text:
Diagnosis of dopa-responsive dystonia and other tetrahydrobiopterin disorders by the study of biopterin metabolism in fibroblasts.cofactor cofactor
An atom, organic molecule, or molecular group that is necessary for the catalytic activity (see catalysis) of many enzymes. A cofactor may be tightly bound to the protein portion of an enzyme and thus be an integral part of its functional structure, or it may for aromatic amino acid mono-oxygenases (Fig. 1) (1). Phenylalanine-3-hydroxylase is responsible for hydroxylation of phenylalanine phenylalanine (fĕn'əlăl`ənēn'), organic compound, one of the 22 α-amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. to tyrosine in the liver. Tyrosine-4-hydroxylase and tryptophan-5-hydroxylase are the rate-limiting enzymes in the biosynthesis Biosynthesis
The synthesis of more complex molecules from simpler ones in cells by a series of reactions mediated by enzymes. The overall economy and survival of the cell is governed by the interplay between the energy gained from the breakdown of compounds of the neurotransmitters dopamine and serotonin. B[H.sub.4] is also a cofactor for the different forms of nitric oxide synthase The nitric oxide synthase (NOS; EC 188.8.131.52) is an enzyme in the body that contributes to transmission from one neuron to another, to the immune system and to dilating blood vessels. .
GTP cyclohydrolase I GTP cyclohydrolase I (GTP 7,8-8,9-dihydrolase, EC 184.108.40.206) is an enzyme that is part of the folate and biopterin biosynthesis pathways. It is responsible for the hydrolysis of guanosine triphosphate (GTP) to form 7,8-dihydroneopterin 3'-triphosphate. (GTPCH; EC 188.8.131.52) catalyzes the first and rate-limiting step in the B[H.sub.4] pathway. It is expressed in peripheral tissues as well as in the brain, and its expression can be induced at the transcriptional level in T lymphocytes, macrophages, and fibroblasts by cytokines such as interferon-[gamma] (IFN-[gamma]) and tumor necrosis factor-[alpha] (TNF-[alpha]) (2, 3). The enzyme converts GTP GTP (guanosine triphosphate): see guanine. to the intermediate 7,8-dihydroneopterin triphosphate triphosphate /tri·phos·phate/ (tri-fos´fat) a salt containing three phosphate radicals.
A salt or ester containing three phosphate groups. (Fig. 1). 6-Pyruvoyltetrahydropterin synthase (PTPS PTPS 6-Pyruvoyl-tetrahydropterin synthase
PTPS Pei Tong Primary School (Singapore)
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PTPS Project Task Planning Sheet ; EC 22.214.171.124) is the second enzyme in the biosynthesis of B[H.sub.4]. It is constitutively expressed in many cell types, including fibroblasts, and catalyzes the synthesis of 6-pyruvoyltetrahydropterin from 7,8-dihydroneopterin triphosphate. The last step in the biosynthesis of B[H.sub.4] is catalyzed by sepiapterin reductase reductase /re·duc·tase/ (-tas) a term used in the names of some of the oxidoreductases, usually specifically those catalyzing reactions important solely for reduction of a metabolite. (SR; EC 18.104.22.168), which reduces 6-pyruvoyltetrahydropterin to B[H.sub.4]. Two enzymes are responsible for the regeneration of B[H.sub.4] following oxidation by the amino acid mono-oxygenases: pterin-4a-carbinolamine dehydratase dehydratase /de·hy·dra·tase/ (de-hi´drah-tas) a common name for a hydro-lyase.
n. (EC 18.104.22.168) and dihydropteridine reductase (DHPR; EC 184.108.40.206; Fig. 1). At least DHPR is widely distributed in animal tissues and constitutively expressed in fibroblasts (4).
Autosomal recessive mutations in the genes coding for GTPCH, PTPS, or DHPR are responsible for B[H.sub.4] deficiencies (5-7). Most of these deficiencies are characterized by neonatal hyperphenylalaninemia, developmental delay, progressive neurological deterioration, hypokinesia, hypersalivation and drooling, swallowing difficulties, truncal truncal /trun·cal/ (trung´k'l) pertaining to the trunk.
1. Of or relating to the trunk of the body.
2. Of or relating to an arterial or nerve trunk. hypotonia hypotonia /hy·po·to·nia/ (-ton´e-ah) diminished tone of the skeletal muscles.
1. Reduced tension or pressure, as of the intraocular fluid in the eyeball.
2. , increased limb tone, myoclonus myoclonus /my·oc·lo·nus/ (mi-ok´lo-nus) shocklike contractions of a muscle or a group of muscles.myoclon´ic
essential myoclonus , and temperature instability, with onset in the first months of life (8). More than 400 patients are tabulated in the international database (7).
Dopa-responsive dystonia dystonia /dys·to·nia/ (-to´ne-ah) dyskinetic movements due to disordered tonicity of muscle.dyston´ic
dystonia musculo´rum defor´mans (DRD DRD Dopa-Responsive Dystonia
DRD Dividends Received Deduction
DRD Drag Rescue Device (firefighter bunker)
DRD Deputy Regional Director
DRD Data Requirements Document
DRD Direct Reading Dosimeter
DRD Department of Redundancy Department ) is a clinical syndrome characterized by childhood-onset dystonia (usually before 12 years of age), with dramatic clinical normalization that is sustained with low doses of L-Dopa (9). The dystonia typically affects the legs, causing gait disturbance and postural instability. Signs and symptoms usually are progressive and worsen later in the evening or after exertion (diurnal fluctuation) (10). Elements of Parkinsonism (rigidity, bradikynesia, and rest tremor) commonly occur, especially in adulthood (11). Early motor development usually is normal, and hyperphenylalaninemia is absent. Recent studies have demonstrated that DRD is caused by autosomal dominant mutations in the coding region of the GCH GCH Gas Central Heating
GCH Gym Class Heroes (band)
GCH Grant Channel
GCH Grand Cross of Hanover (knight)
GCH Gas Collection Header 1 gene, which encodes for GTPCH (12), but only in 40-50% of cases (13). Reduced penetrance penetrance /pen·e·trance/ (pen´i-trins) the frequency with which a heritable trait is manifested by individuals carrying the principal gene or genes conditioning it.
n. and gender-related vulnerability are reported, with females being more affected than males (2.5:1) (14). The clinical phenotypic expression is quite diverse (15), and many cases appear to be sporadic (16).
Today, the diagnosis of B[H.sub.4] deficiencies is based on neonatal screening for phenylketonuria phenylketonuria (fĕn'əlkēt'ənr`ēə) (PKU), inherited metabolic disorder caused by the absence of a specific enzyme (phenylalanine hydroxylase). and urinary pterin analysis (17). DRD is diagnosed mainly by clinical signs and symptoms and by response to L-Dopa/Carbidopa treatment. The examination of pterins and neurotransmitter metabolites in cerebrospinal fluid (CSF Cerebrospinal Fluid (CSF) Analysis Definition
Cerebrospinal fluid (CSF) analysis is a laboratory test to examine a sample of the fluid surrounding the brain and spinal cord. ) requires an invasive procedure, but it is the only way to make a reliable diagnosis of DRD (10, 18). Definitive diagnosis of B[H.sub.4] deficiencies is achieved by enzyme activity determinations and DNA analysis (8).
PTPS and DHPR activities can be measured in liver biopsies (19, 20), erythrocytes (21, 22), and fibroblasts (23, 24), whereas GTPCH is measurable in liver biopsies (25). In mononuclear blood cells, GTPCH can be induced by phytohemagglutinin phytohemagglutinin /phy·to·hem·ag·glu·ti·nin/ (-hem?ah-glldbomact´in-in) a hemagglutinin of plant origin.
Abbr. (26), and reference values have been reported recently by Hibiya et al. (27). However, this test is quite complex and must be performed within 20 h after sample collection (27). In previous studies, fibroblasts were used for indirect measurement of pterin metabolism (28), based on the induction of GTPCH expression with cytokines, but no data are available about nonpathological and pathological values. The possibility of measuring GTPCH activity in cytokine-stimulated fibroblasts was shown by Werner et al. (29). In the present study, we used a similar stimulation procedure for measurement of both pterins and GTPCH activity. To our knowledge, fibroblasts have not been systematically tested for the other B[H.sub.4]-metabolizing enzymes. Cultured skin fibroblasts are easily used and practical for the diagnosis of many metabolic disorders. The samples can be transported at room temperature and stored for long periods after early passages for later analysis.
The aim of the present study was to demonstrate the utility of cultured skin fibroblasts as a diagnostic tool for DRD and B[H.sub.4] deficiencies. Particularly for DRD, the enzyme activity measurement is essential for final diagnosis. B[H.sub.4] metabolism was studied in fibroblasts from a large number of controls and in patients with different forms of B[H.sub.4] deficiency.
Materials and Methods
Primary skin fibroblasts were obtained from 21 patients and 35 controls. Eight patients (four males, four females), 10-45 years of age at the time of the skin biopsy, were affected by DRD (McKusick 128230). Thirteen patients, 2 months to 11 years of age at the time of the skin biopsy, were affected by B[H.sub.4] deficiencies. Of these, three patients (two males, one female) had an autosomal recessive GTPCH deficiency (McKusick 233910), seven (five males, two females) had a PTPS deficiency (McKusick 261640), and three (all males) had a DHPR deficiency (McKusick 261630). The diagnosis was confirmed by mutation analysis in all patients.
Control value biopsies were obtained from 17 subjects (10 males, 7 females) between birth and 1 year of age and 18 subjects (11 males, 7 females) between 1 and 45 years of age. No neurological abnormalities were detected in any of the controls. To establish reference values for prenatal diagnosis, 14 amniocyte samples collected between 15 and 18 weeks of gestational age were also studied. The activities of all of the B[H.sub.4]-metabolizing enzymes, as well as neopterin and biopterin production after stimulation with cytokines, were measured in all samples.
The procedures used were in accordance with the current revision of the Helsinki Declaration of 1975.
Sephadex G-50 Nick spin columns were from Pharmacia. DMEM DMEM Dulbecco's Modified Eagle's Medium (for cell culture growth)
DMEM Design Manufacture and Engineering Management Department , AmnioMax C-100 medium, trypsin/EDTA solution, antibiotics, and fetal calf serum were purchased from Life Technologies. GTP lithium salt, recombinant human IFN-[gamma], and TNF-[alpha] were from Sigma. Calf intestine alkaline phosphatase, NADH NADH the reduced form of NAD.
