United States)

Alvarez, Vanessa; Rodríguez-Sáiz, Marta; de la Fuente, Juan Luis; Gudiña, Eduardo J; Godio, Ramiro P; Martín, Juan F; Barredo, José Luis

2006-04-01

The conversion of beta-carotene into xanthophylls is a subject of great scientific and industrial interest. We cloned the crtS gene involved in astaxanthin biosynthesis from two astaxanthin producing strains of Xanthophyllomyces dendrorhous: VKPM Y2410, an astaxanthin overproducing strain, and the wild type ATCC 24203. In both cases, the ORF has a length of 3166 bp, including 17 introns, and codes for a protein of 62.6 kDa with similarity to cytochrome-P450 hydroxylases. crtS gene sequences from strains VKPM Y2410, ATCC 24203, ATCC 96594, and ATCC 96815 show several nucleotide changes, but none of them causes any amino acid substitution, except a G2268 insertion in the 13th exon of ATCC 96815 which causes a change in the reading frame. A G1470 --> A change in the 5' splicing region of intron 8 was also found in ATCC 96815. Both point mutations explain astaxanthin idiotrophy and beta-carotene accumulation in ATCC 96815. Mutants accumulating precursors of the astaxanthin biosynthetic pathway were selected from the parental strain VKPM Y2410 (red) showing different colors depending on the compound accumulated. Two of them were blocked in the biosynthesis of astaxanthin, M6 (orange; 1% astaxanthin, 71 times more beta-carotene) and M7 (orange; 1% astaxanthin, 58 times more beta-carotene, 135% canthaxanthin), whereas the rest produced lower levels of astaxanthin (5-66%) than the parental strain. When the crtS gene was expressed in M7, canthaxanthin accumulation disappeared and astaxanthin production was partially restored. Moreover, astaxanthin biosynthesis was restored when X. dendrorhous ATCC 96815 was transformed with the crtS gene. The crtS gene was heterologously expressed in Mucor circinelloides conferring to this fungus an improved capacity to synthesize beta-cryptoxanthin and zeaxanthin, two hydroxylated compounds from beta-carotene. These results show that the crtS gene is involved in the conversion of beta-carotene into xanthophylls, being potentially useful to

Phenylketonuria : Tyrosine beyond the phenylalanine-restricted diet

NARCIS (Netherlands)

van Spronsen, FJ; Smit, PGA; Koch, R

Controversies exist on the role of tyrosine in the pathogenesis of phenylketonuria (PKU) and, consequently, on the therapeutic role of tyrosine. This review examines data and theoretical considerations on the role of tyrosine in the pathogenesis and treatment of PKU. It is concluded that treatment

Anti-dopamine beta-hydroxylase immunotoxin-induced sympathectomy in adult rats

Science.gov (United States)

Picklo, M. J.; Wiley, R. G.; Lonce, S.; Lappi, D. A.; Robertson, D.

1995-01-01

Anti-dopamine beta-hydroxylase immunotoxin (DHIT) is an antibody-targeted noradrenergic lesioning tool comprised of a monoclonal antibody against the noradrenergic enzyme, dopamine beta-hydroxylase, conjugated to saporin, a ribosome-inactivating protein. Noradrenergic-neuron specificity and completeness and functionality of sympathectomy were assessed. Adult, male Sprague-Dawley rats were given 28.5, 85.7, 142 or 285 micrograms/kg DHIT i.v. Three days after injection, a 6% to 73% decrease in the neurons was found in the superior cervical ganglia of the animals. No loss of sensory, nodose and dorsal root ganglia, neurons was observed at the highest dose of DHIT. In contrast, the immunotoxin, 192-saporin (142 micrograms/kg), lesioned all three ganglia. To assess the sympathectomy, 2 wk after treatment (285 micrograms/kg), rats were anesthetized with urethane (1 g/kg) and cannulated in the femoral artery and vein. DHIT-treated animals' basal systolic blood pressure and heart rate were significantly lower than controls. Basal plasma norepinephrine levels were 41% lower in DHIT-treated animals than controls. Tyramine-stimulated release of norepinephrine in DHIT-treated rats was 27% of controls. Plasma epinephrine levels of DHIT animals were not reduced. DHIT-treated animals exhibited a 2-fold hypersensitivity to the alpha-adrenergic agonist phenylephrine. We conclude that DHIT selectively delivered saporin to noradrenergic neurons resulting in destruction of these neurons. Anti-dopamine beta-hydroxylase immunotoxin administration produces a rapid, irreversible sympathectomy.

21 CFR 862.1730 - Free tyrosine test system.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Free tyrosine test system. 862.1730 Section 862....1730 Free tyrosine test system. (a) Identification. A free tyrosine test system is a device intended to measure free tyrosine (an amono acid) in serum and urine. Measurements obtained by this device are used in...

Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes

NARCIS (Netherlands)

Twisk, J.; Wit, E.C.M. de; Princen, H.M.G.

1995-01-01

In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase'

Mechanism-based inactivation of benzo[a]pyrene hydroxylase by aryl acetylenes and aryl olefins

International Nuclear Information System (INIS)

Gan, L.S.; Lu, J.Y.L.; Alworth, W.L.

1986-01-01

A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxgenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, 3-ethynylperylene, 2-ethynylfluorene, methyl 1-pyrenyl acetylene, cis- and trans-1-(2-bromovinyl)pyrene, and 1-allylpyrene serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo[a]pyrene hydroxylase, while 1-vinylpyrene and phenyl 1-pyrenyl acetylene do not cause a detectable suicide inhibition of benzo[a]pyrene hydroxylase. The mechanism-based loss of benzo[a]pyrene hydroxylase caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes (suicide destruction). The suicide inhibition by these aryl acetylenes therefore does not involve covalent binding to the heme moiety of the monooxygenase. Nevertheless, in the presence of NADPH, 3 H-labeled 1-ethynylpyrene becomes covalently attached to the cytochrome P-450 protein; the measured stoichiometry of binding is one 1-ethynylpyrene per P-450 heme unit. The authors conclude that the inhibition of benzo[a]pyrene hydroxylase produced by 1-ethynylpyrene may be related to the mechanism of suicide inhibition of P-450 activity by chloramphenicol rather than the mechanism of suicide destruction of P-450 previously described for acetylene and propyne

High Myopia Caused by a Mutation in LEPREL1, Encoding Prolyl 3-Hydroxylase 2

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Mordechai, Shikma; Gradstein, Libe; Pasanen, Annika; Ofir, Rivka; El Amour, Khalil; Levy, Jaime; Belfair, Nadav; Lifshitz, Tova; Joshua, Sara; Narkis, Ginat; Elbedour, Khalil; Myllyharju, Johanna; Birk, Ohad S.

2011-01-01

Autosomal-recessive high-grade axial myopia was diagnosed in Bedouin Israeli consanguineous kindred. Some affected individuals also had variable expressivity of early-onset cataracts, peripheral vitreo-retinal degeneration, and secondary sight loss due to severe retinal detachments. Through genome-wide linkage analysis, the disease-associated gene was mapped to ∼1.7 Mb on chromosome 3q28 (the maximum LOD score was 11.5 at θ = 0 for marker D3S1314). Sequencing of the entire coding regions and intron-exon boundaries of the six genes within the defined locus identified a single mutation (c.1523G>T) in exon 10 of LEPREL1, encoding prolyl 3-hydroxylase 2 (P3H2), a 2-oxoglutarate-dependent dioxygenase that hydroxylates collagens. The mutation affects a glycine that is conserved within P3H isozymes. Analysis of wild-type and p.Gly508Val (c.1523G>T) mutant recombinant P3H2 polypeptides expressed in insect cells showed that the mutation led to complete inactivation of P3H2. PMID:21885030

Euglena mitochondria and chloroplasts form tyrosine-O-sulfate

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Saidha, T.; Hanfstingl, U.; Schiff, J.A. (Brandeis Univ., Waltham, MA (USA))

1989-04-01

Mitochondria from light-grown wild-type Euglena gracilis var. bacillaris Cori or dark-grown mutant W{sub 10}BSmL incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, or with {sup 14}C-tyrosine, non-radioactive sulfate and ATP accumulate a labeled compound in the medium. Since this compound shows exact coelectrophoresis with tyrosine-O-sulfate (TOS) at pH 2.0, 5.8 or 8.0., yields sulfate and tyrosine on acid hydrolysis, and treatment with aryl sulfatase from Aerobacter aerogenes yields sulfate and tyrosine but no tyrosine methyl ester, it is identified as TOS. No TOS is found outside purified developing chloroplasts incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, but both chloroplasts and mitochondria form to {sup 35}S externally when incubated with adenosine 3{prime} phosphate 5{prime}phospho({sup 35}S) sulfate (PAP{sup 35}S). Since no tyrosine need be added, tyrosine is provided from endogenous sources. Although TOS is found in the free pool of Euglena cells it cannot be detected in proteins of cells or mucus ruling our sulfation of tyrosine of protein or incorporation of TOS into proteins. The system forming TOS is membrane-bound and may be involved in tyrosine transport.

Duration-dependence of the effect of treadmill exercise on hyperactivity in attention deficit hyperactivity disorder rats.

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Ji, Eun-Sang; Kim, Chang-Ju; Park, Jun Heon; Bahn, Geon Ho

2014-04-01

Attention-deficit hyperactivity disorder (ADHD) is a common neurobehavioral disorder, and its symptoms are hyperactivity and deficits in learning and memory. Physical exercise increases dopamine synthesis and neuronal activity in various brain regions. In the present study, we investigate the duration-dependence of the treadmill exercise on hyperactivity in relation with dopamine expression in ADHD. Spontaneously hypertensive rats were used for the ADHD rats and Wistar-Kyoto rats were used for the control rats. The rats in the exercise groups were forced to run on a treadmill for 10 min, 30 min, and 60 min once daily for 28 consecutive days. For this experiment, open field test and immunohistochemistry for tyrosine hydroxylase were conducted. The present results revealed that ADHD rats showed hyperactivity, and tyrosine hydroxylase expression in the striatum and substantia nigra were decreased in ADHD rats. Treadmill exercise alleviated hyperactivity and also increased TH expression in ADHD rats. Treadmill exercise for 30 min per day showed most potent suppressing effect on hyperactivity, and this dose of treadmill exercise also most potently inhibited tyrosine hydroxylase expression. The present study suggests that treadmill exercise for 30 min once a day is the most effective therapeutic intervention for ADHD patients.

Radiotherapy of non-small-cell lung cancer in the era of EGFR gene mutations and EGF receptor tyrosine kinase inhibitors.

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Moschini, Ilaria; Dell'Anna, Cristina; Losardo, Pier Luigi; Bordi, Paola; D'Abbiero, Nunziata; Tiseo, Marcello

2015-01-01

Non-small-cell lung cancer (NSCLC) occurs, approximately, in 80-85% of all cases of lung cancer. The majority of patients present locally advanced or metastatic disease when diagnosed, with poor prognosis. The discovery of activating mutations in the EGFR gene has started a new era of personalized treatment for NSCLC patients. To improve the treatment outcome in patients with unresectable NSCLC and, in particular, EGFR mutated, a combined strategy of radiotherapy and medical treatment can be undertaken. In this review we will discuss preclinical data regarding EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) and radiotherapy, available clinical trials investigating efficacy and toxicity of combined treatment (thoracic or whole brain radiotherapy and EGFR-TKIs) and, also, the role of local radiation in mutated EGFR patients who developed EGFR-TKI resistance.

No association between the protein tyrosine phosphatase, receptor-type, Z Polypeptide 1 (PTPRZ1) gene and schizophrenia in the Japanese population.

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Ito, Yoshihito; Yamada, Shinnosuke; Takahashi, Nagahide; Saito, Shinichi; Yoshimi, Akira; Inada, Toshiya; Noda, Yukihiro; Ozaki, Norio

2008-10-05

NRG1-ERBB signaling influences the risk for schizophrenia pathology. A recent study has reported that MAGI1, MAGI2, and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1; located on 7q31.3) gene products regulate the NRG1-ERBB4 signaling pathway, and PTPRZ1 is associated with schizophrenia in a Caucasian population. By applying a gene-based association concept, we analyzed any association between PTPRZ1 tagging SNPs and schizophrenia in the Japanese population (576 schizophrenics and 768 controls). After linkage disequilibrium analysis, 29 single nucleotide polymorphisms (SNPs) were genotyped using a 5'-exonuclease allelic discrimination assay. We found a significant association of one tagging SNP in a genotype-wise analysis (P = 0.007); however, this might be resulted from type I error due to multiple testing (P = 0.17 after SNPSpD correction). No association was observed between schizophrenic patients and controls in either allelic, genotypic, or haplotypic analyses. Our results therefore suggest that PTPRZ1 is unlikely to be related to the development of schizophrenia in the Japanese population.

Protein tyrosine nitration in the cell cycle

International Nuclear Information System (INIS)

Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

2011-01-01

Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

International Nuclear Information System (INIS)

Nakayama, Yuji; Kawana, Akiko; Igarashi, Asae; Yamaguchi, Naoto

2006-01-01

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to ∼200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation

Developmental changes in human dopamine neurotransmission: cortical receptors and terminators

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Rothmond Debora A

2012-02-01

Full Text Available Abstract Background Dopamine is integral to cognition, learning and memory, and dysfunctions of the frontal cortical dopamine system have been implicated in several developmental neuropsychiatric disorders. The dorsolateral prefrontal cortex (DLPFC is critical for working memory which does not fully mature until the third decade of life. Few studies have reported on the normal development of the dopamine system in human DLPFC during postnatal life. We assessed pre- and postsynaptic components of the dopamine system including tyrosine hydroxylase, the dopamine receptors (D1, D2 short and D2 long isoforms, D4, D5, catechol-O-methyltransferase, and monoamine oxidase (A and B in the developing human DLPFC (6 weeks -50 years. Results Gene expression was first analysed by microarray and then by quantitative real-time PCR. Protein expression was analysed by western blot. Protein levels for tyrosine hydroxylase peaked during the first year of life (p O-methyltransferase (p = 0.024 were significantly higher in neonates and infants as was catechol-O-methyltransferase protein (32 kDa, p = 0.027. In contrast, dopamine D1 receptor mRNA correlated positively with age (p = 0.002 and dopamine D1 receptor protein expression increased throughout development (p Conclusions We find distinct developmental changes in key components of the dopamine system in DLPFC over postnatal life. Those genes that are highly expressed during the first year of postnatal life may influence and orchestrate the early development of cortical neural circuitry while genes portraying a pattern of increasing expression with age may indicate a role in DLPFC maturation and attainment of adult levels of cognitive function.

Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: isolation, structure, synthesis, and endocrine activity.

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Mor, A; Chartrel, N; Vaudry, H; Nicolas, P

1994-10-25

Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three members of a family of structurally related peptides, are mainly expressed in the endocrine pancreas, in endocrine cells of the gut, and in the brain, respectively. In the present study, we have isolated a peptide of the pancreatic polypeptide family from the skin of the South American arboreal frog Phyllomedusa bicolor. The primary structure of the peptide was established as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu10-Asp-Ala-Ser-Pro-Glu-Glu- Met-Asn- Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Val-Thr- Arg-Gln-Arg-Tyr-NH2 . This unusual peptide, named skin peptide tyrosine-tyrosine (SPYY), exhibits 94% similarity with PYY from the frog Rana ridibunda. A synthetic replicate of SPYY inhibits melanotropin release from perifused frog neurointermediate lobes in very much the same way as NPY. These results demonstrate the occurrence of a PYY-like peptide in frog skin. Our data also suggest the existence of a pituitary-skin regulatory loop in amphibians.

Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis.

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Beaudoin, Guillaume A W; Facchini, Peter J

2013-02-15

Sanguinarine is a benzo[c]phenenthridine alkaloid with potent antimicrobial properties found commonly in plants of the Papaveraceae, including the roots of opium poppy (Papaver somniferum). Sanguinarine is formed from the central 1-benzylisoquinoline intermediate (S)-reticuline via the protoberberine alkaloid (S)-scoulerine, which undergoes five enzymatic oxidations and an N-methylation. The first four oxidations from (S)-scoulerine are catalyzed by cytochromes P450, whereas the final conversion involves a flavoprotein oxidase. All but one gene in the biosynthetic pathway from (S)-reticuline to sanguinarine has been identified. In this communication, we report the isolation and characterization of (S)-cis-N-methylstylopine 14-hydroxylase (MSH) from opium poppy based on the transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding gene. Along with protopine 6-hydroxylase, which catalyzes the subsequent and penultimate step in sanguinarine biosynthesis, MSH is a member of the CYP82N subfamily of cytochromes P450. The full-length MSH cDNA was expressed in Saccharomyces cerevisiae and the recombinant microsomal protein was tested for enzymatic activity using 25 benzylisoquinoline alkaloids representing a wide range of structural subgroups. The only enzymatic substrates were the N-methylated protoberberine alkaloids N-methylstylopine and N-methylcanadine, which were converted to protopine and allocryptopine, respectively. Copyright © 2013. Published by Elsevier Inc.

 Mutations of noncollagen genes in osteogenesis imperfecta – implications of the gene products in collagen biosynthesis and pathogenesis of disease

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Anna Galicka

2012-06-01

Full Text Available  Recent investigations revealed that the “brittle bone” phenotype in osteogenesis imperfecta (OI is caused not only by dominant mutations in collagen type I genes, but also by recessively inherited mutations in genes responsible for the post-translational processing of type I procollagen as well as for bone formation. The phenotype of patients with mutations in noncollagen genes overlaps with very severe type III and lethal type II OI caused by mutations in collagen genes. Mutations in genes that encode proteins involved in collagen prolyl 3-hydroxylation (P3H1/CRTAP/CyPB eliminated Pro986 hydroxylation and caused an increase in modification of collagen helix by prolyl 4-hydroxylase and lysyl hydroxylase. However, the importance of these disturbances in the disease pathomechanism is not known. Loss of complex proteins’ function as collagen chaperones may dominate the disease mechanism. The latest findings added to the spectrum of OI-causing and collagen-influencing factors other chaperones (HSP47 and FKBP65 and protein BMP-1, which emphasizes the complexity of collagen folding and secretion as well as their importance in bone formation. Furthermore, mutations in genes encoding transcription factor SP7/Osterix and pigment epithelium-derived factor (PEDF constitute a novel mechanism for OI, which is independent of changes in biosynthesis and processing of collagen.

Characterization of the β-Carotene Hydroxylase Gene DSM2 Conferring Drought and Oxidative Stress Resistance by Increasing Xanthophylls and Abscisic Acid Synthesis in Rice1[C][W][OA

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Du, Hao; Wang, Nili; Cui, Fei; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

2010-01-01

Drought is a major limiting factor for crop production. To identify critical genes for drought resistance in rice (Oryza sativa), we screened T-DNA mutants and identified a drought-hypersensitive mutant, dsm2. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative β-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2 mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem II and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2 in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress-related ABA-responsive genes were up-regulated in the overexpression line. DSM2 is a chloroplast protein, and the response of DSM2 to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2 gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice. PMID:20852032

Tyrosine phosphorylation of Grb14 by Tie2

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Dumont Daniel J

2010-10-01

Full Text Available Abstract Background Growth factor receptor bound (Grb proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK. Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported. Results Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106 on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP Ang1. Conclusion Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor.

Tyrosine kinase inhibition: A therapeutic target for the management of chronic-phase chronic myeloid leukemia

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Jabbour, Elias J; Cortes, Jorge E; Kantarjian, Hagop M

2014-01-01

Chronic myeloid leukemia (CML) is a hematologic neoplasm with a progressive, ultimately terminal, disease course. In most cases, CML arises owing to the aberrant formation of a chimeric gene for a constitutively active tyrosine kinase. Inhibition of the signaling activity of this kinase has proved to be a highly successful treatment target transforming the prognosis of patients with CML. New tyrosine kinase inhibitors (TKIs) continue to improve the management of CML, offering alternative options for those resistant to or intolerant of standard TKIs. Here we review the pathobiology of CML and explore emerging strategies to optimize the management of chronic-phase CML, particularly first-line treatment. PMID:24236822

Deoxysarpagine hydroxylase--a novel enzyme closing a short side pathway of alkaloid biosynthesis in Rauvolfia.

Science.gov (United States)

Yu, Bingwu; Ruppert, Martin; Stöckigt, Joachim

2002-08-01

Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at -25 degrees C for >3 months. The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited taxonomic distribution of this enzyme.

Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae

Science.gov (United States)

2013-01-01

Background Xanthophylls, oxygenated derivatives of carotenes, play critical roles in photosynthetic apparatus of cyanobacteria, algae, and higher plants. Although the xanthophylls biosynthetic pathway of algae is largely unknown, it is of particular interest because they have a very complicated evolutionary history. Carotenoid hydroxylase (CHY) is an important protein that plays essential roles in xanthophylls biosynthesis. With the availability of 18 sequenced algal genomes, we performed a comprehensive comparative analysis of chy genes and explored their distribution, structure, evolution, origins, and expression. Results Overall 60 putative chy genes were identified and classified into two major subfamilies (bch and cyp97) according to their domain structures. Genes in the bch subfamily were found in 10 green algae and 1 red alga, but absent in other algae. In the phylogenetic tree, bch genes of green algae and higher plants share a common ancestor and are of non-cyanobacterial origin, whereas that of red algae is of cyanobacteria. The homologs of cyp97a/c genes were widespread only in green algae, while cyp97b paralogs were seen in most of algae. Phylogenetic analysis on cyp97 genes supported the hypothesis that cyp97b is an ancient gene originated before the formation of extant algal groups. The cyp97a gene is more closely related to cyp97c in evolution than to cyp97b. The two cyp97 genes were isolated from the green alga Haematococcus pluvialis, and transcriptional expression profiles of chy genes were observed under high light stress of different wavelength. Conclusions Green algae received a β-xanthophylls biosynthetic pathway from host organisms. Although red algae inherited the pathway from cyanobacteria during primary endosymbiosis, it remains unclear in Chromalveolates. The α-xanthophylls biosynthetic pathway is a common feature in green algae and higher plants. The origination of cyp97a/c is most likely due to gene duplication before divergence of

Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae.

Science.gov (United States)

Cui, Hongli; Yu, Xiaona; Wang, Yan; Cui, Yulin; Li, Xueqin; Liu, Zhaopu; Qin, Song

2013-07-08

Xanthophylls, oxygenated derivatives of carotenes, play critical roles in photosynthetic apparatus of cyanobacteria, algae, and higher plants. Although the xanthophylls biosynthetic pathway of algae is largely unknown, it is of particular interest because they have a very complicated evolutionary history. Carotenoid hydroxylase (CHY) is an important protein that plays essential roles in xanthophylls biosynthesis. With the availability of 18 sequenced algal genomes, we performed a comprehensive comparative analysis of chy genes and explored their distribution, structure, evolution, origins, and expression. Overall 60 putative chy genes were identified and classified into two major subfamilies (bch and cyp97) according to their domain structures. Genes in the bch subfamily were found in 10 green algae and 1 red alga, but absent in other algae. In the phylogenetic tree, bch genes of green algae and higher plants share a common ancestor and are of non-cyanobacterial origin, whereas that of red algae is of cyanobacteria. The homologs of cyp97a/c genes were widespread only in green algae, while cyp97b paralogs were seen in most of algae. Phylogenetic analysis on cyp97 genes supported the hypothesis that cyp97b is an ancient gene originated before the formation of extant algal groups. The cyp97a gene is more closely related to cyp97c in evolution than to cyp97b. The two cyp97 genes were isolated from the green alga Haematococcus pluvialis, and transcriptional expression profiles of chy genes were observed under high light stress of different wavelength. Green algae received a β-xanthophylls biosynthetic pathway from host organisms. Although red algae inherited the pathway from cyanobacteria during primary endosymbiosis, it remains unclear in Chromalveolates. The α-xanthophylls biosynthetic pathway is a common feature in green algae and higher plants. The origination of cyp97a/c is most likely due to gene duplication before divergence of green algae and higher plants

Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

International Nuclear Information System (INIS)

Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

2013-01-01

Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates

Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

Energy Technology Data Exchange (ETDEWEB)

Li, Bin, E-mail: binli@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Eyer, Peter, E-mail: peter.eyer@lrz.uni-muenchen.de [Walther-Straub-Institut Für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, 80336 München (Germany); Eddleston, Michael, E-mail: M.Eddleston@ed.ac.uk [Clinical Pharmacology Unit, University of Edinburgh, Edinburgh (United Kingdom); Jiang, Wei, E-mail: wjiang@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Schopfer, Lawrence M., E-mail: lmschopf@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Lockridge, Oksana, E-mail: olockrid@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States)

2013-06-15

Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

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Fernanda Ledda

2007-01-01

Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

Raman scattering tensors of tyrosine.

Science.gov (United States)

Tsuboi, M; Ezaki, Y; Aida, M; Suzuki, M; Yimit, A; Ushizawa, K; Ueda, T

1998-01-01

Polarized Raman scattering measurements have been made of a single crystal of L-tyrosine by the use of a Raman microscope with the 488.0-nm exciting beam from an argon ion laser. The L-tyrosine crystal belongs to the space group P2(1)2(1)2(1) (orthorhombic), and Raman scattering intensities corresponding to the aa, bb, cc, ab and ac components of the crystal Raman tensor have been determined for each prominent Raman band. A similar set of measurements has been made of L-tyrosine-d4, in which four hydrogen atoms on the benzene ring are replaced by deuterium atoms. The effects of NH3-->ND3 and OH-->OD on the Raman spectrum have also been examined. In addition, depolarization ratios of some bands of L-tyrosine in aqueous solutions of pH 13 and pH 1 were examined. For comparison with these experimental results, on the other hand, ab initio molecular orbital calculations have been made of the normal modes of vibration and their associated polarizability oscillations of the L-tyrosine molecule. On the basis of these experimental data and by referring to the results of the calculations, discussions have been presented on the Raman tensors associated to some Raman bands, including those at 829 cm-1 (benzene ring breathing), 642 cm-1 (benzene ring deformation), and 432 cm-1 (C alpha-C beta-C gamma bending).

Phytanoyl-CoA hydroxylase activity is induced by phytanic acid

NARCIS (Netherlands)

Zomer, A. W.; Jansen, G. A.; van der Burg, B.; Verhoeven, N. M.; Jakobs, C.; van der Saag, P. T.; Wanders, R. J.; Poll-The, B. T.

2000-01-01

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid present in various dietary products such as milk, cheese and fish. In patients with Refsum disease, accumulation of phytanic acid occurs due to a deficiency of phytanoyl-CoA hydroxylase, a peroxisomal enzyme

Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression in U937 cells.