The reduced form of NAD.
n.pr a coenzyme that incorporates niacin and involved in the Krebs cycle. , NADPH NADPH the reduced form of NADP.
The reduced form of NADP.
reduced form of nicotinamide adenine dinucleotide phosphate (NADP) used in a number of reductive synthesis such as fatty , DHPR, peroxidase, leupeptin, and pepstatin were from Roche. Falcon cell culture plasticware was from Becton-Dickinson. y-Globulin and the protein assay dye reagent were from Bio-Rad. Ultrafree-MC filters with PL10 membrane were from Millipore.
Pteridine derivatives (6,7-dimethyltetrahydropterin and sepiapterin) were from Schircks Laboratories. Dihydroneopterin triphosphate was prepared enzymatically as described previously (30), but using recombinant GTPCH (Thony B, Blau N. Enzymatic reactor for synthesis of dihydroneopterin triphosphate. Manuscript in preparation). Recombinant SR was purified in our laboratory (Thbny B, unpublished work). All other chemicals were from Fluka.
Fibroblasts were cultured in DMEM containing 100 mL/L fetal bovine serum Fetal bovine serum ( or foetal bovine serum) is serum taken from the fetuses of cows. Fetal Bovine Serum (or FBS) is the most widely used serum in the culturing of cells. In some papers the expression foetal calf serum is used. , 50 kilounits/L penicillin, and 50 mg/L streptomycin streptomycin (strĕp'tōmī`sĭn), antibiotic produced by soil bacteria of the genus Streptomyces and active against both gram-positive and gram-negative bacteria (see Gram's stain), including species resistant to other . Amniocytes were cultured in AmnioMax C-100 medium. Cells were passaged by trypsinization and studied at low passage numbers (3-12). All cells were Mycoplasma mycoplasma
Any of the bacteria that make up the genus Mycoplasma. They are among the smallest of bacterial organisms. The cell varies from a spherical or pear shape to that of a slender branched filament. negative, as checked by the Hoechst staining method (31). Cultures were kept at 37[degrees]C in humidified air containing 5% C[O.sub.2].
To induce the expression of GTPCH, confluent con·flu·ent
1. Flowing together; blended into one.
2. Merging or running together so as to form a mass, as sores in a rash. cell monolayers in 78-[cm.sup.2] plates were stimulated with recombinant human IFN-[gamma] and TNF-[alpha] at concentrations of 250 and 100 kilounits/L, respectively, in fresh medium. After incubation for 24 h, cells were harvested by trypsinization, washed with phosphate-buffered saline, and immediately lysed for neopterin and biopterin measurement and for GTPCH activity assay. PTPS, DHPR, and SR are constitutively expressed in fibroblasts; therefore, their activities were tested in unstimulated cells. Confluent cells in 78-[cm.sup.2] plates were harvested, washed with phosphate-buffered saline, and kept at -80[degrees]C until analyzed for PTPS, DHPR and SR.
Cytokine-stimulated cells from one confluent 78-[cm.sup.2] plate were lysed in 150 [micro]L of 50 mmol/L Tris-HCl (pH 7.4) containing 1 mmol/L EDTA EDTA: see chelating agents. and 1 mmol/L dithioerythritol by freezing and thawing six times. Lysate ly·sate
The cellular debris and fluid produced by lysis. was centrifuged at 15 000g, and 100 [micro]L of the supernatant was oxidized with 6 [micro]L of 1 mol/L HCl and 20 [micro]L of 10 g/L iodine solution for 15 min at room temperature in the dark. Oxidation was stopped by the addition of 20 [micro]L of 10 g/L ascorbic acid (freshly prepared). Ten microliters of buffer (1 mol/L Tris-HCl, pH 9.6, containing 80 mmol/L Mg[Cl.sub.2]) was added to the sample to adjust to pH 8.0-9.0. Dephosphorylation of neopterin triphosphate was achieved by hydrolysis with alkaline phosphatase (16.8 U) for 60 min at 37[degrees]C. The reaction was stopped by acidification acidification
a technology used by processors to preserve foods by adding acids (such as acetic, citric, phosphoric, propionic and lactic acid) and thereby reduce the risk of growth of harmful bacteria. to pH 2.0 with 7 [micro]L of 2 mol/L HCl. The sample (final volume, 175 [micro]L) was subsequently deproteinized through an Ultrafree-MC filter and analyzed by HPLC HPLC high-performance liquid chromatography.
high performance liquid chromatography.
HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed (32). The intracellular concentrations of neopterin and biopterin after 24-h stimulation with cytokines are expressed as pmol per mg of protein.
The assay was performed as described previously by Hatakeyama and Yoneyama (33), with some modifications. The assay monitors the conversion of the substrate GTP under saturating conditions to neopterin triphosphate, which is detected as neopterin, the oxidized and dephosphorylated product (Fig. 1).
Fibroblasts were analyzed immediately after 24-h incubation with cytokines. Cells from one confluent 78-[cm.sup.2] plate were lysed in 200 [micro]L of freshly prepared homogenization homogenization (həmŏj'ənəzā`shən), process in which a mixture is made uniform throughout. Generally this procedure involves reducing the size of the particles of one component of the mixture and dispersing them evenly buffer (50 mmol/L Tris-HCl, pH 7.5, containing 0.1 mol/L KCI KCI Kansas City International (airport)
KCI Kennel Club of India
KCI Key Club International
KCI Korea Concrete Institute
KCI Kitchener Collegiate Institute
KCI Kids Central, Inc.
KCI The Kitchen Collection, Inc.
KCI Kodak Canada Inc. , 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.2 mmol/L phenylmethylsulfonyl fluoride, 1 [micro]mol/L leupeptin, and 1 [micro]mol/L pepstatin) by freezing and thawing six times and were subsequently centrifuged at 15 000g for 5 min. A 150-[micro]L aliquot aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share) of the supernatant was desalted on a spin column (Nick spin column, Sephadex G50; Pharmacia Biotech), and 50 [micro]L of the filtrate filtrate /fil·trate/ (fil´trat) a liquid or gas that has passed through a filter.
To put or go through a filter.
n. was added to 148 [micro]L of reaction buffer (50 mmol/L Tris-HCl, pH 7.5, containing 0.1 mol/L KCl and 1 mmol/L EDTA) and 2 [micro]L of 100 mmol/L GTP. The mixture was divided into two 100-[micro]L portions; one was incubated for 60 min at 37[degrees]C, whereas the other was immediately oxidized (blank with cell extract). A blank without cell extract (25 [micro]L of homogenization buffer added to 1 [micro]L of 100 mmol/L GTP and to 74 [micro]L of reaction mixture) was incubated together with the sample. The reaction was stopped by cooling the sample on ice and adding 10 [micro]L of oxidizing solution (5 g/L iodine and 10 g/L potassium iodide in 1 mol/L HCl). After oxidation in the dark for 60 min, the reaction was stopped by adding 10 [micro]L of 20 g/L ascorbic acid (freshly prepared). The mixture was adjusted to pH 8.5 by adding 14 [micro]L of 1 mol/L NaOH, and the sample was incubated with 20 [micro]L of alkaline phosphatase solution (300 kU/L calf intestine alkaline phosphatase in 0.1 mol/L Tris-HCl, pH 8.0, containing 1 mmol/L Mg[Cl.sub.2] and 0.1 mmol/L Zn[Cl.sub.2]) for 60 min at 37[degrees]C. Neopterin was measured, after deproteinization through an Ultrafree-MC filter, by HPLC (32). One unit of GTPCH produces 1 [micro]mol of neopterin per minute at 37[degrees]C.
The PTPS activity was measured in extracts from unstimulated cells. The assay was modified according to Shintaku et al. (21). The unstable intermediate metabolite formed from dihydroneopterin triphosphate is converted to B[H.sub.4] by SR in the presence of NADPH. DHPR activity is necessary to stabilize the formation of B[H.sub.4]. B[H.sub.4] is then measured as the oxidized product, biopterin.
Unstimulated cells from one confluent 78-[cm.sup.2] plate were suspended in 150 [micro]l of lysis buffer (10 mmol/L Tris-HCl, pH 7.4, containing 10 mL/L Triton X-100) and lysed by freezing and thawing six times. After centrifugation at 15 000g for 5 min, 50 [micro]L of the supernatant was added to 60 [micro]L of reaction mixture containing 100 mmol/L Tris-HCl, pH 7.4, 10 mmol/L Mg[Cl.sub.2], 1 mmol/L NADPH, 1 mmol/L NADH, 3 mU of SR, 220 mU of DHPR, and 60 [micro]mol/L dihydroneopterin triphosphate in a final volume of 110 [micro]L. A 50-[micro]L aliquot was incubated for 120 min at 37[degrees]C; another 50 [micro]L was used as a blank. A blank without cell extract, containing 50 [micro]L of lysis buffer and 60 [micro]L of reaction mixture, was incubated in the same run. The reaction was stopped by cooling the samples on ice and adding 15 [micro]L of 300 g/L trichloroacetic acid for protein precipitation, followed by centrifugation (5 min at 15 000g) and oxidation. Oxidation was performed by incubation of 50 [micro]L of the supernatant with 10 [micro]L of 10 g/L iodine for 60 min at room temperature in the dark. The same procedure was used for the blanks. After oxidation, 15 [micro]L of 10 g/L ascorbic acid was added to destroy excess iodine. Samples were diluted (1:4 by volume) with water and heated at 60[degrees]C for 2 h before being deproteinized through an Ultrafree-MC filter and analyzed by HPLC (32). One unit of PTPS produces 1 [micro]mol of biopterin per minute at 37[degrees]C.
The activity was assayed according to the method described by Ferre and Naylor (34) with some modifications. The assay monitors the conversion of sepiapterin to B[H.sub.2], which is then measured as the oxidized product, biopterin.
Unstimulated cells from one confluent 78-[cm.sup.2] plate were suspended in 1 mL of lysis buffer (0.1 mol/L potassium phosphate buffer, pH 6.4, containing 0.15 mol/L KCl, 0.68 mol/L glycerol glycerol, glycerin, glycerine, or 1,2,3-propanetriol (prō`pāntrī'ŏl), CH2OHCHOHCH2OH, colorless, odorless, sweet-tasting, syrupy liquid. , and 2.5 mmol/L EDTA), lysed by freezing and thawing six times, and centrifuged for 5 min at 15 000g. A 20-[micro]L portion of the supernatant was incubated in a freshly prepared reaction mixture (final volume, 50 [micro]L) containing 0.1 mol/L potassium phosphate buffer, pH 6.4, 125 [micro]mol/L sepiapterin, and 250 [micro]mol/L NADPH at 37[degrees]C for 30 min in the dark. A blank with cell extract was immediately oxidized, and a blank without cell extract (20 [micro]l of lysis buffer in 50 [micro]l of reaction mixture) was incubated with the samples. The reaction was stopped by adding 10 [micro]L of oxidizing solution (5 g/L iodine and 10 g/L potassium iodide in 1 mol/L HCl) and maintained at room temperature in the dark for 30 min. Excess iodine was removed by adding 10 [micro]L of 10 g/L ascorbic acid solution (freshly prepared). The sample was then deproteinized through an Ultrafree-MC filter and analyzed by HPLC (32). One unit of enzyme activity produces 1 [micro]mol of biopterin per minute at 37[degrees]C.
The activity of this enzyme was assayed, as described previously (24), by monitoring the oxidation of NADH during the reduction of 6,7-dimethyldihydropterin (quinonoid isomer) to 6,7-dimethyltetrahydropterin, catalyzed by DHPR. The pterin derivative 6,7-dimethyltetrahydropterin was oxidized in situ by peroxidase in the presence of H202 to q-6,7-dimethyldihydropterin and thus was recycled for the DHPR reaction. Unstimulated fibroblasts from three confluent 78-[cm.sup.2] plates were suspended in 450 [micro]l of lysis buffer (5 mmol/L Tris-HCl, pH 7.4, containing 0.1 mol/L KCl), lysed by freezing and thawing six times, and centrifuged at 15 000g for 20 min. The supernatant was then analyzed for protein concentration, and an amount corresponding to 120 [micro]g of protein was incubated in the assay. The assay mixture contained 8.8 mmol/L [H.sub.2][O.sub.2], 1 mmol/L NADH, 25 kU/L peroxidase, and reaction buffer (50 mmol/L Tris-HCl, pH 7.0) in a final volume of 1 mL. After a 5-min incubation at 25[degrees]C and before the substrate was added, the absorbance absorbance /ab·sor·bance/ (-sor´bans)
1. in analytical chemistry, a measure of the light that a solution does not transmit compared to a pure solution. Symbol .
2. at 340 nm of the samples and blanks (without cell extract) was measured. Subsequently, 15 [micro]L of substrate (1 mmol/L 6,7-dimethyltetrahydropterin) was added and mixed, and the absorbance was measured at the same wavelength (340 nm). The consumption of NADH was measured against corresponding blanks at 340 nm for 5 min. One unit of DHPR oxidizes 1 [micro]mol of NADH per minute at 25[degrees]C.
Protein concentrations in cell lysates were determined by the Bradford spectrophotometric method (35) using [gama]-globulin as a calibrator. The activities of the various enzymes are expressed as units per mg of protein.
Normal values of neopterin, biopterin, and enzyme activities for controls were expressed as 5th, 50th, and 95th percentiles (Table 1). Medians and minimum and maximum concentrations of the same analytes were used for pathological values because of the limited number of samples (Table 2). Statistical differences between multiple groups (neopterin and biopterin concentrations in controls and patients with different disorders; GTPCH activity in controls and patients with DRD and GTPCH deficiency) were tested using a one-way ANOVA anova
see analysis of variance.
ANOVA Analysis of variance, see there with pairwise comparison according to Tukey. The unpaired Student t-test was used to compare results between two main control groups (amniocytes vs fibroblasts, males vs females, fibroblasts 0-1 year vs fibroblasts >1 year) and between particular enzyme activities in controls and patients with different disorders. Differences were considered significant when P values were <0.05.
After stimulation for 24 h with cytokines, the concentrations of neopterin and biopterin in amniocytes were significantly lower (P <0.001) than in skin fibroblasts collected after birth (Table 1). No differences in the production of neopterin (males, 30-94 pmol/mg; females, 17-99 pmol/mg) and biopterin (males, 189-301 pmol/ mg; females, 153-327 pmol/mg) were found between fibroblasts from males (n = 21) and females (n = 14). GTPCH activity was very low in amniocytes but increased in fibroblasts collected after birth (P <0.01). Fibroblast fibroblast /fi·bro·blast/ (fi´bro-blast)
1. an immature fiber-producing cell of connective tissue capable of differentiating into chondroblast, collagenoblast, or osteoblast.
2. GTPCH activity was higher in females than in males (females, 1.4-7.3 [micro]Umg; males, 1.4-3.9 [micro]U/mg; P = 0.016). No significant difference was observed at different ages (0-1 year, 1.7-4.9 [micro]U/mg; >1 year, 1.3-7.3 [micro]U/mg; P = 0.97; activity 0-10 years vs activity >10 years, P = 0.74).
PTPS activity was higher in amniocytes than in fibroblasts (P <0.001; Table 1). A decrease in enzyme activity was observed in cells collected after the first year of age (0-1 year, 0.5-1.7 [micro]Umg; >1 year, 0.4-0.7 [micro]Umg; P = 0.014). No differences were observed in other age groups after 1 year. Enzyme activities in males (0.4-1.0 [micro]U/mg) vs females (0.4-1.8 [micro]U/mg) showed no significant differences. SR activity was similar in cells collected before and after birth, as well as in males and females (males, 96-202 [micro]U/mg; females, 120-155 [micro]U/mg) and also in different age groups. DHPR activity was also similar in amniocytes and fibroblasts, as well as in males and females (males, 5-7.8 mU/mg; females, 4.2-8.9 mU/mg). A decrease in activity was observed after 1 year of age (0-1 year, 5.4-8.9 mU/mg; >1 year, 4.2-7.0 mU/mg; P = 0.03). No differences were observed in other age groups after 1 year. There were no significant increases in PTPS, SR, and DHPR activities after stimulation with cytokines (data not shown).
Neopterin and biopterin concentrations were significantly different between controls and patients (P <0.001; Table 2). Cytokine-stimulated GTPCH-deficient cells (autosomal recessive form, three cases) showed extremely low concentrations of neopterin and biopterin (Fig. 2) and a median enzyme activity of 0.8 [micro]Umg of protein (30.7% of the 50th percentile for normal activity). Cells from DRD patients (eight cases) also showed reduced neopterin and biopterin concentrations compared with controls (Fig. 2). In both diseases, GTPCH activity was lower than in controls (P <0.001). In DRD patients, the median enzyme activity was 0.4 [micro]U/mg of protein (15.4% of the 50th percentile for normal activity), ranging between 0.1 (3.8%) and 0.6 [micro]U/mg (23%). GTPCH activity in the autosomal recessive form was higher than in the autosomal dominant form (GTPCH deficiency vs DRD, P <0.01). PTPS-deficient cells (seven cases) contained extremely high concentrations of neopterin (median, 475 pmol/mg; range, 334-734 pmol/mg), and no measurable biopterin (Fig. 2) after stimulation with cytokines for 24 h. Enzyme activity, measured in unstimulated cells, was always below the detection limit (<0.05 [micro]U/mg, 7% of 50th percentile for controls). DHPR-deficient cells (three cases) showed intracellular pterin concentrations within reference values after stimulation (for neopterin, median 49 pmol/mg; range 29-81 pmol/mg; for biopterin, median, 179 pmol/mg; range, 99-181 pmol/mg; Fig. 2), with biopterin concentrations significantly lower than in controls (P <0.001). Enzyme activity was not detectable in unstimulated fibroblasts from the same patients (<0.3 mU/mg, 5% of 50th percentile for controls).
Autosomal recessive B[H.sub.4] deficiencies are characterized by the presence of neonatal hyperphenylalaninemia with abnormal urinary pterin excretion. The differential diagnosis between phenylketonuria and B[H.sub.4] deficiencies is extremely important because B[H.sub.4]-deficient patients do not respond to low phenylalanine dietary treatment. However, they benefit from early substitution therapy with B[H.sub.4] and neurotransmitter precursors (L-Dopa and 5-hydroxytryptophan) (36, 37).
DRD is diagnosed mainly by the typical clinical signs and symptoms and by the responsiveness to therapy with L-Dopa/Carbidopa. Hyperphenylalaninemia usually is not present in these patients; however, the oral phenylalanine loading test is consistent with a partial deficiency of B[H.sub.4] in the liver. Plasma phenylalanine/tyrosine ratios increase significantly 4-6 h after the challenge (38). Unfortunately, false-positive results with this test are possible; in fact, heterozygous het·er·o·zy·gous
1. Having different alleles at one or more corresponding chromosomal loci.
2. Of or relating to a heterozygote. phenylketonuric subjects show the same abnormal phenylalanine/tyrosine profiles (39), whereas a small number of genetically confirmed DRD subjects showed no abnormalities with this test (40). A lumbar puncture for CSF examination is invasive but most informative for the diagnosis of both recessive B[H.sub.4] deficiencies (41) and DRD (10). Low CSF concentrations of homovanillic acid and 5-hydroxyindoleacetic acid, metabolites of dopamine and serotonin, respectively, are found in all variants of B[H.sub.4] deficiencies. Furthermore, both neopterin and biopterin are very low in GTPCH deficiency, neopterin is high and biopterin low in PTPS deficiency, and biopterin concentrations are very high in DHPR deficiency (8, 41). CSF examinations in DRD patients revealed low concentrations of homovanillic acid, indicating a lack of dopamine synthesis, and low neopterin and biopterin concentrations are consistent with a GTPCH deficiency (10).
The final diagnosis of B[H.sub.4] deficiencies is achieved by enzyme activity determinations and, with some limitations, by detection of mutations. PTPS and DHPR activities are measurable in many different tissues and cells such as erythrocytes (21, 22), fibroblasts (23, 24), and liver (19, 20). Dried blood spots are routinely used for DHPR deficiency screening among hyperphenylalaninemic newborns (22). Measurement of GTPCH activity is rather difficult because this enzyme is not expressed in blood cells and fibroblasts. Blau et al. (26) described an assay in phytohemagglutinin-stimulated mononuclear blood cells that was used to measure reduced activity in heterozygous and homozygous ho·mo·zy·gous
Having the same alleles at one or more gene loci on homologous chromosome segments.