Science.gov (United States)

Chihara, Kazuyasu; Kato, Yuji; Yoshiki, Hatsumi; Takeuchi, Kenji; Fujieda, Shigeharu; Sada, Kiyonao

2017-09-13

The adaptor protein c-Abl SH3 domain binding protein-2 (3BP2) is tyrosine phosphorylated by Syk in response to cross-linking of antigen receptors, which in turn activates various immune responses. Recently, a study using the mouse model of cherubism, a dominant inherited disorder caused by mutations in the gene encoding 3BP2, showed that 3BP2 is involved in the regulation of phagocytosis mediated by Fc receptor for IgG (FcγR) in macrophages. However, the molecular mechanisms underlying 3BP2-mediated regulation of phagocytosis and the physiological relevance of 3BP2 tyrosine phosphorylation remains elusive. In this study, we established various gene knockout U937 cell lines using the CRISPR/Cas9 system and found that 3BP2 is rapidly tyrosine phosphorylated by Syk in response to cross-linking of FcγRI. Depletion of 3BP2 caused significant reduction in the Fc receptor γ chain (FcRγ)-mediated phagocytosis in addition to the FcγRI-mediated induction of chemokine mRNA for IL-8, CCL3L3 and CCL4L2. Syk-dependent tyrosine phosphorylation of 3BP2 was required for overcoming these defects. Finally, we found that the PH and SH2 domains play important roles on FcγRI-mediated tyrosine phosphorylation of 3BP2 in HL-60 cells. Taken together, these results indicate that Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression.

Tyrosine phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

Science.gov (United States)

Zhong, Li; Li, Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.; Weigel-Van Aken, Kirsten A.; Hobbs, Jacqueline A.; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun

2008-01-01

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ~68% and ~74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which leads to ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy. PMID:18834608

Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

International Nuclear Information System (INIS)

Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

2008-01-01

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

[Recommendations for the diagnosis and treatment of classic forms of 21-hydroxylase-deficient congenital adrenal hyperplasia].

Science.gov (United States)

Rodríguez, Amparo; Ezquieta, Begoña; Labarta, José Igancio; Clemente, María; Espino, Rafael; Rodriguez, Amaia; Escribano, Aranzazu

2017-08-01

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is an autosomal recessive disorder caused by mutations in the CYP21A2 gene. Cortisol and aldosterone synthesis are impaired in the classic forms (adrenal insufficiency and salt-wasting crisis). Females affected are virilised at birth, and are at risk for genital ambiguity. In this article we give recommendations for an early as possible diagnosis and an appropriate and individualised treatment. A patient and family genetic study is essential for the diagnosis of the patient, and allows genetic counselling, as well as a prenatal diagnosis and treatment for future pregnancy. Copyright © 2016 Asociación Española de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.

Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

Science.gov (United States)

Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

2013-01-01

Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. PMID:23566956

Rat-liver cholesterol 7α-hydroxylase. Pt. 1

International Nuclear Information System (INIS)

Cantfort, J. van; Renson, J.; Gielen, J.

1975-01-01

A new assay is described to measure the activity of cholesterol 7α-hydroxylase and compared to the conventional 14 C method used by other investigators. This method is based on the mechanism of the enzymic hydroxylation, i.e. a direct and stereospecific substitution of the 7α-hydrogen by a hydroxyl group. [7α- 3 H]cholesterol is incubated at 37 0 C and in the presence of molecular O 2 , in a medium buffered by potassium phosphate at pH 7.4 and containing liver microsomes (or 9,000 x g supernatant), NADPH, MgCl 2 and cysteamine. Tween-80 (1.5 mg/ml) is used to introduce enough substrate (300 μM) in the incubation mixture to saturate the ezyme (K(m) = 100 μM). Under these conditions the tritiated water released into the incubation medium reflects accurately the enzymic activity. The results obtained with this method are similar to the one obtained with a [4- 14 C]cholesterol technique (r = 0.96; P 3 H]cholesterol method is a complete independence from further metabolism of the first enzymic product, the 7α-hydroxycholesterol, the tritiated water representing the entire cholesterol 7α-hydroxylase activity. (orig.) [de

Combinatorial analysis of enzymatic bottlenecks of L-tyrosine pathway by p-coumaric acid production in Saccharomyces cerevisiae.

Science.gov (United States)

Mao, Jiwei; Liu, Quanli; Song, Xiaofei; Wang, Hesuiyuan; Feng, Hui; Xu, Haijin; Qiao, Mingqiang

2017-07-01

To identify new enzymatic bottlenecks of L-tyrosine pathway for further improving the production of L-tyrosine and its derivatives. When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l -1 , respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l -1 ) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain. Combinatorial metabolic engineering provides a new strategy for further improvement of L-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

Influence of neonatal and adult hyperthyroidism on behavior and biosynthetic capacity for norepinephrine, dopamine and 5-hydroxytryptamine in rat brain.

Science.gov (United States)

Rastogi, R B; Singhal, R L

1976-09-01

In neonatal rats, administration of l-triiodothyronine (10 mug/100 g/day) for 30 days presented signs of hyperthyroidism which included accelerated development of a variety of physical and behavioral characteristics accompanying maturation. The spontaneous motor activity was increased by 69%. Exposure of developing rats to thyroid hormone significantly increased the endogenous concentration of striatal tyrosine and the activity of tyrosine hydroxylase as well as the levels of dopamine in several brain regions. The concentration of striatal homovanillic acid and 3,4-dihydroxyphenylacetic acid, the chief metabolites of dopamine, was also increased and the magnitude of change was greater than the rise in dopamine. Despite increases in the activity of tyrosine hydroxylase and the availability of the substrate tyrosine, the steady-state levels of norepinephrine remained unaltered in various regions of brain except in cerebellum. Futhermore, neonatal hyperthyroidism significantly increased the levels of midbrain tryptophan and tryptophan hydroxylase activity but produced no change in 5-hydroxytryptamine levels of several discrete brain regions, except hypothalamus and cerebellum where its concentration was slightly decreased. However, the 5-hydroxyindoleacetic acid levels were enhanced in hypothalamus, ponsmedulla, midbrain, striatum and hippocampus. The elevated levels of 5-hydroxyindoleacetic acid did not seem to be due to increased intraneuronal deamination of 5-hydroxytryptamine since monoamine oxidase activity was not affected in cerebral cortex and midbrain of hyperthyroid rats. The data demonstrate that hyperthyroidism significantly increased the synthesis as well as the utilization of catecholamines and 5-hydroxytryptamine in maturing brain. Since the mature brain is known to respond differently to thyroid hormone action than does the developing brain, the effect of L-triiodothyronine treatment on various putative neurohumors also was examined in adult rats

Conversion of p-tyrosine to p-tyramine in the isolated perfused rat kidney: Modulation by perfusate concentrations of p-tyrosine

International Nuclear Information System (INIS)

Brier, M.E.; Bowsher, R.R.; Henry, D.P.; Mayer, P.R.

1991-01-01

The authors used the isolated perfused rat kidney to evaluate the role of renal decarboxylation of p-tyrosine as the source of urinary p-tyramine. Kidneys were perfused with concentrations of p-tyrosine ranging from 0.02 mM to 2.0 mM. p-Tyramine was measured by a sensitive and specific radioenzymatic assay. An increase in the perfusate concentration of p-tyrosine resulted in a significant increase in p-tyramine production that was blocked by the addition of NSD-1015, an inhibitor of aromatic-1-amino decarboxylase (AADC). They conclude p-tyrosine is the precursor for the renal production of p-tyramine, renal AADC catalyzes the formation of urinary p-tyramine, synthesized p-tyramine is predominantly excreted in the urine, and p-tyramine synthesis is modulated by the arterial delivery of p-tyrosine to the kidney

Kinetic mechanism and isotope effects of Pseudomonas cepacia 3-hydroxybenzoate-t-hydroxylase

International Nuclear Information System (INIS)

Wang, L.H.; Yu, Y.; Hamzah, R.Y.; Tu, S.C.

1986-01-01

The kinetic mechanism of Pseudomonas cepacia 3-hydroxybenzoate-6-hydroxylase has been delineated. Double reciprocal plots of initial rate versus m-hydroxybenzoate concentration at a constant level of oxygen and several fixed concentrations of NADH yielded a set of converging lines. Similar reciprocal plots of velocity versus NADH concentration at a constant oxygen level and several fixed m-hydroxybenzoate concentrations also showed converging lines. In contrast, double reciprocal plots of initial rate versus NADH concentration at a fixed m-hydroxybenzoate level and several oxygen concentrations showed a series of parallel lines. Parallel lines were also obtained from double reciprocal plots of initial rate versus m-hydroxybenzoate concentration at a fixed NADH level and varying oxygen concentrations. These results suggest a sequential binding of m-hydroxybenzoate and NADH by the hydroxylase. The enzyme-bound FAD is reduced and NAD is released. The reduced enzyme subsequently reacts with oxygen leading to the formation of other products. This hydroxylase exhibited a primary isotope effect of /sup D/V = 3.5 for (4R)-[4- 2 H] NADH but no isotope effect was observed with (4S)-[4- 2 H]NADH. An isotope effect of /sup T/V/K = 5.0 was also observed using (4R)-[4- 3 H]NADH. This tritium isotope effect was apparently independent of m-hydroxybenzoate concentration

Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

International Nuclear Information System (INIS)

Devaux, Patricia; Messling, Veronika von; Songsungthong, Warangkhana; Springfeld, Christoph; Cattaneo, Roberto

2007-01-01

The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Our study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein

Large daily fluctuations in plasma tyrosine in treated patients with phenylketonuria

NARCIS (Netherlands)

vanSpronsen, FJ; vanDijk, T; Smit, GPA; vanRijn, M; Reijngoud, DJ; Berger, Ruud; Heymans, HSA

1996-01-01

In patients with phenylketonuria (PKU), extra tyrosine supplementation is advocated in addition to tyrosine-enriched amino acid mixtures. PKU patients have low fasting plasma tyrosine concentrations, but little is known about tyrosine fluctuations during the day. Plasma tyrosine concentrations were

Dopaminergic Neuron-Specific Deletion of p53 Gene Attenuates Methamphetamine Neurotoxicity.

Science.gov (United States)

Lu, Tao; Kim, Paul P; Greig, Nigel H; Luo, Yu

2017-08-01

p53 plays an essential role in the regulation of cell death in dopaminergic (DA) neurons and its activation has been implicated in the neurotoxic effects of methamphetamine (MA). However, how p53 mediates MA neurotoxicity remains largely unknown. In this study, we examined the effect of DA-specific p53 gene deletion in DAT-p53KO mice. Whereas in vivo MA binge exposure reduced locomotor activity in wild-type (WT) mice, this was significantly attenuated in DAT-p53KO mice and associated with significant differences in the levels of the p53 target genes BAX and p21 between WT and DAT-p53KO. Notably, DA-specific deletion of p53 provided protection of substantia nigra pars reticulata (SNpr) tyrosine hydroxylase (TH) positive fibers following binge MA, with DAT-p53KO mice having less decline of TH protein levels in striatum versus WT mice. Whereas DAT-p53KO mice demonstrated a consistently higher density of TH fibers in striatum compared to WT mice at 10 days after MA exposure, DA neuron counts within the substantia nigra pars compacta (SNpc) were similar. Finally, supportive of these results, administration of a p53-specific inhibitor (PFT-α) provided a similarly protective effect on MA binge-induced behavioral deficits. Neither DA specific p53 deletion nor p53 pharmacological inhibition affected hyperthermia induced by MA binge. These findings demonstrate a specific contribution of p53 activation in behavioral deficits and DA neuronal terminal loss by MA binge exposure.

The tyrosine B10 hydroxyl is crucial for oxygen avidity of Ascaris hemoglobin.

Science.gov (United States)

Kloek, A P; Yang, J; Mathews, F S; Frieden, C; Goldberg, D E

1994-01-28

The parasitic nematode Ascaris suum has a gene encoding a two-domain hemoglobin with remarkable oxygen avidity. The strong interaction with oxygen is a consequence of a particularly slow oxygen off-rate. The single polypeptide chain consists of two domains, each of which can be expressed separately in Escherichia coli as a globin-like protein exhibiting oxygen binding characteristics comparable with the native molecule. Site-directed mutagenesis was performed on the gene segment encoding domain one. The E7 position, involved in forming a hydrogen bond with the liganded oxygen in vertebrate globins, is a glutamine in both Ascaris domains. Conversion of this residue to leucine or alanine produced a hemoglobin variant with an oxygen off-rate 5- or 60-fold faster than that of unaltered domain one. Replacement of the tyrosine B10 with either phenylalanine or leucine (as found in vertebrate globins) yielded hemoglobin mutants with oxygen off-rates 280- or 570-fold faster, approaching rates found with vertebrate myoglobins. The data suggest that the distal glutamine hydrogen bonds with the liganded oxygen and that the tyrosine B10 hydroxyl contributes an additional hydrogen bond that appears substantially responsible for the extreme oxygen avidity of Ascaris hemoglobin.

Characterization and Two-Dimensional Crystallization of Membrane Component AlkB of the Medium-Chain Alkane Hydroxylase System from Pseudomonas putida GPo1

OpenAIRE

Alonso, Hernan; Roujeinikova, Anna

2012-01-01

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the no...

Importance of tyrosine phosphorylation in receptor kinase complexes.

Science.gov (United States)

Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

2015-05-01

Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

Science.gov (United States)

Lu, D; Yang, H; Raizada, M K

1996-12-01

Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene.

Science.gov (United States)

Park, Jeong-Woong; Song, Ki-Duk; Kim, Nam Young; Choi, Jae-Young; Hong, Seul A; Oh, Jin Hyeog; Kim, Si Won; Lee, Jeong Hyo; Park, Tae Sub; Kim, Jin-Kyoo; Kim, Jong Geun; Cho, Byung-Wook

2017-10-01

Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase ( AXL ) gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form) in equine skeletal muscle to gain insight(s) into the role of each alternative transcript during exercise. We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR). Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D) prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3) and immunoglobin (Ig) domain was different between two alternative isoforms. It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s) and significance of the alternative splicing isoforms in race horse skeletal muscle.

Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene

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Jeong-Woong Park

2017-10-01

Full Text Available Objective Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase (AXL gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form in equine skeletal muscle to gain insight(s into the role of each alternative transcript during exercise. Methods We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR, and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR. Results Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3 and immunoglobin (Ig domain was different between two alternative isoforms. Conclusion It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s and significance of the alternative splicing isoforms in race horse skeletal muscle.

Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy.