Identical genes controlling a specified inherited trait. GTPCH subjects, and subsequently in DRD patients (12). The test must be performed with fresh blood samples, within 20 h after sample collection (27). Bezin et al. (42) suggested that the stimulation of lymphoblasts could lead to incorrect determinations of the actual activity of GTPCH in DRD patients.
Because not all GTPCH-deficient DRD patients have GCH1 mutations in the coding region or in the splice sites, which are detectable by the current genomic DNA sequencing of GCH1 (43), and because of the high occurrence of sporadic mutations in this gene (14), DNA testing for the autosomal dominant DRD is not suitable for routine clinical practice. Suzuki et al. (44) reported that in 40% of DRD patients no mutation was found in the GCH1 gene but that some of them showed reduced GTPCH activity in phytohemagglutinin-stimulated mononuclear blood cells. Therefore, measurement of enzyme activity is extremely important for the diagnosis of DRD. Recent studies demonstrated that cytokine-stimulated fibroblasts may be used for investigating B[H.sub.4] pathway integrity (28), by measuring intracellular neopterin and biopterin production patterns, and for GTPCH activity measurement (29). Here, we adapted these methods to investigate in cytokine-stimulated fibroblasts both pterin production patterns and GTPCH activity, with a diagnostic purpose. We first determined the reference values of intracellular neopterin and biopterin in fibroblasts and amniocytes after stimulation with cytokines. Under these conditions, amniocytes appeared to produce rather low amounts of pterins, consistent with their low GTPCH activity (Table 1). Therefore, these cells are not suitable for prenatal diagnosis of GTPCH deficiency. On the other hand, prenatal diagnosis of PTPS and DHPR deficiency should be possible because the activities of these two enzymes are well detectable in amniocytes (Table 1).
We observed that cells collected in the first year of life showed higher PTPS and DHPR activities than cells collected at a later age. GTPCH activity was slightly but not significantly higher when cells were collected during the first year of life. Because Hibiya et al. (27) reported a higher GTPCH activity in mononuclear blood cells in the first 10 years of life, we compared fibroblasts of these two control groups and found no significant difference. Only GTPCH activity was different between males and females, with higher activities in females. In contrast, lower activities in females were reported by Ichinose et al. (12), and no difference was found by Hibiya et al. (27) in mononuclear blood cells. The analysis of a larger collection of controls is probably needed to better define these differences in fibroblasts; however, this is not of diagnostic importance.
The study of pathological samples allowed us to define the utility of cultured skin fibroblasts in the diagnosis of B[H.sub.4] defects and DRD. Except for SR, for which no patients have as yet been reported, all the studied enzymes exhibited very low activities in patient samples compared with controls (Table 2). Indeed, PTPS and DHPR activities in unstimulated fibroblasts from the corresponding patients were below the detection limit (<7% and <5% of the 50th percentile of normal activity, respectively). PTPS deficiency was characterized by a typical pattern of pterin production in stimulated fibroblasts, with high neopterin concentrations and no measurable biopterin. The same pattern was observed in urine and CSF (8). DHPR-deficient patients showed normal intracellular neopterin concentrations after stimulation, whereas biopterin concentrations were lower than in controls. Milstien et al. (28) suggested that the possible pattern of pterin production in these cells could be characterized by increased neopterin because of the lack of feedback inhibition of GTPCH by B[H.sub.4], and reduced biopterin because of the fast transport rates of the oxidized forms of pterins out of the cells. This hypothesis would explain the differences of pterin patterns between the cell extracts and the extracellular fluids (plasma, urine, CSF), where increased biopterin is characteristic of the disease (41).
GTPCH activity was lower in stimulated fibroblasts from patients with the autosomal dominant form than in those from patients with the autosomal recessive form. Previous measurements in phytohemagglutinin-stimulated mononuclear blood cells showed a reduction of GTPCH activity in DRD patients to <20% (12), which was comparable to our finding, and a reduction of activity to <3% in autosomal recessive GTPCH deficiency (26), in contrast to our results in stimulated fibroblasts (Table 2). A dominant-negative effect of the mutated subunit on the activity of the wild-type subunit in DRD was suggested by Hirano and Ueno (45). Indeed, cotransfection experiments with two mutant GTPCH alleles, identified in DRD patients, demonstrated that mutant proteins inhibit the activity of the wild-type enzyme. In contrast, cotransfection of the mutant allele found in a patient with autosomal recessive GTPCH deficiency failed to inhibit the wild-type activity, producing much higher GTPCH activity (46). The molecular mechanism of this dominant-negative effect and the different phenotype, which is more severe in recessive GTPCH deficiency despite higher enzyme activity in vitro, remain to be explained. Recently, Suzuki et al. (44) reported that a reduction in the amount of enzymatic protein could be responsible for the lower GTPCH activity in DRD compared with the autosomal recessive GTPCH deficiency. A dominant-negative effect might still be acting at an earlier stage, at either the translational or the transcriptional level. Regardless, GTPCH activity measurement in cytokine-stimulated fibroblasts was diagnostic for both disorders.
In conclusion, skin cultured fibroblast assays are confirmatory for all suspected B[H.sub.4] deficiencies. Our studies now provide for the first time reference values for direct and indirect (intracellular neopterin and biopterin) estimation of enzyme activity. The study of B[H.sub.4] metabolism is particularly useful in DRD patients because it allows an etiologic diagnosis when other tests are inconclusive. This study was supported by the Swiss National Science Foundation The Swiss National Science Foundation is a science research support organization mandated by the Swiss Federal Government. The SNSF was established in 1952 as a foundation under private law. Its secretariat is based in Berne. , Grant 31-54183.98 (to N.B.). We are grateful to M. Killen for editorial work and Dr. L. Molinari for help with statistical analyses. We acknowledge the following colleagues who provided fibroblasts from their patients: Dr J. Van Hove, Department of Pediatrics, University Hospital Gasthuisberg, Leuven, Belgium; Dr. M. Poppe Poppe is a surname, and may refer to:
- Erik Poppe
- Nils Poppe
- Ulrike Poppe
- Walter Poppe
This page or section lists people with the surname Poppe. , University Children's Hospital, Dresden, Germany; Dr. H. Steinbbck, St. Anna Children's Hospital, Wien, Austria; Dr. S. Scheibenreiter, University Children's Hospital, Wien, Austria; Dr. B. Bartholome, University Children's Hospital, Bochum, Germany; Dr. J.F. Hansen, Juliane Marie Centret Neuropediatric Clinic, Copenhagen, Denmark; Dr. B.T. Poll-The, UMC UMC United Methodist Church
UMC United Microelectronics Corporation
UMC University Medical Center
UMC United Microelectronics Corp (Republic of China)
UMC University of Missouri-Columbia , Utrecht, The Netherlands; Dr. J. Herwig, University Children's Hospital, Frankfurt, Germany; Dr. S. Winter, Medical Genetics, Madera, CA; Dr. T. Coskun, Hacettepe University, Ankara, Turkey; Dr. B. Leroux, American Memorial Hospital, Reims, France, Dr. A.B. Burlina, Department of Pediatrics, University of Padua History
The university was founded in 1222 when a large group of students and professors left the University of Bologna in search of more academic freedom. The first subjects to be taught were jurisprudence and theology. , Italy; Dr. I. Dianzani, University of Turin The University of Turin (Italian Università degli Studi di Torino, UNITO) is a university in the city of Turin in the Piedmont region of north-western Italy. It has 12 faculties and 55 departments. , Italy; and Dr. P.T. Clayton, Institute of Child Health, University College London "UCL" redirects here. For other uses, see UCL (disambiguation).
University College London, commonly known as UCL, is the oldest multi-faculty constituent college of the University of London, one of the two original founding colleges, and the first British , United Kingdom.
Received September 26, 2000; accepted December 4, 2000.
(1.) Kaufman S. Tetrahydrobiopterin-basic biochemistry and role in human disease. Baltimore: The Johns Hopkins University Johns Hopkins University, mainly at Baltimore, Md. Johns Hopkins in 1867 had a group of his associates incorporated as the trustees of a university and a hospital, endowing each with $3.5 million. Daniel C. Press, 1997:31-234.
(2.) Schoedon G, Troppmair J, Fontana A, Huber C, Curtius HC, Niederwieser A. Biosynthesis and metabolism of pterins in peripheral blood mononuclear cells and leukemia lines of man and mouse. Eur J Biochem 1987;166:303-10.
(3.) Werner ER, Werner Felmayer G, Wachter H. Tetrahydrobiopterin and cytokines. Proc Soc Exp Biol Med 1993;203:1-12.
(4.) Thony B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration, and functions. Biochem J 2000;347:1-26.
(5.) Thony B, Blau N. Mutations in the GTP cyclohydrolase I and 6-pyruvoyl-tetrahydropterin synthase genes. Hum Mutat 1997;10: 11-20.
(6.) Smooker PM, Cotton RGH RGH Rochester General Hospital (New York)
RGH Rawalpindi General Hospital (Rawalpindi, Pakistan) . Molecular basis of dihydropteridine reductase deficiency. Hum Mutat 1995;5:279-84.
(7.) Blau N, Thony B, Dianzani I. DIOMDB. Database of mutations causing tetrahydrobiopterin deficiency. http://www.BH4.org/ biomdbl.html, updated for 2000.
(8.) Blau N, Thony B, Cotton RGH, Hyland K. Disorders of tetrahydrobiopterin and related biogenic biogenic /bi·o·gen·ic/ (-jen´ik) having origins in biological processes.
having the property of originating in a biological process. amines. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B, eds. The metabolic and molecular bases of inherited disease, 8th ed. New York: McGrawHill, 2001:1275-776.
(9.) Segawa M, Nomura Y. Hereditary progressive dystonia with marked diurnal fluctuation and dopa-responsive dystonia: pathognomonic pathognomonic /pa·thog·no·mon·ic/ (path?ug-no-mon´ik) specifically distinctive or characteristic of a disease or pathologic condition; denoting a sign or symptom on which a diagnosis can be made. clinical features. In: Segawa M, Nomura Y, eds. Age related dopamine dependent disorders. Basel: Karger, 1995:1024.