Science.gov (United States)

Moyle, Louise A; Blanc, Eric; Jaka, Oihane; Prueller, Johanna; Banerji, Christopher Rs; Tedesco, Francesco Saverio; Harridge, Stephen Dr; Knight, Robert D; Zammit, Peter S

2016-11-14

Facioscapulohumeral muscular dystrophy (FSHD) involves sporadic expression of DUX4, which inhibits myogenesis and is pro-apoptotic. To identify target genes, we over-expressed DUX4 in myoblasts and found that the receptor tyrosine kinase Ret was significantly up-regulated, suggesting a role in FSHD. RET is dynamically expressed during myogenic progression in mouse and human myoblasts. Constitutive expression of either RET9 or RET51 increased myoblast proliferation, whereas siRNA-mediated knockdown of Ret induced myogenic differentiation. Suppressing RET activity using Sunitinib, a clinically-approved tyrosine kinase inhibitor, rescued differentiation in both DUX4-expressing murine myoblasts and in FSHD patient-derived myoblasts. Importantly, Sunitinib also increased engraftment and differentiation of FSHD myoblasts in regenerating mouse muscle. Thus, DUX4-mediated activation of Ret prevents myogenic differentiation and could contribute to FSHD pathology by preventing satellite cell-mediated repair. Rescue of DUX4-induced pathology by Sunitinib highlights the therapeutic potential of tyrosine kinase inhibitors for treatment of FSHD.

Disruption of Ttll5/stamp gene (tubulin tyrosine ligase-like protein 5/SRC-1 and TIF2-associated modulatory protein gene) in male mice causes sperm malformation and infertility.

Science.gov (United States)

Lee, Geun-Shik; He, Yuanzheng; Dougherty, Edward J; Jimenez-Movilla, Maria; Avella, Matteo; Grullon, Sean; Sharlin, David S; Guo, Chunhua; Blackford, John A; Awasthi, Smita; Zhang, Zhenhuan; Armstrong, Stephen P; London, Edra C; Chen, Weiping; Dean, Jurrien; Simons, S Stoney

2013-05-24

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp(tm/tm)) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp(tm/tm) sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp(tm/tm) males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.

Disruption of Ttll5/Stamp Gene (Tubulin Tyrosine Ligase-like Protein 5/SRC-1 and TIF2-associated Modulatory Protein Gene) in Male Mice Causes Sperm Malformation and Infertility*

Science.gov (United States)

Lee, Geun-Shik; He, Yuanzheng; Dougherty, Edward J.; Jimenez-Movilla, Maria; Avella, Matteo; Grullon, Sean; Sharlin, David S.; Guo, Chunhua; Blackford, John A.; Awasthi, Smita; Zhang, Zhenhuan; Armstrong, Stephen P.; London, Edra C.; Chen, Weiping; Dean, Jurrien; Simons, S. Stoney

2013-01-01

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamptm/tm) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamptm/tm sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamptm/tm males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility. PMID:23558686

SH3 domain tyrosine phosphorylation--sites, role and evolution.

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Zuzana Tatárová

Full Text Available BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F. This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

Therapeutic Potential of a Prolyl Hydroxylase Inhibitor FG-4592 for Parkinson’s Diseases in Vitro and in Vivo: Regulation of Redox Biology and Mitochondrial Function

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Xuan Li

2018-04-01

Full Text Available As the main transcription factor that regulates the cellular responses to hypoxia, Hypoxia-inducible factor-1α (HIF-1α plays an important role in the pathogenesis of Parkinson’s disease (PD. HIF-1α is normally degraded through ubiquitination after hydroxylation by prolyl hydroxylases (PHD. Emerging evidence has suggested that HIF PHD inhibitors (HIF-PHI may have neuroprotective effects on PD through increasing HIF-1α levels. However, the therapeutic benefit of HIF-PHI for PD remains poorly explored due to the lack of proper clinical compounds and understanding of the underlying molecular mechanisms. In this study, we examined the therapeutic benefit of a new HIF-PHI, FG-4592, which is currently in phase 3 clinical trials to treat anemia in patients with chronic kidney diseases (CKD in PD models. FG-4592 attenuates MPP+ -induced apoptosis and loss of tyrosine hydroxylase (TH in SH-SY5Y cells. Pretreatment with FG-4592 mitigates MPP+-induced loss of mitochondrial membrane potential (MMP, mitochondrial oxygen consumption rate (OCR, production of reactive oxygen species (ROS and ATP. Furthermore, FG-4592 counterbalances the oxidative stress through up-regulating nuclear factor erythroid 2 p45-related factor 2 (Nrf-2, heme oxygenase-1 (HO-1 and superoxide dismutase 2 (SOD2. FG-4592 treatment also induces the expression of Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α through increasing the phosphorylation of AMP-activated protein kinase (AMPK. In MPTP-treated mice, FG-4592 protects against MPTP-induced loss of TH-positive neurons of substantia nigra and attenuates behavioral impairments. Collectively, our study demonstrates that FG-4592 is a promising therapeutic strategy for PD through improving the mitochondrial function under oxidative stress.

Lack of Association between Dopamine Beta-Hydroxylase (DBH 19-bp Insertion/Deletion Polymorphism and Risk of Schizophrenia

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Mansour shakiba

2016-12-01

Full Text Available Objective: Interaction between genetic and environmental factors is considered as major factors in Schizophrenia (SCZ. It has been shown that dopaminergic and noradrenergic neurotransmission dysfunction play an essential role in the SCZ pathogenesis.This study aimed to find the impact of functional 19-bp insertion/deletion (ins/del polymorphism in dopamine beta-hydroxylase (DBH gene on SCZ risk in a sample of Iranian population.Method: This case-control study was conducted on 109 SCZ patients and 116 matched healthy subjects. Genomic DNA samples were extracted from peripheral blood cells using salting out method. Genotyping of 19-bp ins/del DBH polymorphism was done using Polymerase Chain Reaction (PCR method.Results: Neither the overall chi-square comparison of cases and controls (

Positron Emission Tomography Imaging Demonstrates Correlation between Behavioral Recovery and Correction of Dopamine Neurotransmission after Gene Therapy

International Nuclear Information System (INIS)

Leriche, L.; Besret, L.; Gregoire, M.C.; Deglon, N.; Hantraye, Ph.; Leriche, L.; Besret, L.; Gregoire, M.C.; Deglon, N.; Hantraye, Ph.; Bjorklund, T.; Breysse, N.; Carlsson, T.; Kirik, D.; Dolle, F.; Mandel, R.J.; Kirik, D.

2009-01-01

In vivo gene transfer using viral vectors is an emerging therapy for neuro-degenerative diseases with a clinical impact recently demonstrated in Parkinson's disease patients. Recombinant adeno-associated viral (rAAV) vectors, in particular, provide an excellent tool for long-term expression of therapeutic genes in the brain. Here we used the [ 11 C]raclopride [(S)-(-)-3, 5-dichloro-N-((1-ethyl-2-pyrrolidinyl)methyl)-2-hydroxy- 6-methoxybenzamide] micro-positron emission tomography (PET) technique to demonstrate that delivery of the tyrosine hydroxylase (TH) and GTP-cyclohydrolase 1 (GCH1) enzymes using an rAAV5 vector normalizes the increased [ 11 C]raclopride binding in hemi-parkinsonian rats. Importantly, we show in vivo by micro-PET imaging and postmortem by classical binding assays performed in the very same animals that the changes in [ 11 C]raclopride after viral vector-based enzyme replacement therapy is attributable to a decrease in the affinity of the tracer binding to the D2 receptors, providing evidence for reconstitution of a functional pool of endogenous dopamine in the striatum. Moreover, the extent of the normalization in this non-invasive imaging measure was highly correlated with the functional recovery in motor behavior. The PET imaging protocol used in this study is fully adaptable to humans and thus can serve as an in vivo imaging technique to follow TH+GCH1 gene therapy in PD patients and provide an additional objective measure to a potential clinical trial using rAAV vectors to deliver L-3, 4-dihydroxyphenylalanine in the brain. (authors)

Genes up-regulated during red coloration in UV-B irradiated lettuce leaves.

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Park, Jong-Sug; Choung, Myoung-Gun; Kim, Jung-Bong; Hahn, Bum-Soo; Kim, Jong-Bum; Bae, Shin-Chul; Roh, Kyung-Hee; Kim, Yong-Hwan; Cheon, Choong-Ill; Sung, Mi-Kyung; Cho, Kang-Jin

2007-04-01

Molecular analysis of gene expression differences between green and red lettuce leaves was performed using the SSH method. BlastX comparisons of subtractive expressed sequence tags (ESTs) indicated that 7.6% of clones encoded enzymes involved in secondary metabolism. Such clones had a particularly high abundance of flavonoid-metabolism proteins (6.5%). Following SSH, 566 clones were rescreened for differential gene expression using dot-blot hybridization. Of these, 53 were found to overexpressed during red coloration. The up-regulated expression of six genes was confirmed by Northern blot analyses. The expression of chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and dihydroflavonol 4-reductase (DFR) genes showed a positive correlation with anthocyanin accumulation in UV-B-irradiated lettuce leaves; flavonoid 3',5'-hydroxylase (F3',5'H) and anthocyanidin synthase (ANS) were expressed continuously in both samples. These results indicated that the genes CHS, F3H, and DFR coincided with increases in anthocyanin accumulation during the red coloration of lettuce leaves. This study show a relationship between red coloration and the expression of up-regulated genes in lettuce. The subtractive cDNA library and EST database described in this study represent a valuable resource for further research for secondary metabolism in the vegetable crops.

Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

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Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

2014-01-01

Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthesis of 14C-labelled α-methyl tyrosine

International Nuclear Information System (INIS)

Rajagopal, S.; Venkatachalam, T.K.; Conway, T.; Diksic, M.

1992-01-01

A new route for the preparation of radioactively labelled α-methyl L-tyrosine is described. The labelling at the α position has been successfully achieved with 14 C-, 11 C- (very preliminary, unpublished), and 3 H-labelled methyl iodide. A detailed report on 14 C-labelling at the α position and the hydrolysis of 4-methoxy α-methyl phenylalanine is presented. The alkylation proceeds via the methylation of the carbanion of N-benzylidene 4-methoxy phenylalanine methyl ester 2. Hydrolysis of 4-O methyl tyrosine to tyrosine by HBr and HI were analysed and used in the optimization of the hydrolysis conditions of 4. Enantiomeric purity of the isolated L-isomer has been found to be 99% as judged by HPLC. Pseudo first-order rate constant for the hydrolysis of 14 C-labelled α-methyl 4-methoxy phenyl alanine methyl ester was determined. Preliminary findings of the 3 H- and 11 C-radiolabelled α-methyl tyrosine (methyl labelled) are also mentioned. For the first time it was shown that α-methyl D,L-tyrosine can be separated into enantiomerically pure α-methyl D- and L-tyrosine using a CHIRALPAK WH column. (author)

Crosstalk between G protein-coupled receptors (GPCRs and tyrosine kinase receptor (TXR in the heart after morphine withdrawal

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Pilar eAlmela

2013-12-01

Full Text Available G protein-coupled receptors (GPCRs comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signalling, resulting in high expression of protein kinase (PK A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK, one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl-5-isoquinolinesulfonamide (HA-1004, a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

Double labelling immunohistochemical characterization of autonomic sympathetic neurons innervating the sow retractor clitoridis muscle

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L Ragionieri

2009-08-01

Full Text Available Retrograde neuronal tracing and immunohistochemical methods were used to define the neurochemical content of sympathetic neurons projecting to the sow retractor clitoridis muscle (RCM. Differently from the other smooth muscles of genital organs, the RCM is an isolated muscle that is tonically contracted in the rest phase and relaxed in the active phase. This peculiarity makes it an interesting experimental model. The fluorescent tracer fast blue was injected into the RCM of three 50 kg subjects. After a one-week survival period, the ipsilateral paravertebral ganglion S1, that in a preliminary study showed the greatest number of cells projecting to the muscle, was collected from each animal. The co-existence of tyrosine hydroxylase with choline acetyltransferase, neuronal nitric oxide synthase, calcitonin gene-related peptide, leuenkephalin, neuropeptide Y, substance P and vasoactive intestinal polypeptide was studied under a fluorescent microscope on cryostat sections. Tyrosine hydroxylase was present in about 58% of the neurons projecting to the muscle and was found to be co-localized with each of the other tested substances.Within fast blue-labelled cells negative to the adrenergic marker, small populations of neurons singularly containing each of the other enzymatic markers or peptides were also observed. The present study documents the complexity of the neurochemical interactions that regulate the activity of the smooth myocytes of the RCM and their vascular components.

Lack of tryptophan hydroxylase-1 in mice results in gait abnormalities.

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Suidan, Georgette L; Duerschmied, Daniel; Dillon, Gregory M; Vanderhorst, Veronique; Hampton, Thomas G; Wong, Siu Ling; Voorhees, Jaymie R; Wagner, Denisa D

2013-01-01

The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (-/-) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system.

Formation of tyrosine isomers in aqueous phenylalanine solutions by gamma irradiation

International Nuclear Information System (INIS)

Aflaki, F.; Salahinejad, M.; Roozbehani, A.

2009-01-01

Ortho-tyrosine detection method can be used for detection of irradiated protein rich foods. Tyrosine isomers produced by gamma radiation of aqueous phenylalanine solutions at wide dose levels (0.1-50 k Gy) were examined to obtain basic information for o-tyrosine detection method of irradiated foods. Determination of tyrosines produced in aqueous phenylalanine solutions were carried out by high performance liquid chromatography and fluorescence detection. The detection limit of o-tyrosine was 0.01 ppm and the linear range of calibration and the relative standard deviation of analysis was 50 ng and 4-13%, respectively. The amounts of the tyrosines increased with the irradiation level up to 10 k Gy and no further tyrosine formation was observed when the dose level was increased. At a constant dose level, the yield of tyrosines initially increased with the phenylalanine concentration, while with further increase of phenylalanine concentration no effect on increase of tyrosine yield was observed. When the dose rate was varying from 2.3 k Gy/h to 1.2 k Gy/h with a total amount of 10 k Gy in each case, there was no significant effect on tyrosine isomers formation was observed. Also the results showed that tyrosine yield was affected by temperature, p H and the presence of oxygen

Tyrosine phosphorylation of WW proteins

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Reuven, Nina; Shanzer, Matan

2015-01-01

A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

Dinucleotide repeat polymorphism in Fms-like tyrosine kinase-1 (Flt-1 gene is not associated with preeclampsia

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Park So-Yeon

2008-07-01

Full Text Available Abstract Background Preeclampsia is a major cause of maternal and perinatal mortality and morbidity. The etiology of preeclampsia remains unclear. Recently, it was shown that misregulation of fms-like tyrosine kinase-1 (Flt-1 in the peripheral blood mononuclear cells of pregnant women results in over-expression of the soluble splice variant of Flt-1, sFlt-1, producing an additional (extra-placental source of sFlt-1 that can contribute to the etiology of preeclampsia. The aim of this study was to investigate the relationship between preeclampsia and a dinucleotide (threonine-glycine; TGn repeat polymorphism in the 3' non-coding region of the Flt-1 gene. Methods The number of the d(TGn repeats was analyzed in 170 patients with preeclampsia and in 202 normotensive pregnancies. The region containing the dinucleotide repeat polymorphism of the Flt-1 gene was amplified by polymerase chain reaction (PCR from the DNA samples and was analyzed by direct PCR sequencing. Results We found 10 alleles of the dinucleotide repeat polymorphism and designated these as allele*12 (A1 through allele*23 (A12 according to the number of the TG repeats, from 12 to 23. The frequency of the 14-repeat allele (A3 was most abundant (63.82% in preeclampsia and 69.06% in controls, followed by the 21-repeat allele (A10; 28.53% in preeclampsia and 23.76% in controls. There was no significant difference in the allele frequency between patients with preeclampsia and normal controls. The most common genotype in preeclamptic and normotensive pregnancies was heterozygous (TG14/(TG21 (41.76% and homozygous (TG14/(TG14 (45.05%, respectively. However, the genotype frequencies were not significantly different between preeclamptic patients and controls. Conclusion This is the first study to characterize the dinucleotide repeat polymorphism of the Flt-1 gene in patients with preeclampsia. We found no differences in the allele or genotype frequencies between patients with preeclampsia and

Phenylketonuria : tyrosine supplementation in phenylalanine-restricted diets

NARCIS (Netherlands)

van Spronsen, FJ; van Rijn, M; Bekhof, J; Koch, R; Smit, PGA

Treatment of phenylketonuria (PKU) consists of restriction of natural protein and provision of a protein substitute that lacks phenylalanine but is enriched in tyrosine. Large and unexplained differences exist, however, in the tyrosine enrichment of the protein substitutes. Furthermore, some

Rapid enzymatic analysis of plasma for tyrosine.

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Shimizu, H; Taniguchi, K; Sugiyama, M; Kanno, T

1990-01-01

In this rapid, simple, and convenient enzymatic method for measurement of tyrosine in plasma, tyrosine is converted to tyramine by action of tyrosine decarboxylase (EC 4.1.1.25) and the tyramine produced is oxidized to p-hydroxybenzyl aldehyde and hydrogen peroxide by action of tyramine oxidase (EC 1.4.3.9). The hydrogen peroxide is reacted with 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine in the presence of peroxidase (EC 1.11.1.7) to obtain quinoneimine dye, the absorbance of which is measured at 570 nm. Thus tyrosine is measured in the visible range. The CV was 4.6% or less, and the measurement was unaffected by other amino acids, except for phenylalanine. The values obtained (y) correlated well with those obtained with an amino acid analyzer (x): y = 0.902x + 3.92 mumol/L (Syx = 12.3; r = 0.985; n = 54).

Evaluation of o-[11C]methyl-L-tyrosine and o-[18F]fluoromethyl-L-tyrosine as tumor imaging tracers by PET

International Nuclear Information System (INIS)

Ishiwata, Kiichi; Kawamura, Kazunori; Wang Weifang; Furumoto, Shozo; Kubota, Kazuo; Pascali, Claudio; Bogni, Anna; Iwata, Ren

2004-01-01

We investigated the potential of O-[ 11 C]methyl-L-tyrosine and O-[ 18 F]fluoromethyl-L-tyrosine as positron-emitting tracers for tumor imaging. The two tracers had similar distribution patterns in rats bearing AH109A hepatoma, with pancreas and, on a lesser extent, AH109A showing the highest uptake. Uptake of both tracers in the AH109A and uptake ratios of AH109A-to-tissues (with the exception of AH109A-to-bone) gradually increased for 60 min. O-[ 11 C]methyl-L-tyrosine was metabolically stable, whereas a negligible low amount of metabolites was observed for O-[ 18 F]fluoromethyl-L-tyrosine. Both tracers showed the potential for tumor imaging

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity

DEFF Research Database (Denmark)

Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno

2004-01-01

or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone......, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX......[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570...

The Influence of LepR Tyrosine Site Mutations on Mouse Ovary Development and Related Gene Expression Changes

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Tu, Xiaoyu; Kuang, Zhichao; Gong, Xia; Shi, Yan; Yu, Lin; Shi, Huijuan; Wang, Jian; Sun, Zhaogui

2015-01-01

Leptin exerts many biological functions, such as in metabolism and reproduction, through binding to and activating the leptin receptor, LepRb, which is expressed in many regions of the brain. To better understand the roles of LepR downstream signaling pathways, Y123F mice, which expressed mutant leptin receptors with phenylalanine (F) substituted for three tyrosines (Y) (Tyr985, Tyr1077 and Tyr1138), were generated. The body weight and abdominal fat deposits of Y123F homozygous mice (HOM) were higher than those of wild-type mice (WT). HOM ovaries were atrophic and the follicles developed abnormally; however, the HOM ovaries did not exhibit polycystic phenotypes. Moreover, Y123F HOM adults had no estrous cycle and the blood estrogen concentration remained stable at a low level below detection limit of 5 pg/ml. LepR expression in HOM ovaries was higher than in WT ovaries. Using cDNA Microarrays, the mRNA expressions of 41 genes were increased, and 100 were decreased in HOM vs. WT ovaries, and many signaling pathways were evaluated to be involved significantly. The expressions of 19 genes were validated by real-time quantitative PCR, most of which were consistent with the microarray results. Thus, Y123F HOM mice were suggested as a new animal model of PCOS for research that mainly emphasizes metabolic disorders and anovulation, but not the polycystic phenotype. Meanwhile, using the model, we found that JAK-STAT and hormone biosynthesis pathways were involved in the follicular development and ovulation disorders caused by LepR deficiency in ovaries, although we could not exclude indirect actions from the brain. PMID:26529315

Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides

DEFF Research Database (Denmark)

Winterbourn, Christine C; Parsons-Mair, Helena N; Gebicki, Silvia

2004-01-01

Superoxide reacts rapidly with other radicals, but these reactions have received little attention in the context of oxidative stress. For tyrosyl radicals, reaction with superoxide is 3-fold faster than dimerization, and forms the addition product tyrosine hydroperoxide. We have explored structural...... requirements for hydroperoxide formation using tyrosine analogues and di- and tri-peptides. Superoxide and phenoxyl radicals were generated using xanthine oxidase, peroxidase and the respective tyrosine derivative, or by gamma-radiation. Peroxides were measured using FeSO4/Xylenol Orange. Tyrosine and tyramine...... formed stable hydroperoxides, but N-acetyltyrosine and p-hydroxyphenylacetic acid did not, demonstrating a requirement for a free amino group. Using [14C]tyrosine, the hydroperoxide and dityrosine were formed at a molar ratio of 1.8:1. Studies with pre-formed hydroperoxides, and measurements of substrate...

Mechanism of Cytochrome P450 17A1-Catalyzed Hydroxylase and Lyase Reactions

DEFF Research Database (Denmark)

Bonomo, Silvia; Jorgensen, Flemming Steen; Olsen, Lars

2017-01-01

Cytochrome P450 17A1 (CYP17A1) catalyzes C17 hydroxylation of pregnenolone and progesterone and the subsequent C17–C20 bond cleavage (lyase reaction) to form androgen precursors. Compound I (Cpd I) and peroxo anion (POA) are the heme-reactive species underlying the two reactions. We have characte...... the concept that the selectivity of the steroidogenic CYPs is ruled by direct interactions with the enzyme, in contrast to the selectivity of drug-metabolizing CYPs, where the reactivity of the substrates dominates....... characterized the reaction path for both the hydroxylase and lyase reactions using density functional theory (DFT) calculations and the enzyme–substrate interactions by molecular dynamics (MD) simulations. Activation barriers for positions subject to hydroxylase reaction have values close to each other and span...

25-Hydroxyvitamin D3 1-Alpha-Hydroxylase-Dependent Stimulation of Renal Klotho Expression by Spironolactone

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Ioana Alesutan

2013-11-01

Full Text Available Background: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1 by FGF23. Conversely, 1,25(OH2D3 stimulates, by activating the vitamin D3 receptor (Vdr, the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. Methods: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293 or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR or of mineralocorticoid receptor (NR3C2. Results: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours and mice (8 hours or 5 days. Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours. Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. Conclusion: Besides blocking the effects of

Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides.

Science.gov (United States)

Mitsuhashi, Jun; Nakayama, Tsutomu; Narai-Kanayama, Asako

2015-01-01

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media at 30 °C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, L-tyrosine ethyl ester, even at 5mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes L-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate. Copyright © 2015 Elsevier Inc. All rights reserved.

Plasma Catecholamines (CA) and Gene Expression of CA Biosynthetic Enzymes in Adrenal Medulla and Sympathetic Ganglia of Rats Exposed to Single or Repeated Hypergravity

Science.gov (United States)

Petrak, J.; Jurani, M.; Baranovska, M.; Hapala, I.; Frollo, I.; Kvetnansky, R.

2008-06-01

The aim of this study was to evaluate plasma epinephrine (EPI) and norepinephrine (NE) levels in blood collected directly during a single or 8-times repeated centrifugation at hypergravity 4G, using remote controlled equipment. Plasma EPI levels showed a huge hypergravity-induced increase. After the last blood collection during hypergravity, the centrifuge was turned off and another blood sampling was performed immediately after the centrifuge decelerated and stopped (10 min). In these samples plasma EPI showed significantly lower levels compared to centrifugation intervals. Plasma NE levels showed none or small changes. Repeated exposure to hypergravity 4G (8 days for 60 min) eliminated the increase in plasma EPI levels at the 15 min interval but did not markedly affect plasma NE levels. To explain these findings we measured mRNA levels of CA biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla (AM) and stellate ganglia (SG) of rats exposed to continuous hypergravity (2G) up to 6 days. In AM, TH, DBH and PNMT mRNA levels were significantly increased in intervals up to 3 days, however, after 6 day hypergravity exposure, no significant elevation was found. In SG, no significant changes in gene expression of CA enzymes were seen both after a single or repeated hypergravity. Thus, our data show that hypergravity highly activates the adrenomedullary system, whereas the sympathoneural system is not significantly changed. In conclusion, our results demonstrate that during repeated or continuous exposure of the organism to hypergravity the adrenomedullary system is adapted, whereas sympathoneural system is not affected.

Essential roles of insulin, AMPK signaling and lysyl and prolyl hydroxylases in the biosynthesis and multimerization of adiponectin.

Science.gov (United States)

Zhang, Lin; Li, Ming-Ming; Corcoran, Marie; Zhang, Shaoping; Cooper, Garth J S

2015-01-05

Post-translational modifications (PTMs) of the adiponectin molecule are essential for its full bioactivity, and defects in PTMs leading to its defective production and multimerization have been linked to the mechanisms of insulin resistance, obesity, and type-2 diabetes. Here we observed that, in differentiated 3T3-L1 adipocytes, decreased insulin signaling caused by blocking of insulin receptors (InsR) with an anti-InsR blocking antibody, increased rates of adiponectin secretion, whereas concomitant elevations in insulin levels counteracted this effect. Adenosine monophosphate-activated protein kinase (AMPK) signaling regulated adiponectin production by modulating the expression of adiponectin receptors, the secretion of adiponectin, and eventually the expression of adiponectin itself. We found that lysyl hydroxylases (LHs) and prolyl hydroxylases (PHs) were expressed in white-adipose tissue of ob/ob mice, wherein LH3 levels were increased compared with controls. In differentiated 3T3-L1 adipocytes, both non-specific inhibition of LHs and PHs by dipyridyl, and specific inhibition of LHs by minoxidil and of P4H with ethyl-3,4-dihydroxybenzoate, caused significant suppression of adiponectin production, more particularly of the higher-order isoforms. Transient gene knock-down of LH3 (Plod3) caused a suppressive effect, especially on the high molecular-weight (HMW) isoforms. These data indicate that PHs and LHs are both required for physiological adiponectin production and in particular are essential for the formation/secretion of the HMW isoforms. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Overexpression of parkin in rat nigrostriatal dopamine system protects against methamphetamine neurotoxicity

Science.gov (United States)

Liu, Bin; Traini, Roberta; Killinger, Bryan; Schneider, Bernard; Moszczynska, Anna

2013-01-01

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum, sparing other striatal terminals and cell bodies. We previously detected a deficit in parkin after binge METH in rat striatal synaptosomes. Parkin is an ubiquitin-protein E3 ligase capable of protecting dopamine neurons from diverse cellular insults. Whether the deficit in parkin mediates the toxicity of METH and whether parkin can protect from toxicity of the drug is unknown. The present study investigated whether overexpression of parkin attenuates degeneration of striatal dopaminergic terminals exposed to binge METH. Parkin overexpression in rat nigrostriatal dopamine system was achieved by microinjection of adeno-associated viral transfer vector 2/6 encoding rat parkin (AAV2/6-parkin) into the substantia nigra pars compacta. The microinjections of AAV2/6-parkin dose-dependently increased parkin levels in both the substantia nigra pars compacta and striatum. The levels of dopamine synthesizing enzyme, tyrosine hydroxylase, remained at the control levels; therefore, tyrosine hydroxylase immunoreactivity was used as an index of dopaminergic terminal integrity. In METH-exposed rats, the increase in parkin levels attenuated METH-induced decreases in striatal tyrosine hydroxylase immunoreactivity in a dose-dependent manner, indicating that parkin can protect striatal dopaminergic terminals against METH neurotoxicity. PMID:23313192

The neurotropic parasite Toxoplasma gondii increases dopamine metabolism.