(10.) Nygaard TG. Dopa-responsive dystonia. Delineation of the clinical syndrome and clues to pathogenesis. Adv Neurol 1993;60:577-85.
(11.) Nygaard TG, Wilhelmsen KC, Riesch NJ, Brown DL, Trugman JM, Gilliam TC, et al. Linkage mapping of dopa-responsive dystonia (DRD) to chromosome 14q. Nat Genet 1993;5:386-91.
(12.) Ichinose H, Ohye T, Takahashi E, Seki N, Hori T, Segawa M, et al. Hereditary progressive dystonia with marked diurnal fluctuation caused by mutation in the GTP cyclohydrolase I gene. Nat Genet 1994;8:236-41.
(13.) Furukawa Y, Kish SJ. Dopa-responsive dystonia: recent advances and remaining issues to be addressed. Mov Disord 1999;14: 709-15.
(14.) Furukawa Y, Lang AE, Trugman JM, Bird TD, Hunter A, Sadeh M, et al. Gender-related penetrance and de novo GTP cyclohydrolase I gene mutations in dopa-responsive dystonia. Neurology 1998;50: 1015-20.
(15.) Steinberger D, Weber Y, Korinthenberger R, Deuschl G, Benecke R, Martinius J, Muller U. High penetrance and pronounced variation in expressivity expressivity /ex·pres·siv·i·ty/ (eks?pres-siv´i-te) in genetics, the extent to which an inherited trait is manifested by an individual. of GCH1 mutations in five families with Dopa-responsive dystonia. Ann Neurol 1998;43:634-9.
(16.) Nygaard TG, Marsden CD, Fahn S. Dopa-responsive dystonia: long term treatment response and prognosis. Neurology 1991;41:174.
(17.) Dhondt JL. Strategy for the screening of tetrahydrobiopterin deficiency among hyperphenylalaninaemic patients: 15-years experience. J Inherit Metab Dis 1991;14:117-27.
(18.) Fink JK, Barton N, Cohen cohen
or kohen
(Hebrew: "priest") Jewish priest descended from Zadok (a descendant of Aaron), priest at the First Temple of Jerusalem. The biblical priesthood was hereditary and male. W, Lovenberg W, Burns RS, Hallett M. Dystonia with marked diurnal variation associated with biopterin deficiency. Neurology 1988;38:707-11.
(19.) Niederwieser A, Leimbacher W, Curtius HC, Ponzone A, Rey F, Leupold D. Atypical phenylketonuria with "dihydrobiopterin synthetase synthetase /syn·the·tase/ (-the-tas) a term used in the names of some of the ligases, no longer favored because of its similarity to synthase and its emphasis on reaction products.
n. " deficiency: absence of phosphate-eliminating enzyme activity demonstrated in liver. Eur J Pediatr 1985;144:13-6.
(20.) Kaufman S, Holtzman NA, Milstien S, Butler U, Krumholz A. Phenylketonuria due to a deficiency of dihydropteridine reductase. N Engl J Med 1975;293:785-90.
(21.) Shintaku H, Niederwieser A, Leimbacher W, Curtius HC. Tetrahydrobiopterin deficiency: assay for 6-pyruvoyl-tetrahydropterin synthase activity in erythrocytes, and detection of patients and heterozygous carriers. Eur J Pediatr 1988;147:15-9.
(22.) Arai N, Narisawa K, Hayakawa H, Tada K. Hyperphenylalaninemia due to dihydropteridine reductase deficiency: diagnosis by enzyme assays on dried blood spots. Pediatrics 1982;70:426-30.
(23.) Guzman J, Blau N. 6-Pyruvoyl-tetrahydropterin synthase in human tissues and cell lines. Pteridines 1992:3:43-5.
(24.) Milstien S, Holtzman NA, O'Flynn ME, Thomas GH, Butler IJ, Kaufman S. Hyperphenylalaninemia due to dihydropteridine reductase deficiency. Assay of the enzyme in fibroblasts from affected infants, heterozygotes, and in normal amniotic fluid cells. J Pediatr 1976;89:763-6.
(25.) Blau N, Niederwieser A. Guanosine triphosphate cyclohydrolase I assay in human and rat liver using high-performance liquid chromatography of neopterin phosphates and guanine guanine (gwä`nēn), organic base of the purine family. It was reported (1846) to be in the guano of birds; later (1879–84) it was established as one of the major constituents of nucleic acids. nucleotides. Anal Biochem 1983;128:446-52.
(26.) Blau N, Joller P, Atares M, Cardesa-Garcia J, Niederwieser A. Increase of GTP cyclohydrolase I activity in mononuclear blood cells by stimulation: detection of heterozygotes of GTP cyclohydrolase I deficiency. Clin Chim Acta 1985;148:47-52.
(27.) Hibiya M, Ichinose H, Ozaki N, Fujita K, Nishimoto T, Yoshikawa T, et al. Normal values and age-dependent changes in GTP cyclohydrolase I activity in stimulated mononuclear blood cells measured by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 2000;740:35-42.
(28.) Milstien S, Kaufman S, Sakai N. Tetrahydrobiopterin biosynthesis defects examined in cytokine-stimulated fibroblasts. J Inherit Metab Dis 1993;16:975-81.
(29.) Werner ER, Werner-Felmayer G, Fuchs D, Hausen A, Reibnegger G, Wachter H. Parallel induction of tetrahydrobiopterin biosynthesis and indoleamine 2,3-dioxygenase activity in human cells and cell lines by interferon-[gamma]. Biochem J 1989;262:861-6.
(30.) Blau N, Niederwieser A, Redweik U, Schoedon G, Leimbacher W. Immobilization Immobilization Definition
Immobilization refers to the process of holding a joint or bone in place with a splint, cast, or brace. This is done to prevent an injured area from moving while it heals. of GTP cyclohydrolase I and 6-pyruvoyl-tetrahydropterin synthase to different affinity supports. In: Levine RA, Milstien S, Kuhn DM, Curtius HC, eds. Pterins and biogenic amines in neuropsychiatry neuropsychiatry /neu·ro·psy·chi·a·try/ (noor?o-si-ki´ah-tre) the combined specialties of neurology and psychiatry.
n. , pediatrics and immunology. Grosse Pointe: Lakeshore Publishing, 1989:97-105.
(31.) Kucherlapati RS, Hilwig I, Gropp A, Ruddle rud·dle also red·dle or rad·dle
Red ocherous iron ore, used in dyeing and marking.
tr.v. rud·dled, rud·dling, rud·dles
To dye or mark with or as if with red ocher: FH. Mammalian chromosome identification in interspecific in·ter·spe·cif·ic
Arising or occurring between species.
interspecific also interspecies
Arising or occurring between species.
Adj. 1. hybride cells using "Hoechst 33258". Humangenetik 1975;27:9-14.
(32.) Curtius HC, Blau N, Kuster T. Pterins. In: Hommes FA, ed. Techniques in diagnostic human biochemical genetics. New York: Wiley-Liss, 1991:377-96.
(33.) Hatakeyama K, Yoneyama T. A sensitive assay for the enzymatic activity of GTP cyclohydrolase I. In: Titheradge MA, ed. Nitric oxide protocols. Totowa, NJ: Humana Press, 1998:265-72.
(34.) Ferre J, Naylor EW. Sepiapterin reductase in human amniotic amniotic /am·ni·ot·ic/ (am?ne-ot´ik) pertaining to or developing an amnion.
pertaining to the amnion.
amniotic fluid and skin fibroblasts, chorionic villi, and various blood fractions. Clin Chim Acta 1988;174:271-82.
(35.) Bradford MM. A rapid and sensitive method for the quantitation of microgram microgram /mi·cro·gram/ (µg) (mi´kro-gram) one millionth (10-6) of a gram.
Abbr. quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.
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(39.) Saraiva JM, Seakins JWT JWT J. Walter Thompson (agency)
JWT Java Windows Terminal
JWT Just War Theory
JWT Jim Wolf Technology
JWT Java Workflow Tooling
JWT John Wayne Trail (US Army Yakima Training Center)
JWT Justified War Theory , Smith I. Plasma phenylalanine and tyrosine levels revisited in heterozygotes for hyperphenylalaninemia. J Inherit Metab Dis 1993;16:105-9.
(40.) Saunders-Pullman RJ, Raymond D, Hyland K. Markers of disease in dopa-responsive dystonia [Abstract]. Mov Disord 1998;13:285.
(41.) Blau N, Hoffmann GF. Differential diagnosis of disorders of biogenic amines metabolism. Eur J Pediatr Neurol 1998;2:210-20.
(42.) Bezin L, Nygaard TG, Neville JD, Shen H, Levine RA. Reduced lymphoblast lymphoblast /lym·pho·blast/ (lim´fo-blast) a morphologically immature lymphocyte, representing an activated lymphocyte that has been transformed in response to antigenic stimulation. neopterin detects GTP cyclohydrolase dysfunction in dopa-responsive dystonia. Neurology 1998;50:1021-7.
(43.) Bandmann 0, Nygaard TG, Surtees R, Marsden CD, Wood NW, Harding AE. Dopa-responsive dystonia in British patients-new mutations of the GTP cyclohydrolase I gene and evidence for genetic heterogeneity. Hum Mol Genet 1996;5:403-6.
(44.) Suzuki T, Ohye T, Nagatsu T, Ichinose H. Characterization of wild type and mutants of recombinant human GTP cyclohydrolase I: relationship to etiology of Dopa-responsive dystonia. J Neurochem 1999;73:2510-6.
(45.) Hirano M, Yanagihara T, Ueno S. Dominant negative effect of GTP cyclohydrolase I mutations in dopa-responsive hereditary progressive dystonia. Ann Neurol 1998;44:365-71.
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[1] Nonstandard abbreviations: B[H.sub.4], tetrahydrobiopterin; GTPCH, GTP cyclohydrolase 1; IFN-[gamma], interferon-[gamma]; TNF-[alpha], tumor necrosis factor-[alpha]; PTPS, 6-pyruvoyltetrahydropterin synthase; SR, sepiapterin reductase; DHPR, dihydropteridine reductase; DRD, Dopa-responsive dystonia; and CSF, cerebrospinal fluid.
LUISA BONAFE, BEAT THONY, WALTER LEIMBACHER, LUCJA KIERAT, and NENAD BLAU *
Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zurich History
The University of Zurich was founded in 1833 with existing colleges of theology (founded by Huldrych Zwingli in 1525), law and medicine merged together with a new faculty of Philosophy. , Steinwiesstrasse 75, 8032 Zurich, Switzerland.
* Author for correspondence. Fax 41-1-266-7169; e-mail email@example.com.
Table 1. Reference values for neopterin and biopterin production and GTPCH activity in cytokine-stimulated control cells, and PTPS, SR, and DHPR activities in unstimulated control cells. Amniocytes Fibroblasts Percentile (n = 14) (n = 35) Neopterin, pmol/mg 5 0 18 50 4 43 95 14 98 Biopterin, pmol/mg 5 0 154 50 23 232 95 115 303 GTPCH activity, [micro]U/mg 5 0.0 1.4 50 0.2 2.6 95 1.8 6.5 PTPS activity, (a) [micro]U/mg 5 1.0 0.4 50 3.0 0.7 95 3.4 1.6 SR activity, (a) [micro]U/mg 5 89 99 50 143 138 95 313 185 DHPR activity, (a) [micro]U/mg 5 5.6 4.5 50 7.5 6.7 95 9.6 8.3 (a) Activity measured in unstimulated cells. Table 2. Neopterin and biopterin production and enzyme activity in fibroblasts of patients with DRD and autosomal recessive [BH.sub.4] deficiencies. (a) Neopterin, Biopterin, Enzyme activity, pmol/mg pmol/mg [micro] U/mg (%) DRD (n = 8) Median 0 84 0.4 (15.4) Range 0-27 0-139 0.1-0.6(3.8-23) GTPCH deficiency (n = 3) Median 0 24 0.8 (30.7) Range 0-26 0-74 0.7-1.0(26.9-38.5) PTPS deficiency (n = 7) Median 475 0 <0.05 (<7) (b) Range 334-734 0 <0.05 (<7) (b) DHPR deficiency (n = 3) Median 49 179 <0.3 (<5) (b,c) Range 29-81 98-181 <0.3 (<5) (b,c) Controls (n = 35) 43 232 (100.00) (18-98) (154-303) (a) Neopterin and biopterin concentrations (in all patients) and GTPCH activity (in patients with DRD and autosomal recessive GTPCH deficiency) were measured in cytokine-stimulated fibroblasts. PTPS and DHPR activities (in PTPS- and DHPR deficient patients, respectively) were measured in unstimulated fibroblasts. (b) Activity measured in unstimulated fbroblasts. (c) mU/mg.
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County's cold weather shelter program opened at alternate sites on Dec. 6, then at armories on Dec. 13.
County homeless shelters
The Santa Ana armory is at 612 E. Warner. The Fullerton armory is at 400 S. Brookhurst.
Bus pick up locations are in Anaheim and Santa Ana.
For information call Michael Escarcida at 714-836-7188 ext. 108 or e-mail firstname.lastname@example.com regarding Santa Ana site; or call James Brooks at 714-836-7188 ext. 107 or e-mail firstname.lastname@example.com regarding Fullerton site.
Source: Mercy House and County of Orange
Last month, when a cold chill pierced our normally sunny Orange County bubble, I could think of only one thing, and despite the approaching holidays it had nothing to do with visions of sugar plums or yuletide festivities.
I had the homeless on my mind, all 21,479 of them. That's the official county estimate for those expected to become homeless over the course of a year in Orange County.
Hidden away at night in civic center parking structures, buildings and constructions sites, would these men, women and children be able to withstand temperatures that have dipped into the 40s in Santa Ana?
Last year, the county government's Cold Weather Armory Emergency Shelter Program began operating on Dec. 1. But this year, Dec. 1 came and went and all was dark.
The cold weather shelter program wasn't scheduled to start until Dec. 16, according to Larry Haynes, the executive director of the Santa Ana-based nonprofit Mercy House, which is contracted by the county to operate the program.
Dec. 1, Dec. 16 – it might seem like I'm quibbling for just a few weeks, but imagine: It's just you, some blankets and a cold, concrete floor on a December night. Even a one-day delay feels inhumane.
So what was the hold up? Right now, the county is at the mercy of the California National Guard, which allows the county to operate the cold weather shelter program out of its armories in Santa Ana and Fullerton.
This year, on Nov. 22, the National Guard gave the county notice that the armories wouldn't be available until Dec. 15 because both facilities were needed for a soldier readiness training program.
It's not uncommon for the shelter program to have dark nights throughout the season for this same reason, and Mercy House and previous program operators have turned to alternate sites, such as churches, on those dark nights.
So, with the Nov. 22 notice, why didn't Mercy House find an alternate location and open on Dec. 1 regardless of the delay? The weather may have been unseasonably warm this weekend, but the week of Nov. 22, temperatures in Santa Ana dipped into the 40s.
Haynes says Mercy House didn't want to open in mid-December and that the temperatures were a driving force for why his staff scrambled to find alternative sites at two churches. But the soonest they could open was Dec. 6. On Dec. 13, the program transitioned to the two armories, which can shelter a combined 400 people.
He points out that the armory operation is a "huge logistical feat," with a lot of moving parts, and that this is the first year any operator has opened at an alternate site.
"The opening had nothing to do with funding, nothing to do with anybody's priorities, had nothing to do with anything other than the National Guard needed to use the site for military operations," says Haynes, whose staff contacted 54 churches to find an alternate location.
In years past, the cold weather program would open a week before Thanksgiving because as its name implies, it's meant to house the homeless when temperatures fall.
But this year's delay is just a symptom of the underlying problem, which is that our county lacks a true year-round emergency homeless shelter at a permanent location.
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text: The New Americans Changing ü Before the 2 nd Patterns Industrial Revolution, most immigrants came from Britain, Germany, and Scandinavia. ü Very similar to the Americans already here. ü Most were Protestant and many spoke English. ü Settled in rural areas Changing ü After 2nd Industrial Patterns cont'd Revolution, most new immigrants came from southern and eastern Europe ü Italy, Poland, Russia, Greece, etc.. ü Came for jobs from the 2nd Industrial Revolution ü Many were escaping political and religious persecution Changing ü Had many unfamiliar Patterns cont'd cultural customs ü Religiously diverse Catholics , Jews, Eastern Orthodox Christians, etc) ü Spoke many different languages and dialects Class Task Turn to pages 613614. Read the sections labeled Leaving Troubles Behind, Seeking Fairness, and Seeking Opportunity. After you have finished reading, compare the reasons for immigration to the U.S. today with those in the late 1800s. Are immigrants' reasons for coming to The journey • Trip took between 6 days to one month over • Most immigrants traveled in the cheapest 3 rd class (steerage) • Steerage was below deck, crowded, and dirty • Diseases spread rapidly Class Task Read the poem, The New Colossus, by Emma Lazarus and complete the handout. ü 1892 Ellis Island Arrival in opens in New York America Harbor ü Contained hospitals, dorms, processing centers ü Received thousands of immigrants a day ü 1907 Peak year for receiving immigrants (over 1 million arrived) ü April 17, 1907 (Peak day 11,747 arrived) ü Medical inspection Ellis Island first no contagious diseases ü If they pass the medical exam, they go to the Great Hall to be processed ü Immigrants had to answer 29 questions about age, background, criminal history, job, and money, etc. ü If no medical issues, whole Insert clip about Ellis Island/Statue of Liberty Becoming ü "Streets paved with Americans gold" ü Immigrants wanted better lives in America ü Most settled in cities 3 out of 9 stayed in NYC Immigrant ü Entire families had to work to survive even Work children ü Most jobs were unskilled, dangerous, long hours and low paying ü 16 hour days, 6 days a week ü Avg $11.55 a day; women earned less, children even less ü Common forms of work sewing factories, steel mills, meat packing plants, coal mines Immigrant Life ü Settled in neighborhoods with other immigrants of from same countries ü Little Italy, Chinatown, Little Greece ü Formed benevolent societies to help each other in times of need ü Wanted to assimilate as much as possible to provide a better future for their children ü Often worked during days, school at night Immigrant ü Most immigrants settled in cities Living ü Cities were unprepared for massive waves of immigrants ü Very little housing, terrible health/sanitation services, lots of crime ü Public Tenements were built to house immigrants ü Very poor conditions Primary Resource Analysis Read the passage from Jacob Riis', How the Other Half Lives, and answer the following questions: 1.Why does Riis describe the tenements in which many immigrants live as "dens of death"? 2.What were living conditions like in many tenements as described by Riis? Use at Photograph Analysis With your group, analyze the photograph about tenement life by completing the activity on the Photograph Analysis: 1. Describe what is going on in your photograph. 2. Write TWO ideas you have about tenement living using the picture. (Start with, "Tenement life was __________. I know this because ________________." Opposition to ü Many people thought that the Immigration poverty and lack of education of the immigrants would hurt US society ü There was much racial and religious prejudice against immigrants ü Many workers opposed immigration bc they thought the Reading for Information Read the passage labeled, "No Longer Welcome" with your partner and answer the following questions: 1.Why did many Americans oppose immigration? 2.What business practice did workers' unions oppose? Why? Chinese Exclusion Act üBanned Chinese (1882) people from coming to the US for 10 years (and was later extended into 1900s ) Immigration üWanted all Restriction immigrants to prove they were League (1894) literate in some language üCongress passed literacy law in 1897 but president vetoed the bill for being "unAmerican." Primary Resource Analysis Turn to page 616 in your textbook and find the box labeled, Interpreting Political Cartoons. With your partner, answer the following questions: 1. How are the successful Americans treating the new immigrant? 2. If the Americans looked backward, as the title suggests, what would they see? What is the cartoonist saying? Fitting In ü Immigrants became "Americanized" (Assimilate) ü America becomes known as "The Melting Pot" ü Changed their language, clothing, food in an effort to assimilate to American culture ü Embraced ideas of democracy, rights, and freedom While immigrants assimilated and became Americanized, they also changed America and its people. This wave of immigration led to massive changes in our culture, cities, and businesses. Effects on ü Food American ü Clothing Culture ü Language ü Art ü Music Effects on ü Massive waves of American immigrants led to rapid growth of cities Cities ü This is known as urbanization ü Cities often grew too fast ü Not enough police, firefighters, hospitals, sanitation workers ü Overcrowding and lack of sanitation ü Many suffered from disease and poor Settlement ü Private citizens wanted to help the poor in the Houses cities ü Started settlement houses community centers set in poor neighborhoods that offered education, recreation, and social activities) ü Hull House Jane Addams and Ellen Gates Starr) ü Served the needs of poor and immigrant Hull House Urban Reform ü Terrible conditions led to call for urban reform ü Laws passed that improve cities: ü Safety codes for buildings ü Illegal to build tenements ü Street paving ü Sanitation (garbage and sewer) ü Water supply ü Pollution ü These improvements gave many urban Workplace ü Terrible conditions in Reform factories, mills, and mines led to calls for reform ü Thousands of immigrants were hurt or killed at work due to bad conditions ü Workers fight for shorter workdays, better pay, safer working conditions ü Want to end child labor ü Labor unions formed bc the gov't wasn't doing enough to help workers Labor Unions ü A labor union is when a group of workers join together to work towards a common goal to get what they want Union üMother Jones (United Mine Leaders Workers) üTerrence Powderly (Knights of Labor) üSamuel Gompers (American Federation of Labor) üThese leaders organized workers to fight for their rights Primary Resource Analysis Read the excerpt from the speech by Samuel Gompers and answer the following questions: 1. According to the passage, what do the workers want? 2. What point is Gompers trying to make in this speech? Primary Resource Analysis We want eight hours (i.e., an 8 hour work day) and nothing less. We have been accused of being selfish, and it has been said that we will want more; that last year we got an advance (raise in pay) of ten cents and now we want more. We do want more. You will find that man generally wants more. Go and ask a tramp (homeless person) what he wants, and if he doesn't want a drink he will want a good square meal. You ask a workingman, who is getting two dollars a day, and he will say that he wants ten cents more. Ask a man who gets five dollars a day and he will want fifty cents more. The man who receives five thousand dollars a year wants six thousand dollars a year, and the man who owns eight or nine hundred thousand dollars will want a hundred thousand more to make it a million, while the man who has his millions will want every thing he can lay is hands on and then raise his voice (yell) against the poor devil who wants ten cents more a day. Bread & Roses Listen to the song, Bread & Roses . With a partner, answer the following questions: 1.) What does bread symbolize? What do roses symbolize? 2.) What risks did these women face by striking? Essential Question How has immigration impacted the United States? Give at least three examples.