Directory of Open Access Journals (Sweden)

Emese Prandovszky

Full Text Available The highly prevalent parasite Toxoplasma gondii manipulates its host's behavior. In infected rodents, the behavioral changes increase the likelihood that the parasite will be transmitted back to its definitive cat host, an essential step in completion of the parasite's life cycle. The mechanism(s responsible for behavioral changes in the host is unknown but two lines of published evidence suggest that the parasite alters neurotransmitter signal transduction: the disruption of the parasite-induced behavioral changes with medications used to treat psychiatric disease (specifically dopamine antagonists and identification of a tyrosine hydroxylase encoded in the parasite genome. In this study, infection of mammalian dopaminergic cells with T. gondii enhanced the levels of K+-induced release of dopamine several-fold, with a direct correlation between the number of infected cells and the quantity of dopamine released. Immunostaining brain sections of infected mice with dopamine antibody showed intense staining of encysted parasites. Based on these analyses, T. gondii orchestrates a significant increase in dopamine metabolism in neural cells. Tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, was also found in intracellular tissue cysts in brain tissue with antibodies specific for the parasite-encoded tyrosine hydroxylase. These observations provide a mechanism for parasite-induced behavioral changes. The observed effects on dopamine metabolism could also be relevant in interpreting reports of psychobehavioral changes in toxoplasmosis-infected humans.

Insulin growth factors regulate the mitotic cycle in cultured rat sympathetic neuroblasts

International Nuclear Information System (INIS)

DiCicco-Bloom, E.; Black, I.B.

1988-01-01

While neuronal mitosis is uniquely restricted to early development, the underlying regulation remains to be defined. The authors have now developed a dissociated, embryonic sympathetic neuron culture system that uses fully defined medium in which cells enter the mitotic cycle. The cultured cells expressed two neuronal traits, tyrosine hydroxylase and the neuron-specific 160-kDa neurofilament subunit protein, but were devoid of glial fibrillary acidic protein, a marker for non-myelin-forming Schwann cells in ganglia. Approximately one-third of the tyrosine hydroxylase-positive cells synthesized DNA in culture, specifically incorporating [ 3 H]thymidine into their nuclei. They used this system to define factors regulating the mitotic cycle in sympathetic neuroblasts. Members of the insulin family of growth factors, including insulin and insulin-like growth factors I and II, regulated DNA synthesis in the presumptive neuroblasts. Insulin more than doubled the proportion of tyrosine hydroxylase-positive cells entering the mitotic cycle, as indicated by autoradiography of [ 3 H]thymidine incorporation into nuclei. Scintillation spectrometry was an even more sensitive index of DNA synthesis. In contrast, the trophic protein nerve growth factor exhibited no mitogenic effect, suggesting that the mitogenic action of insulin growth factors is highly specific. The observations are discussed in the context of the detection of insulin growth factors and receptors in the developing brain

Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

Science.gov (United States)

Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

2014-02-01

Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects. © 2013 Institute of Zoology, Chinese Academy of Sciences.

A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

Science.gov (United States)

Shen, S H; Bastien, L; Posner, B I; Chrétien, P

1991-08-22

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.

Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance.

Science.gov (United States)

Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward

2018-01-17

Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.

Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects.

Science.gov (United States)

Batthyány, Carlos; Bartesaghi, Silvina; Mastrogiovanni, Mauricio; Lima, Analía; Demicheli, Verónica; Radi, Rafael

2017-03-01

"Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function. Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation. The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.

Regulation of melanogenesis: the role of cAMP and MITF

Directory of Open Access Journals (Sweden)

Michał Otręba

2012-01-01

Full Text Available The article presents the melanogenesis pathway and the role of cyclic adenosine monophosphate (cAMP and microphthalmia transcription factor (MITF in regulation of this process. Products of melanogenesis are eu- and/or pheomelanins synthesized in a multistage process of tyrosine oxidation and polymerization. The conversions require the presence of tyrosinase (TYR, key enzyme, tyrosine hydroxylase isoform I (THI and tyrosinase related proteins (TRP1 and TRP2. Many types of signal molecules and transcription factors participate in regulation of melanin synthesis, but the most important are cAMP and MITF. cAMP is the second messenger in the intracellular signal cascade, which is synthesized from adenosine triphosphate (ATP by adenylyl cyclase, activated among others by the melanocortin receptor and the αS subunit of G protein. The signal molecule cAMP regulates MITF, TYR, THI, GTP-cyclohydroxylase I (GTP-CHI transcription and phenylalanine hydroxylase (PAH phosphorylation at Ser16 by protein kinase A (PKA. Mutations of genes encoding proteins belonging to the cAMP signal cascade may lead to McCune-Albright and Carney syndromes. MITF is one of the most important nuclear transcription factors regulating melanogenesis. Currently 10 isoforms of human MITF are known, but in melanocytes only MITF-M, MITF-Mdel, MITF-A and MITF-H occur. MITF transcription factor regulates melanogenesis by activation of tyrosinase, TRP1 and TRP2 transcription. It also affects expression of other factors regulating melanosome maturation, biogenesis and transport. Moreover, it regulates melanocyte proliferation and protection against apoptosis. Mutations of the MITF gene may lead to hereditary diseases: Waardenburg type II and Tietz syndromes.

The role of GH receptor tyrosine phosphorylation in Stat5 activation

DEFF Research Database (Denmark)

Hansen, J A; Hansen, L H; Wang, X

1997-01-01

Stimulation of GH receptors leads to rapid activation of Jak2 kinase and subsequent tyrosine phosphorylation of the GH receptor. Three specific tyrosines located in the C-terminal domain of the GH receptor have been identified as being involved in GH-stimulated transcription of the Spi 2.1 promoter....... Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA......-binding activity, whereas the GH receptor mutant lacking all intracellular tyrosines was not. Synthetic tyrosine phosphorylated peptides corresponding to the GH receptor sequence around the three tyrosines inhibited Stat5 DNA-binding activity while their non-phosphorylated counterparts were ineffective. Tyrosine...

Expression and purification of the metal-containing monooxygenases tryptophan hydroxylase and dopamine β-hydroxylase

DEFF Research Database (Denmark)

Karlsen, Pernille Efferbach

-hyperactive disorder (ADHD) among others. Since all these diseases are the cause of huge economical and personal costs it is very important to gain more knowledge of TPH and DβH since these two enzymes could be possible targets for medicine against the diseases mentioned above. TPH a three-domain, iron......-containing enzyme which belongs to the aromatic amino acid hydroxylase (AAAH) family. It exist in two isoforms, TPH1 and TPH2, which are expressed in different tissues and have different properties. TPH is known as a very diffcult protein to work with especially due to instability and only truncated forms of TPH1...... have been purified and crystallized. This project concern the human neuronal TPH or TPH2. In an attempt to overcome the problems with recombinant TPH two stability and solubility optimized variants of TPH2 are designed. Escherichia coli (E. coli) expression strains for these variants and full length...

Origin and characterization of retrograde labeled neurons supplying the rat urethra using fiberoptic confocal fluorescent microscopy in vivo and immunohistochemistry.

Science.gov (United States)

Lee, Keon-Cheol; Sharma, Seema; Tuttle, Jeremy B; Steers, William D

2010-10-01

Autonomic innervation of urethral smooth muscle may influence urinary continence after prostatectomy. It is unclear whether the cavernous nerves carry fibers that influence continence. Using a retrograde axonal tracer combined with real-time in vivo imaging and ex vivo immunohistochemistry we determined the course and type of neurons supplying urethral smooth muscle distal to the prostate in the rat. We injected the retrograde axonal tracers cholera toxin B fragment-Alexa Fluor 488 and Fast Blue in the distal urethral smooth muscle in 10 rats each. Five days later the cavernous nerves and pelvic ganglion were imaged using fiberoptic confocal fluorescence microscopy (cholera toxin B fragment-Alexa Fluor 488) or harvested for immunohistochemistry (Fast Blue). Dual immunofluorescence of Fast Blue neurons with tyrosine hydroxylase or neuronal nitric oxide synthase was done to characterize neurons as noradrenergic or nitrergic. To ascertain whether the cavernous nerves contain fibers to the urethra that originate in the pelvic ganglia we cut the cavernous nerves with their ancillary branches in 3 rats and imaged them for Fast Blue. Fluorescent neurons and axons were detected in cavernous nerves and the pelvic ganglion. Few neurons were seen in rats with cavernous nerve section. Of urethral neurons 53.1% showed neuronal nitric oxide synthase positivity while 40.6% were immunoreactive for tyrosine hydroxylase. About 6.2% of urethral neurons failed to show tyrosine hydroxylase or neuronal nitric oxide synthase immunoreactivity. Most of the autonomic innervation to the urethra beyond the prostatic apex travels in the cavernous nerves. Many nerves may be parasympathetic based on neuronal nitric oxide synthase immunoreactivity. Nerves supplying the urethra outside the cavernous nerves may course posterior to the prostate. Along with afferent fibers, tyrosine hydroxylase immunoreactivity expressing neuron fibers, ie noradrenergic nerves, traveling in the cavernous nerves may

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells.

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Keisuke Tabara

Full Text Available Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs. However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2 or BIBW2992 (pan-TKI of EGFR family proteins. Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

Protein Tyrosine Nitration : Selectivity, Physicochemical and Biological Consequences, Denitration, and Proteomics Methods for the Identification of Tyrosine-Nitrated Proteins

NARCIS (Netherlands)

Abello, Nicolas; Kerstjens, Huib A. M.; Postma, Dirkje S.; Bischoff, Rainer

Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO(2)). In the present article, we review the main nitration reactions and

Selective expression of a protein-tyrosine kinase, p56lyn, in hematopoietic cells and association with production of human T-cell lymphotropic virus type I

International Nuclear Information System (INIS)

Yamanashi, Yuji; Mori, Shigeo; Inoue, Kazushi; Yamamoto, Tadashi; Toyoshima, Kumao; Yoshida, Mitsuaki; Kishimoto, Tadamitsu

1989-01-01

This paper reports the identification of the lyn gene product, a member of the src-related family of protein-tyrosine kinases, and its expression in hematopoietic cells. A lyn-specific sequence (Arg-25 to Ala-119 of the protein) was expressed in Escherichia coli as a fusion protein with β-galactosidase. Antiserum raised against the fusion protein immunoprecipitated a 56-kDa protein from human B lymphocytes. Incubation of the immunoprecipitate with [γ- 32 P]ATP resulted in the phosphorylation of this protein at tyrosine residues. Immunohistological and immunoblotting analyses showed that the lyn gene product was expressed in lymphatic tissues (spleen and tonsil) and in adult lung, which contains many macrophages. Furthermore, both the transcripts and the protein products of the lyn gene accumulated in macrophages/monocytes, platelets, and B lymphocytes but were not expressed appreciably in granulocytes, erythrocytes, or T lymphocytes, suggesting that lyn gene products function primarily in certain differentiated cells of lymphoid and myeloid lineages

Role of Mitochondrial DNA Mutations in Cellular Vulnerability to Mitochondria-Specific Environmental Toxins

National Research Council Canada - National Science Library

Hirsch, Etienne C

2005-01-01

.... To test such a hypothesis in Parkinson's disease we proposed to: 1) develop an animal model with accumulated mtDNA mutations in catecholaminergic neurons by creating a transgenic mouse containing a tyrosine hydroxylase (TH...

Determination of o-tyrosine in irradiated chicken

International Nuclear Information System (INIS)

Zoller, O.; Schoeni, D.; Zimmerli, B.

1991-01-01

The author explains his method to determine O-Tyrosine in irradiated chickens with a high-performance liquid chromatography. The method is simple and fast, but a proper chromatographic separation is difficult. The detection limit with a high sensitive detector is about 0.05-0.1 mg O-Tyrosine/kg meat (9 refs)

The dynamic landscape of gene regulation during Bombyx mori oogenesis.

Science.gov (United States)

Zhang, Qiang; Sun, Wei; Sun, Bang-Yong; Xiao, Yang; Zhang, Ze

2017-09-11

Oogenesis in the domestic silkworm (Bombyx mori) is a complex process involving previtellogenesis, vitellogenesis and choriogenesis. During this process, follicles show drastic morphological and physiological changes. However, the genome-wide regulatory profiles of gene expression during oogenesis remain to be determined. In this study, we obtained time-series transcriptome data and used these data to reveal the dynamic landscape of gene regulation during oogenesis. A total of 1932 genes were identified to be differentially expressed among different stages, most of which occurred during the transition from late vitellogenesis to early choriogenesis. Using weighted gene co-expression network analysis, we identified six stage-specific gene modules that correspond to multiple regulatory pathways. Strikingly, the biosynthesis pathway of the molting hormone 20-hydroxyecdysone (20E) was enriched in one of the modules. Further analysis showed that the ecdysteroid 20-hydroxylase gene (CYP314A1) of steroidgenesis genes was mainly expressed in previtellogenesis and early vitellogenesis. However, the 20E-inactivated genes, particularly the ecdysteroid 26-hydroxylase encoding gene (Cyp18a1), were highly expressed in late vitellogenesis. These distinct expression patterns between 20E synthesis and catabolism-related genes might ensure the rapid decline of the hormone titer at the transition point from vitellogenesis to choriogenesis. In addition, we compared landscapes of gene regulation between silkworm (Lepidoptera) and fruit fly (Diptera) oogeneses. Our results show that there is some consensus in the modules of gene co-expression during oogenesis in these insects. The data presented in this study provide new insights into the regulatory mechanisms underlying oogenesis in insects with polytrophic meroistic ovaries. The results also provide clues for further investigating the roles of epigenetic reconfiguration and circadian rhythm in insect oogenesis.