text:
What's NewiOS 7 compatibility
Fix for audio sometimes cutting out when returning from backgrounding
App Description3.5/4 stars in the USA Today!
Featured in the Huffington Post!
Come aboard the ChuggaBugga train and ignite your imagination with an interactive train engine and four boxcars that transform into unique playgrounds -accompanied by the adorable ChuggaBugs! A lovingly crafted game built by a father for his kids, ChuggaBugga engages your children while encouraging learning and creativity. Make the train engine whistle and shout, explore a construction site bursting with fun, learn counting and watch a ChuggaBug go high-diving into a pool, create music and colors on an accordion, change shapes into 3D objects with a robot and much more. Designed for all ages, even the youngest children can enjoy this experience and discover the playful interactions and surprise animations waiting to be explored on the ChuggaBugga train.
App Changes
September 26, 2013 New version 1.02
April 20, 2013 Price Decrease: $1.99 -> $0.99
March 23, 2013 New version 1.01
March 21, 2013 Initial Release


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Lotsa good stuff, but especially the piece that begins "Our baptismal call is not to "ministry." (Amy, attempting to cultivate a Garbo-like aura of inaccessibility, still does not have functional links, so you will have to scroll down till you find it.)
The piece reminds me of yet another fine piece of work by the St. Catherine of Siena Institute: The Parish: Mission or Maintenance?
Priests have houses of formation called seminaries. Laypeople do too–or should. They are called "parishes". At parishes, we are supposed to be formed to be disciples and apostles. At the altar, the priest is to preside so that, in the world, we are equipped to preside. The mission of the Church is to be done, not by laypeople hankering to fill quasi-priestly roles around the parish, but by laypeople using their charisms out in the world for the building up of the body of Christ, the evangelization of our neighbors, and the renewal of the face of the earth.
This article lays out the authentic Catholic vision better than anything else I know of.
Read. It.
And if you haven't had Siena out to do a "Called and Gifted Workshop" at your parish, what are you waiting for?


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Protocol translator and Gateway for LonWorks, Modbus, BACnet, Metasys, EtherNet IP
Modbus, BACnet, LonWorks®, EtherNet/IP, Metasys by Johnson Controls, KNX, M-Bus and Fire Alarm Panel protocol gateways from FieldServer Technologies can prepare your infrastructure for the future! Critical infrastructure device and network integration results in efficient building and plant operation, while conserving resources, maximizing returns on assets, and saving energy. With FieldServer gateways, you can combine existing trusted devices and systems with new technologies and services - optimizing your building and process control environments. There is no need to compromise when FieldServer lets you tie it all together to meet the demanding performance and interoperabilty efficiencies of the future. When systems talk, you save energy and resources!
FieldServer Technologies is a wholly owned division of Sierra Monitor Corporation, a publically traded company traded under OTCQB:SRMC.
Technical Support is provided by FieldServer's proven technical support team or one of the trained Approved Integrators. Product and protocol driver manuals, utility software and technical assistance is available on-line.
The QuickServer BAS Gateway can now be ordered online via the new FieldServer Shopping Cart. Visit the store, select the QuickServer that fits your application, choose from available profiles and accessory kit and place your order. Just another way FieldServer makes it easy to solve your needs.
The new Advanced FieldServer Toolbox utility contains all of the files you need to Detect, Connect and Configure your FieldServer. This includes setting IP address, setting device time, monitor the individual FieldServers on the network, diagnostics, status check and management of the configuration.
FieldServer has the protocol gateways you need to meet your interconnectivity needs. Whether interfacing a single device to another, or integrating multiple devices with multiple protocols to a plant-wide network FieldServer has the solution meet your needs.
FieldServer's extensive 100+ protocol library provides easy solutions for users and integrators to interface devices to modern open systems.
- BACnet - FieldServer's BTL Marked gateways interface to BACnet
- LonWorks - LonMark Certified gateways connect devices to LonWorks networks
- KNX - KNX Gateway to interface to Europe's popular protocol networks
- M-Bus - New M-Bus gateways for various meters in building automation
- Modbus - Modbus Compliance Tested gateways for building automation and industrial controls
- Metasys N2 by JCI - A major protocol in buildings, the FieldServer gateways can interface to Metasys N2
- EtherNet/IP - Gateways for Allen Bradley EtherNet/IP networks


text:
The Electro-Groom Vacuum with Booster Power is the world's most powerful portable vacuum cleaner. The Electro-Groom® Vacuum thoroughly cleans, is easy to operate and to dispose of hair. Quiet so horses won't spook. Gets horses clean without getting any of that dirt on you! Cuts down grooming time to increase your riding time.
Rounders® Peppermint horse treats are irresistible healthy treats that contain vitamins and minerals. A great reward that is good for your horse. 30 oz.
Perfect Prep EQ Training Day™ has four main active ingredients: magnesium, taurine, thiamine, inositol. Also has B Vitamins and amino acids enhanced by synergistic support of essential trace minerals. Start administering 3 days before event. 2 lb powder.
An excellent, quality mask that fits over a bridle, Rider's International Fly Mask with Ears offers effective protection and is great for turnout. Keeps pests away from your horse's sensitive face and ears.
An equine powder, Cosequin® Optimized with MSM maintains horses' joint health. Contains 7200 mg glucosamine HCL, 1200 mg chondroitin sulfate, 210 mg ascorbic acid, 32 mg manganese, plus 5,000 mg MSM per dose. 1400 gm powder (84-day supply).
Case of 12 FirstIce® Packs for Ice Horse™ products. Only requires 30-second massage to activate a loose, flexible cold source. Refreezable. Stays cold for over two hours. Great to use with horses after a competitive class or intensive training. Also works for heat!
Total Joint Care Supplement by Ramard is a highly effective synergistic blend of the most powerful joint care products available. Addresses the functionality of the entire joint; synovial fluid, cartilage, soft tissue, tendons and ligaments all need to be healthy to achieve peak performance.


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Category:American lobbyists
From Wikipedia, the free encyclopedia
This category has only the following subcategory.
- ► Pennsylvania lobbyists (17 P)
Pages in category "American lobbyists"
The following 200 pages are in this category, out of 284 total. This list may not reflect recent changes (learn more).(previous 200) (next 200)
(previous 200) (next 200)


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Sadiqa Basiri Saleem
Global Leadership Awards
As a refugee living in Pakistan, Sadiqa Basiri Saleem was close to earning a medical degree when the Taliban shut down her Afghan-run school. When she returned home to Wardak province after the fall of the Taliban, she found 150,000 girls with no hope for an education — for years, the regime had forbidden girls over the age of eight from attending school.
So Sadiqa and three other women pooled their money. They provided 36 girls with uniforms, supplies and funding to study in an abandoned mosque.
Outcries against "superfluous" women's education and anonymous threats poured in, but the Oruj Learning Center flourished. The Center now educates over 2,700 girls in six schools and more than 200 women at four literacy centers.
Sadiqa has also established the Family Welfare Center


text:
Read my fishing gear reviews
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I am located in Rochester Hills, Michigan approximately 40 miles north of Detroit.
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text:
BPL Community Profile
G H (NotLight) - BPL Forum Member
You cannot send this user a personal message (PM) because they have not specified a PM email address yet.
All Forum Posts by G H
Showing 1 - 10 of 12 | Page: 1 2
What if you can't push the PLB button?
I bought the Inreach SE this summer. I really like it. Even with the superior specs, I did not think the ResQlink made the most sense for solo hiking. Seems to me, if you are real trouble, y...
Ziploc Twistlocs like Valerie Says
I agree with Valerie. The Ziploc twistlocs from the grocery store work great. You can also get the sheets of craft foam for 99 cents at Michaels arts and crafts to use for a cozy/make it so you c...
Re: Therm-a-rest Xtherm - What size?
I have an x-lite regular and x-therm large. For me personally, neither are comfortable. Size is not the issue - it's the horizontal bubble baffles that are just not comfortable for me. I...