Tetrahydrobiopterin precursor sepiapterin provides protection against neurotoxicity of 1-methyl-4-phenylpyridinium in nigral slice cultures

DEFF Research Database (Denmark)

Madsen, Jakob Torp; Jansen, Pernille; Hesslinger, Christian

2003-01-01

Complex-I inhibition and oxidative processes have been implicated in the loss of nigral dopamine neurones in Parkinson's disease and the toxicity of MPTP and its metabolite MPP+. Tetrahydrobiopterin, an essential cofactor for tyrosine hydroxylase, may act as an antioxidant in dopaminergic neurones...... and protects against the toxic consequences of glutathione depletion. Here we studied the effects of manipulating tetrahydrobiopterin levels on MPP+ toxicity in organotypic, rat ventral mesencephalic slice cultures. In cultures exposed to 30 micro m MPP+ for 2 days, followed by 8 days 'recovery' in control...... medium, we measured dopamine and its metabolites in the tissue and culture medium by HPLC, lactate dehydrogenase release to the culture medium, cellular uptake of propidium iodide and counted the tyrosine hydroxylase-immunoreactive neurones. Inhibition of tetrahydrobiopterin synthesis by 2,4-diamino-6...

Growth and gene expression are predominantly controlled by distinct regions of the human IL-4 receptor.

Science.gov (United States)

Ryan, J J; McReynolds, L J; Keegan, A; Wang, L H; Garfein, E; Rothman, P; Nelms, K; Paul, W E

1996-02-01

IL-4 causes hematopoietic cells to proliferate and express a series of genes, including CD23. We examined whether IL-4-mediated growth, as measured by 4PS phosphorylation, and gene induction were similarly controlled. Studies of M12.4.1 cells expressing human IL-4R truncation mutants indicated that the region between amino acids 557-657 is necessary for full gene expression, which correlated with Stat6 DNA binding activity. This region was not required for 4PS phosphorylation. Tyrosine-to-phenylalanine mutations in the interval between amino acids 557-657 revealed that as long as one tyrosine remained unmutated, CD23 was fully induced. When all three tyrosines were mutated, the receptor was unable to induce CD23. The results indicate that growth regulation and gene expression are principally controlled by distinct regions of IL-4R.

Conformational Clusters of Phosphorylated Tyrosine.

Science.gov (United States)

Abdelrasoul, Maha; Ponniah, Komala; Mao, Alice; Warden, Meghan S; Elhefnawy, Wessam; Li, Yaohang; Pascal, Steven M

2017-12-06

Tyrosine phosphorylation plays an important role in many cellular and intercellular processes including signal transduction, subcellular localization, and regulation of enzymatic activity. In 1999, Blom et al., using the limited number of protein data bank (PDB) structures available at that time, reported that the side chain structures of phosphorylated tyrosine (pY) are partitioned into two conserved conformational clusters ( Blom, N.; Gammeltoft, S.; Brunak, S. J. Mol. Biol. 1999 , 294 , 1351 - 1362 ). We have used the spectral clustering algorithm to cluster the increasingly growing number of protein structures with pY sites, and have found that the pY residues cluster into three distinct side chain conformations. Two of these pY conformational clusters associate strongly with a narrow range of tyrosine backbone conformation. The novel cluster also highly correlates with the identity of the n + 1 residue, and is strongly associated with a sequential pYpY conformation which places two adjacent pY side chains in a specific relative orientation. Further analysis shows that the three pY clusters are associated with distinct distributions of cognate protein kinases.

Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

Directory of Open Access Journals (Sweden)

Alessandra Pasquo

Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

Protein tyrosine phosphatase receptor type R deficient mice exhibit increased exploration in a new environment and impaired novel object recognition memory

NARCIS (Netherlands)

Erkens, M.; Bakker, B.; Duijn, L.M. van; Hendriks, W.J.A.J.; Zee, C.E.E.M. van der

2014-01-01

Mouse gene Ptprr encodes multiple protein tyrosine phosphatase receptor type R (PTPRR) isoforms that negatively regulate mitogen-activated protein kinase (MAPK) signaling pathways. In the mouse brain, PTPRR proteins are expressed in cerebellum, olfactory bulb, hippocampus, amygdala and perirhinal

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity.

Science.gov (United States)

Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno; Stensballe, Allan; Jensen, Ole N; Carter-Su, Christin

2004-06-01

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.

Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain.

Directory of Open Access Journals (Sweden)

Mun Chiang Chan

Full Text Available As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs. Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs. Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4 induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

A novel spleen tyrosine kinase inhibitor blocks c-Jun N-terminal kinase-mediated gene expression in synoviocytes

NARCIS (Netherlands)

Cha, Hoon-Suk; Boyle, David L.; Inoue, Tomoyuki; Schoot, Reineke; Tak, Paul P.; Pine, Polly; Firestein, Gary S.

2006-01-01

Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor engagement. However, the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor-alpha (TNFalpha)-stimulated fibroblast-like

Measurement of protein synthesis: in vitro comparison of (68)Ga-DOTA-puromycin, [ (3)H]tyrosine, and 2-fluoro-[ (3)H]tyrosine.

Science.gov (United States)

Eigner, Sebastian; Beckford Vera, Denis R; Fellner, Marco; Loktionova, Natalia S; Piel, Markus; Melichar, Frantisek; Rösch, Frank; Roß, Tobias L; Lebeda, Ondrej; Henke, Katerina Eigner

2013-01-01

Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using (68)Ga-DOTA-puromycin ((68)Ga-DOTA-Pur), [(3)H]tyrosine, and 2-fluoro-[(3)H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine. DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. (68)Ga was obtained from a (68)Ge/(68)Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741-1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [(3)H]Tyrosine and 2-fluoro-[(3)H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). (68)Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20-25 MBq (68

Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone

International Nuclear Information System (INIS)

Gajewski, E.; Dizdaroglu, M.

1990-01-01

Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A γ-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone γ-irradiated in N 2 O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J·kg -1 ). This yield amounted to 0.05 nmol·J -1 . Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed

Eating to stop: Tyrosine supplementation enhances inhibitory control but not response execution

NARCIS (Netherlands)

Colzato, L.S.; Jongkees, B.J.; Sellaro, R.; van den Wildenberg, W.P.M.; Hommel, B.

2014-01-01

Animal studies and research in humans have shown that the supplementation of tyrosine, or tyrosine-containing diets, increase the plasma tyrosine and enhance brain dopamine (DA). However, the strategy of administering tyrosine (and the role of DA therein) to enhance cognition is unclear and heavily

ROLE OF TYROSINE-SULFATED PROTEINS IN RETINAL STRUCTURE AND FUNCTION

Science.gov (United States)

Kanan, Y.; Al-Ubaidi, M.R.

2014-01-01

The extracellular matrix (ECM) plays a significant role in cellular and retinal health. The study of retinal tyrosine-sulfated proteins is an important first step toward understanding the role of ECM in retinal health and diseases. These secreted proteins are members of the retinal ECM. Tyrosine sulfation was shown to be necessary for the development of proper retinal structure and function. The importance of tyrosine sulfation is further demonstrated by the evolutionary presence of tyrosylprotein sulfotransferases, enzymes that catalyze proteins’ tyrosine sulfation, and the compensatory abilities of these enzymes. Research has identified four tyrosine-sulfated retinal proteins: fibulin 2, vitronectin, complement factor H (CFH), and opticin. Vitronectin and CFH regulate the activation of the complement system and are involved in the etiology of some cases of age-related macular degeneration. Analysis of the role of tyrosine sulfation in fibulin function showed that sulfation influences the protein's ability to regulate growth and migration. Although opticin was recently shown to exhibit anti-angiogenic properties, it is not yet determined what role sulfation plays in that function. Future studies focusing on identifying all of the tyrosine-sulfated retinal proteins would be instrumental in determining the impact of sulfation on retinal protein function in retinal homeostasis and diseases. PMID:25819460

Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea.

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Bramwell G Lambrus

Full Text Available Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP, or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.

Manganese (II) induces chemical hypoxia by inhibiting HIF-prolyl hydroxylase: Implication in manganese-induced pulmonary inflammation

International Nuclear Information System (INIS)

Han, Jeongoh; Lee, Jong-Suk; Choi, Daekyu; Lee, Youna; Hong, Sungchae; Choi, Jungyun; Han, Songyi; Ko, Yujin; Kim, Jung-Ae; Mi Kim, Young; Jung, Yunjin

2009-01-01

Manganese (II), a transition metal, causes pulmonary inflammation upon environmental or occupational inhalation in excess. We investigated a potential molecular mechanism underlying manganese-induced pulmonary inflammation. Manganese (II) delayed HIF-1α protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1α hydroxylation and subsequent von Hippel-Lindau(VHL)-dependent HIF-1α degradation. HPH inhibition by manganese (II) was neutralized significantly by elevated dose of iron. Consistent with this, the induction of cellular HIF-1α protein by manganese (II) was abolished by pretreatment with iron. Manganese (II) induced the HIF-1 target gene involved in pulmonary inflammation, vascular endothelial growth factor (VEGF), in lung carcinoma cell lines. The induction of VEGF was dependent on HIF-1. Manganese-induced VEGF promoted tube formation of HUVEC. Taken together, these data suggest that HIF-1 may be a potential mediator of manganese-induced pulmonary inflammation

Tyrosine dephosphorylation regulates AMPAR internalisation in mGluR-LTD.

Science.gov (United States)

Gladding, Clare M; Collett, Valerie J; Jia, Zhengping; Bashir, Zafar I; Collingridge, Graham L; Molnár, Elek

2009-02-01

Long-term depression (LTD) can be induced at hippocampal CA1 synapses by activation of either NMDA receptors (NMDARs) or group I metabotropic glutamate receptors (mGluRs), using their selective agonists NMDA and (RS)-3,5-dihydroxyphenylglycine (DHPG), respectively. Recent studies revealed that DHPG-LTD is dependent on activation of postsynaptic protein tyrosine phosphatases (PTPs), which transiently dephosphorylate tyrosine residues in AMPA receptors (AMPARs). Here we show that while both endogenous GluR2 and GluR3 AMPAR subunits are tyrosine phosphorylated at basal activity, only GluR2 is dephosphorylated in DHPG-LTD. The tyrosine dephosphorylation of GluR2 does not occur in NMDA-LTD. Conversely, while NMDA-LTD is associated with the dephosphorylation of GluR1-serine-845, DHPG-LTD does not alter the phosphorylation of this site. The increased AMPAR endocytosis in DHPG-LTD is PTP-dependent and involves tyrosine dephosphorylation of cell surface AMPARs. Together, these results indicate that the subunit selective tyrosine dephosphorylation of surface GluR2 regulates AMPAR internalisation in DHPG-LTD but not in NMDA-LTD in the hippocampus.

Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway

International Nuclear Information System (INIS)

Matsumoto, Ken-ichi; Minamitani, Takeharu; Orba, Yasuko; Sato, Mami; Sawa, Hirofumi; Ariga, Hiroyoshi

2004-01-01

The results of our previous study showed that tumor invasion and metastasis are promoted in extracellular matrix (ECM) tenascin-X-deficient (TNX-/-) mice via increased expression of matrix metalloproteinases (MMPs). However, little is known about the relationship between TNX deficiency and activation of MMP genes. In this study, we investigated the molecular mechanism by which TNX deficiency activates the MMP-2 gene. We examined the intracellular signaling pathways that regulate gene expression of the proteinase in isolated fibroblasts. Results of gelatin zymography showed that MMP-2 was induced to a greater extent in TNX-/- fibroblasts embedded in type I collagen than in wild-type fibroblasts. RT-PCR analysis revealed that the increased level of MMP-2 expression was caused at the transcription level. Conversely, stable overexpression of TNX in a fibroblast cell line reduced MMP-2 expression and suppressed MMP-2 promoter activity. In addition, treatment of TNX-/- fibroblasts with SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and genistein, a tyrosine kinase inhibitor, suppressed the increased level of proMMP-2 and increased MMP-2 promoter activity in TNX-/- fibroblasts. Furthermore, increased activation of JNK and tyrosine phosphorylation of certain proteins were observed in TNX-/- fibroblasts. These findings suggest that induction of MMP-2 by TNX deficiency is mediated, at least in part, through the JNK and protein tyrosine kinase phosphorylation pathway

Behavioral and cognitive effects of tyrosine intake in healthy human adults

NARCIS (Netherlands)

Hase, Adrian; Jung, Sophie E.; aan het Rot, Marije

2015-01-01

The amino acid tyrosine is the precursor to the catecholamine neurotransmitters dopamine and norepinephrine. Increasing tyrosine uptake may positively influence catecholamine-related psychological functioning. We conducted a systematic review to examine the effects of tyrosine on behavior and

Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

OpenAIRE

Garcia-Morales, P; Minami, Y; Luong, E; Klausner, R D; Samelson, L E

1990-01-01

Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this ...