+1 Flex Skillet
+1 again on the MSR flex skillet. * Has high sides for boiling water - you can one pot it using only the skillet. * Fits over the lid of my old bear vault (but not my new ones, curiously)...
Re: 15 miles
Oh thank you my long lost brother for relieving my guilt of not constantly hydrating myself. Yeah, up to about half marathon you really want to just drink and have your slow burn carbs before hand...
Boiling and steam escape
Above about 185F most of your heat loss is from steam escaping. You want to cover your pot to prevent that steam from escaping. I don't think it's a percent-covered thing - as soon as you...
Old thread. But, I really like the MSR flexlite. Ti fry pans just don't work. Flexlite is just thick enough. A little bit of foil is enough for a diffuser. The thing about the flexlite vs ...
Re: most not lightweight thing on the trail
The most ridiculous overweight thing I've seen on the trail is myself :(
For reference: TNF replaced my 10 year old tent fly for free, no questions asked. Big Agnes repaired my tent that I told them I bought used for free, no questions asked.
Snow Peak Solo Cook Kit
I think the Snow Peak Solo Cook kit posted above by Mike W is a very good place for you to start. Everything WILL nest together in a small package, with your stove and fuel inside the main tall po...
All Reader Reviews by G H
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text:
The number has stayed near 14-year lows
Congress wouldn't dare get rid of the program
Join the NASDAQ Community today and get free, instant access to portfolios, stock ratings, real-time alerts, and more!
On Feb 14, Real estate investment trust (REIT)
Vornado Realty Trust
) declared that it has inked a deal to sell Broadway Mall for $94
million. The sale of the mall located on Route 106 in Hicksville,
Long Island, New York is anticipated to close in first-quarter
Through this deal, Vornado will reap around $92 million in net
proceeds (after closing costs). Notably, non-cash impairment loss
of $13.4 million related to the Broadway Mall will be reflected
in the company's fourth-quarter 2013 financial results.
The Broadway Mall, previously known as Mid-Island Plaza,
commenced operations in Oct 1956 and was acquired by Vornado in
2005. Currently, the mall has an anchor tenant base that includes
Macy's, Target, Forever 21, and Long Island's only IKEA store.
The latest move reflects Vornado's strategy to streamline its
mall business. Broadway is the last shopping complex owned by
Vornado in Long Island. In Jan 2013, the company disposed another
shopping mall - Green Acres Mall - to Santa Monica,
The Macerich Company
) for $500 million.
We expect these concerted efforts to enhance Vornado's financial
flexibility and pave the way for more profitable investment
opportunities. Also, these would strengthen its Class A office
properties portfolio that is concentrated in select high-rent,
high barrier-to-entry geographic markets, and usually fare better
amid challenging economic conditions.
Vornado currently carries a Zacks Rank #3 (Hold). Some
better-ranked REITs include
Sabra Health Care REIT, Inc.
) and
Chatham Lodging Trust
). Sabra Health Care holds a Zacks Rank #1 (Strong Buy) while
Chatham Lodging carries a Zacks Rank #2 (Buy).
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- Browse properties
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- Jost Van Dyke
- VRBO Listing #97510
VRBO is part of the HomeAway family, the world leader in vacation rentals with over 1 million listings. We offer the largest selection of properties for any travel occasion and every budget. We're committed to helping families and friends find a perfect vacation rental to create unforgettable travel experiences together. Learn More
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Anyone have a family crest for Jachens?
Alos Looking for info on Meta Jachens b Dec 12, 1855 GER,Leuchtenburg,Kirchspiel Lesum to Hinrich Jachens, and Beke Kimm.
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text:
From 399€ per room per night
Celebrate New Year's Eve with us!
Bed and breakfast with New Year's Eve Gala, party, including three drinks.
Type of board Breakfast included
From 31/Dec to 31/Dec Choose dates
It includes
Room with Sea View
only in our Website
- New Year´s Eve Gala Dinner
- 3 free drinks
- Resopon
- Parking
- Late check out 2:00 pm
1.- Choose arrival date
Dates when offer applies


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As some of you know I am an anti-apple and hate it to death.
But this has impressed me with regards to managing apps.
Meraki Systems Manager
Plus its free!!
Printable View
As some of you know I am an anti-apple and hate it to death.
But this has impressed me with regards to managing apps.
Meraki Systems Manager
Plus its free!!
Can't frown at free !! Thanks for the post
Oooo .. this might be the thing I've been looking for .. well, that and the holy grail .. but you know .. 1 out of 2 .. :)
*bump* for anybody that missed.
Very cool and for pc's too seems ideal for staff laptops.
Will investigate this tomorrow - it's been on my todo list to look at for a while.
Just installed it on a client pc and so far it hasn't checked in with the meraki dashboard wondering if maybe somethign in smoothie is blocking it but can't find any info on the meraki pages about what ports etc... it uses.
Looks like it also used vnc for remote desktop access to clients.
Ah it just worked here - But we have all outbound access open and no proxy.
Should be fairly easy in the smoothie to determine what it is trying to do though.
Dunno the meraki documentation is non existent.
If it does what it says on the tin and it is free then ace one! Can't believe they'd give something like this away free.
@plexer - if you know the IP of the ipad just stick it in the web-filter live log and see if it is blocking anything - if nothing is hitting it there then under firewall should do it.
Ipad yuck this was a client laptop :)
Done that and nothing is displayed that looks relevant to the meraki nothing at all under firewall but then as this is SWG-1200 there probably wouldn't./
I restarted the meraki service just to see if it would kick anything off.
Local firewall should prevent outbound connections though surely?


text:
Publishers are hawking more gory and gross books to appeal to an elusive market: boys and teachers and parents desperate to make them more literate. Take a look at some popular offerings. (See related article.)
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• "Captain Underpants and the Attack of the Talking Toilets" • Written and illustrated by Dav Pilkey • Publisher: Scholastic (February 1999) • Ages 7 to 10 | 144 pages
Research from Scholastic, the U.S. publisher of the Harry Potter series, shows that interest in reading declines sharply starting at age eight and continues to fall as kids grow into their teens, especially among boys.
In battling for those boys, many in the industry consider Scholastic's "Captain Underpants'' series a major victory. First published in 1997, the series, with plenty of toilet humor and pictures, has 37 million copies in print.
• "Oh, Yuck!: The Encyclopedia of Everything Nasty" • Written by Joy Masoff, illustrated by Terry Sirrell • Publisher: Workman Publishing Company • Published: January 2000 • Age Range: 8 to 12 | 224 pages
This nonfiction encyclopedia covers everything that is bad – from why vomit smells to where dandruff comes from – with hundreds of illustrations and photographs.
Workman Publishing Company
• "Sir Fartsalot Hunts the Booger" • Written by Kevin Bolger, illustrated by Stephen Gilpin • Publisher: Penguin Group (May 2008) • Ages 9 to 12 | 224 pages
Sir Fartsalot is on a quest to solve the riddle of the Foul West Wind – a horrible odor that turns up whenever danger lurks – and rid the Kingdom of Armpit of the dreaded Booger.
Kevin Bolger, an elementary school teacher in Ottawa, Canada, wrote "Sir Fartsalot Hunts the Booger'' after having his students read "Captain Underpants." Pearson's Penguin Group published the book in May, and it is already in its second printing with 55,000 copies total.
Penguin Group
• "Vlad the Impaler: The Real Count Dracula"
• Written by Enid A. Goldberg and Norman Itzkowitz, illustrated by Mark Summers • Publisher: Scholastic Library Publishing (September 2007) • Grades: 6 to 9 | 128 pages
"Vlad the Impaler" from the "Wicked History" series offers a brief biography of Vlad, who had a violent childhood and was a prisoner of the Turks for many years.
Eleven-year-old Yathrib Aryanpure, who just finished sixth grade in Tuscaloosa, Ala., said he loved the book, especially when the tyrant was assassinated, ending up with his own severed head on a stick. "Vlad the Impaler went on a killing rampage," he said. "In the end, he got a taste of his own medicine.''
• "It's Disgusting and We Ate It: True Food Facts from Around the World and Throughout History" • Written by James Solheim, illustrated by Eric Brace • Publisher: Simon & Schuster Children's Publishing • Published: May 1998 • Age Range: 5 to 10 | 48 pages
The book includes medieval England's garbage stew and information about the amount of protein in a spider.
Simon & Schuster Children's Publishing
• "Help! What's Eating My Flesh? Runaway Staph and Strep Infections!" • By Thomasine E. Lewis Tilden • Publisher: Scholastic Library Publishing (September 2007) • Ages: Young adult | 64 pages
Part of the "24/7: Science Behind the Scenes" series, this book describes, among other things, how germs invade a body and how a hospital visit turns deadly.
Scholastic's "Wicked History" and "24/7 Science Behind the Scenes" series already have more than 300,000 copies in print since they were first published almost a year ago.
• "Do You Know Where Your Water Has Been? The Disgusting Story Behind What You're Drinking" • By Kelly Regan Barnhill • Publisher: Capstone Press • Published: Coming in Fall 2008 • Grades: 3 to 9 | 32 pages
Capstone Press is publishing a new series called "Sanitation Investigation" that goes behind the scenes of the sanitation system – from the history of toilets to a look at tap water.
Last year, U.S. publishers released 261 new works of juvenile fiction aimed at boys, more twice the number in 2003, according to Bowker's Books in Print database. There were 20 nonfiction entries for boys, compared to only four in 2003.
Capstone Press


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CHARLOTTE, N.C. (AP) When Bobcats coach Mike Dunlap looks at point guard Kemba Walker he sees the NBA's next coming of Chris Paul.
EL SEGUNDO, Calif.
ORLANDO, Fla. (AP) Carmelo Anthony scored 16 of his 40 points in the fourth quarter, helping the New York Knicks top the Orlando Magic 114-106 on Saturday night.
LOS ANGELES (AP) Chris Paul had 27 points and nine assists, Blake Griffin added 20 points and the Los Angeles Clippers set a franchise record with their 12th straight home victory, 115-89 over th
LOS ANGELES (AP) Chris Paul had 30 points and 13 assists, Blake Griffin added 24 points, and the Los Angeles Clippers led all the way in beating the Lakers 107-102 on Friday night.