Robotic synthesis of L-[1-11C]tyrosine

International Nuclear Information System (INIS)

Luurtsema, Gert; Medema, Jitze; Elsinga, P.H.; Visser, G.M.; Vaalburg, Willem

1994-01-01

L-[1- 11 C]tyrosine promises to become an important tracer for determination of the protein synthesis rate (PSR) in tumor tissue and brain. The commercially available Anatech RB-86 robotic system is utilized for the automation of the L-[1- 11 C]tyrosine production via the isocyanide method as reported by Bolster et al. (Eur. J. Nucl. Med. 12, 321-324, 1986). The total synthesis time, including HPLC-purification and enantiomeric separation is 60 min. With a practical yield of 20 mCi L-[1- 11 C]tyrosine at a specific activity > 1000 Ci/mmol. (author)

SUBSTRATE-SPECIFICITY OF THE ALKANE HYDROXYLASE SYSTEM OF PSEUDOMONAS-OLEOVORANS GPO1

NARCIS (Netherlands)

van Beilen, J.B.; Kingma, Jacob; Witholt, Bernard

1994-01-01

We have studied the hydroxylation of a wide range of linear, branched and cyclic alkanes and alkylbenzenes by the alkane hydroxylase system of Pseudomonas oleovorans GPo1 in vivo and in vitro. In vivo hydroxylation was determined with whole cells of the recombinant PpS8141; P. putida PpS81 carrying

Adrenal scan in 17-alpha-hydroxylase deficiency: false indication of adrenal adenoma

International Nuclear Information System (INIS)

Shore, R.M.; Lieberman, L.M.; Newman, T.J.; Friedman, A.; Bargman, G.J.

1981-01-01

A patient who was thought to have testicular feminization syndrome and primary aldosteronism had an adrenal scan that suggested an adrenal adenoma. After later diagnosis of 17-alpha-hydroxylase deficiency, she was treated with glucocorticoids rather than surgery. Her clinical course and a repeat adrenal scan confirmed she did not have a tumor

Immunochemically identical hydrophilic and amphiphilic forms of the bovine adrenomedullary dopamine beta-hydroxylase

DEFF Research Database (Denmark)

Bjerrum, Ole Jannik; Helle, K B; Bock, Elisabeth Marianne

1979-01-01

. The dopamine beta-hydroxylases of the buffer and membrane fractions were antigenically identical, but differed in their amphiphilicity, as demonstrated by the change in precipitation patterns on removal of Triton X-100 from the gel, on charge-shift crossed immunoelectrophoresis and on crossed hydrophobic...

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

Science.gov (United States)

Shak, S; Goldstein, I M

1985-09-01

Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.

Tyrosine transport in winter flounder intestine: Interaction with Na+-K+-2Cl- cotransport

International Nuclear Information System (INIS)

Musch, M.W.; McConnell, F.M.; Goldstein, L.; Field, M.

1987-01-01

Tyrosine absorption across the brush border of the intestinal epithelium of the winter flounder Pseudopleuronectes americanus was studied in Ussing chambers modified to determine early rates of uptake. At 0.1 mM tyrosine, the 4-min rate of uptake (influx) of tyrosine across the brush border averaged 37.5 nmol·cm -2 ·h -1 . Omission of Na decreased influx by 60%, indicting that tyrosine influx occurs, at least in part, by a Na-coupled process. Ouabain inhibited influx by 80%. Inhibition of brush border Na + -K + -2Cl - cotransport by bumetanide, 8-bromo-cyclic GMP, or Cl replacement stimulated tyrosine influx 2.5- to 4-fold. However, atriopeptin III, which also inhibits Na + -K + -2Cl - cotransport, did not stimulate tyrosine influx. Cyclic AMP, which does not appear to inhibit ion cotransport, did not stimulate tyrosine influx. Both cyclic GMP and bumetanide also stimulated the net mucosa-to-serosa tyrosine flux (43 and 29%, respectively) and increased the cellular concentration of tyrosine by 50%. Thus tyrosine's influx is increased to a greater extent than is its transmural flux or its cellular concentration, suggesting that the main change occurs at the brush border and represents large increases in both influx and efflux of tyrosine across this membrane

Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate

International Nuclear Information System (INIS)

Hylemon, P.B.; Studer, E.J.; Pandak, W.M.; Heuman, D.M.; Vlahcevic, Z.R.; Chiang, J.Y.

1989-01-01

The HPLC-spectrophotometric method for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous [4- 14 C]cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous [4- 14 C]-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase

Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

Science.gov (United States)

Thompson, Robert E.; Liu, Xuyu; Ripoll-Rozada, Jorge; Alonso-García, Noelia; Parker, Benjamin L.; Pereira, Pedro José Barbosa; Payne, Richard J.

2017-09-01

Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

[Alternation of proteins in brain of Parkinson's disease model rats after the transplantation of TH-NTN gene modified bone marrow mesenchymal stem cells].

Science.gov (United States)

Huang, Yue; Chang, Cheng; Zhang, Jie-wen; Gao, Xiao-qun

2012-09-04

To explore the effects of tyrosine hydroxylase-neurturin (TH-NTN) gene modified bone marrow mesenchymal stem cell (BMSC) transplantation in Parkinson's disease (PD) model rats and the alternations of correlated proteins. The PD rat model was established by the 2-point injection of 6-hydroxydopamine (6-OHDA) into unilateral (right) striatum. Successful modeling rats were separated into PD, BMSC and TH-NTN-BMSC groups. BMSC and TH-NTN-BMSC groups were transplanted into BMSCs and TH-NTN gene modified BMSC cells separately into right striatum. After transplantation, ethology detection in all groups was made with an intraperitoneal injection of apomorphine (APO). Dopamine (DA) and Dihydroxyphenylacetic Acid (DOPAC) in striatum were detected by high performance liquid electrochemical analysis. TH and NTN proteins in right striatum were also analyzed by immunohistochemistry and Western blot. Finally the density of dopamine receptors in post synaptic density of dopaminergic synapses of corpus striatum were compared between each group by post-embedding immunogold electron microscopy. After an injection of APO, rotation frequency decreased in TH-NTN-BMSC group, i.e. (5.7 ± 1.3) circles/min versus (10.8 ± 2.2), (9.9 ± 1.2) circles/min in PD and BMSC groups (P TH-NTN-BMSC group versus (923 ± 132)/µm(2) in PD and (860 ± 116)/µm(2) in BMSC groups was also found. The combined therapy of TH and NTN genes increases the synthesis of DA and also protects the dopaminergic neurons to achieve double therapeutic effects. It may provide potential innovations of PD genetic therapy.

Vomeronasal inputs to the rodent ventral striatum.

Science.gov (United States)

Ubeda-Bañon, I; Novejarque, A; Mohedano-Moriano, A; Pro-Sistiaga, P; Insausti, R; Martinez-Garcia, F; Lanuza, E; Martinez-Marcos, A

2008-03-18

Vertebrates sense chemical signals through the olfactory and vomeronasal systems. In squamate reptiles, which possess the largest vomeronasal system of all vertebrates, the accessory olfactory bulb projects to the nucleus sphericus, which in turn projects to a portion of the ventral striatum known as olfactostriatum. Characteristically, the olfactostriatum is innervated by neuropeptide Y, tyrosine hydroxylase and serotonin immunoreactive fibers. In this study, the possibility that a structure similar to the reptilian olfactostriatum might be present in the mammalian brain has been investigated. Injections of dextran-amines have been aimed at the posteromedial cortical amygdaloid nucleus (the putative mammalian homologue of the reptilian nucleus sphericus) of rats and mice. The resulting anterograde labeling includes the olfactory tubercle, the islands of Calleja and sparse terminal fields in the shell of the nucleus accumbens and ventral pallidum. This projection has been confirmed by injections of retrograde tracers into the ventral striato-pallidum that render retrograde labeling in the posteromedial cortical amygdaloid nucleus. The analysis of the distribution of neuropeptide Y, tyrosine hydroxylase, serotonin and substance P in the ventral striato-pallidum of rats, and the anterograde tracing of the vomeronasal amygdaloid input in the same material confirm that, similar to reptiles, the ventral striatum of mammals includes a specialized vomeronasal structure (olfactory tubercle and islands of Calleja) displaying dense neuropeptide Y-, tyrosine hydroxylase- and serotonin-immunoreactive innervations. The possibility that parts of the accumbens shell and/or ventral pallidum could be included in the mammalian olfactostriatum cannot be discarded.

Electronic structure description of the cis-MoOS unit in models for molybdenum hydroxylases.

Science.gov (United States)

Doonan, Christian J; Rubie, Nick D; Peariso, Katrina; Harris, Hugh H; Knottenbelt, Sushilla Z; George, Graham N; Young, Charles G; Kirk, Martin L

2008-01-09

The molybdenum hydroxylases catalyze the oxidation of numerous aromatic heterocycles and simple organics and, unlike other hydroxylases, utilize water as the source of oxygen incorporated into the product. The electronic structures of the cis-MoOS units in CoCp2[TpiPrMoVOS(OPh)] and TpiPrMoVIOS(OPh) (TpiPr = hydrotris(3-isopropylpyrazol-1-yl)borate), new models for molybdenum hydroxylases, have been studied in detail using S K-edge X-ray absorption spectroscopy, vibrational spectroscopy, and detailed bonding calculations. The results show a highly delocalized Mo=S pi* LUMO redox orbital that is formally Mo(dxy) with approximately 35% sulfido ligand character. Vibrational spectroscopy has been used to quantitate Mo-Ssulfido bond order changes in the cis-MoOS units as a function of redox state. Results support a redox active molecular orbital that has a profound influence on MoOS bonding through changes to the relative electro/nucleophilicity of the terminal sulfido ligand accompanying oxidation state changes. The bonding description for these model cis-MoOS systems supports enzyme mechanisms that are under orbital control and dominantly influenced by the unique electronic structure of the cis-MoOS site. The electronic structure of the oxidized enzyme site is postulated to play a role in polarizing a substrate carbon center for nucleophilic attack by metal activated water and acting as an electron sink in the two-electron oxidation of substrates.

Aberrant expression of the tyrosine kinase receptor EphA4 and the transcription factor twist in Sézary syndrome identified by gene expression analysis.

Science.gov (United States)

van Doorn, Remco; Dijkman, Remco; Vermeer, Maarten H; Out-Luiting, Jacoba J; van der Raaij-Helmer, Elisabeth M H; Willemze, Rein; Tensen, Cornelis P

2004-08-15

Sézary syndrome (Sz) is a malignancy of CD4+ memory skin-homing T cells and presents with erythroderma, lymphadenopathy, and peripheral blood involvement. To gain more insight into the molecular features of Sz, oligonucleotide array analysis was performed comparing gene expression patterns of CD4+ T cells from peripheral blood of patients with Sz with those of patients with erythroderma secondary to dermatitis and healthy controls. Using unsupervised hierarchical clustering gene, expression patterns of T cells from patients with Sz were classified separately from those of benign T cells. One hundred twenty-three genes were identified as significantly differentially expressed and had an average fold change exceeding 2. T cells from patients with Sz demonstrated decreased expression of the following hematopoietic malignancy-linked tumor suppressor genes: TGF-beta receptor II, Mxi1, Riz1, CREB-binding protein, BCL11a, STAT4, and Forkhead Box O1A. Moreover, the tyrosine kinase receptor EphA4 and the potentially oncogenic transcription factor Twist were highly and selectively expressed in T cells of patients with Sz. High expression of EphA4 and Twist was also observed in lesional skin biopsy specimens of a subset of patients with cutaneous T cell lymphomas related to Sz, whereas their expression was nearly undetectable in benign T cells or in skin lesions of patients with inflammatory dermatoses. Detection of EphA4 and Twist may be used in the molecular diagnosis of Sz and related cutaneous T-cell lymphomas. Furthermore, the membrane-bound EphA4 receptor may serve as a target for directed therapeutic intervention.

Are tyrosine residues involved in the photoconversion of the water-soluble chlorophyll-binding protein of Chenopodium album?

Science.gov (United States)

Takahashi, S; Seki, Y; Uchida, A; Nakayama, K; Satoh, H

2015-05-01

Non-photosynthetic and hydrophilic chlorophyll (Chl) proteins, called water-soluble Chl-binding proteins (WSCPs), are distributed in various species of Chenopodiaceae, Amaranthaceae, Polygonaceae and Brassicaceae. Based on their photoconvertibility, WSCPs are categorised into two classes: Class I (photoconvertible) and Class II (non-photoconvertible). Chenopodium album WSCP (CaWSCP; Class I) is able to convert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton under light in the presence of molecular oxygen. Potassium iodide (KI) is a strong inhibitor of the photoconversion. Because KI attacks tyrosine residues in proteins, tyrosine residues in CaWSCP are considered to be important amino acid residues for the photoconversion. Recently, we identified the gene encoding CaWSCP and found that the mature region of CaWSCP contained four tyrosine residues: Tyr13, Tyr14, Tyr87 and Tyr134. To gain insight into the effect of the tyrosine residues on the photoconversion, we constructed 15 mutant proteins (Y13A, Y14A, Y87A, Y134A, Y13-14A, Y13-87A, Y13-134A, Y14-87A, Y14-134A, Y87-134A, Y13-14-87A, Y13-14-134A, Y13-87-134A, Y14-87-134A and Y13-14-87-134A) using site-directed mutagenesis. Amazingly, all the mutant proteins retained not only chlorophyll-binding activity, but also photoconvertibility. Furthermore, we found that KI strongly inhibited the photoconversion of Y13-14-87-134A. These findings indicated that the four tyrosine residues are not essential for the photoconversion. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

Science.gov (United States)

Catarsi, S; Drapeau, P

1997-08-01

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

Protein-Tyrosine Phosphorylation in Bacillus subtilis

DEFF Research Database (Denmark)

Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

2005-01-01

phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

25-Hydroxyvitamin D depletion does not exacerbate MPTP-induced dopamine neuron damage in mice.