We present rapid and sensitive assay of tryptophan hydroxylase 2 enzyme activity based on the fluorescence of the complex of 5-hydroxytryptophan (5-HTP) with
o
-phthalic aldehyde. This method was ...compared with the standard method based on chromatographic isolation of 5-HTP followed by its quantification using an electrochemical detector. High sensitivity of the developed fluorometric method and similarity of the results obtained by fluorometric and chromatographic methods were demonstrated. The use of this rapid, cheap, and effective fluorometric method can simplify and facilitate measurements of tryptophan hydroxylase 2 activity and can make this assay available for a wide range of neurochemical and pharmacological laboratories.
The enzyme tryptophan hydroxylase 2 (TPH2) catalyzes the hydroxylation of L-tryptophan to L-5-hydroxytryptophan (5-HTP), the first and the key step in 5-HT synthesis in the mammalian brain. Mutations ...in the human
Tph2
gene reducing enzyme activity increase the risk of psychopathology. Pharmacological chaperones are small molecules that can specifically bind to mutant protein molecules, restore their disturbed 3D structure to the native state, and increase their stability and functional activity. The chaperone activity of (R)-2-amino-6-(1R,2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one (BH
4
) is expressed by increasing the
in vitro
thermal stability of mutant tyrosine hydroxylase and phenylalanine hydroxylase molecules which are similar to TPH2 in their structure and characteristics. The
P447R
substitution in the mouse TPH2 molecule results in a 2-fold decrease in enzyme activity in their brains. We studied the effect of this mutation on the TPH2 thermal stability, as well as on the ability of BH
4
and its 8 structural analogues to increase the thermal stability of the mutant TPH2 from midbrain extracts of BALB/C mice. Temperature stability was studied by the decrease in enzyme activity during its heating for 2 min at increasing temperatures and was evaluated by the T
50
value that is the temperature at which the enzyme activity decreased by half. For the mutant TPH2, the T
50
value was decreased compared to the wild type enzyme. BH
4
and its closest structural analogue, 6-methyl-5,6,7,8-tetrahydropterin, increased the T
50
value,
i.e.
, exhibited chaperone activity. Other close BH
4
analogs, 6,7-dimethyl-5,6,7,8-tetrahydropterin and folic acid, were not effective. It can be assumed that BH
4
can be effective in the treatment of mental disorders caused by mutations in the
Tph2
gene.
We studied the effect of reduced tryptophan hydroxylase (TPH) activity and short daylight exposure on the behavior and the 5-HT system of the brain in
D. rerio
. Male and female
D. rerio
were exposed ...for 30 days to standard (12:12 h light:dark) and short (4:20 h light:dark) photoperiods in the presence or absence of TPH inhibitor (
p
-chlorophenylalanine, pCPA, 5 mg/liter). On day 31, the fish behavior in the “novel tank diving” test, their sex and body weight were determined, and the levels of pCPA, 5-HT, and its metabolite 5-HIAA were measured by HPLC; the levels of the key genes encoding metabolism enzymes (
Tph1a
,
Tph1b
,
Tph2
, and
Mao
) and receptors of 5-HT (
Htr1aa
,
Htr2aa
) were assessed by real-time PCR with reverse transcription. The short daylight exposure caused masculinization of females, reduced body weight, and motor activity in the “novel tank diving” test, but did not affect the 5-HT system of the brain. Long-term pCPA treatment had no effect on sex and body weight, significantly reduced the 5-HIAA level, but increased
Tph1a
and
Tph2
gene expression in the brain. No effects of the interaction of short daylight and pCPA exposure on the sex, body weight, behavior, and 5-HT system of the brain were found. Thus, a moderate decrease in TPH activity cannot potentiate the negative effects of short daylight exposure on the sex, body weight, behavior, and 5-HT system of
D. rerio
.
The central dopaminergic system is implicated in the regulation of various physiological processes and behavioral responses, including social behavior. Although long-term individual housing of ...rodents is well known to alter their behavioral and neurochemical parameters, data interpretation remains ambiguous. In this work, we studied the effects of long-term social isolation on the behavior and state of the central dopaminergic system in male C57Bl/6 mice. The animals of the experimental group, aged 40–42 days, were housed singly in individual cages for six weeks, while the age-matched mice of the control group were housed in a group. Isolation did not affect locomotor and exploratory activity in the open field test compared to group housing. At the same time, singly-housed animals demonstrated a longer duration of social contacts in a resident–intruder model, as well as a weakened stereotypical behavior in the marble burying test compared to group-housed rats. These behavioral changes were accompanied by an increase in striatal mRNA levels of the genes encoding dopamine D1 and D2 receptors. Moreover, the level of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) decreased in the hypothalamus and increased in the frontal cortex in singly-housed compared to group-housed mice. The results provide a better insight into the effects of long-term social isolation on the behavior and dopaminergic system in mice.
—
Tryptophan hydroxylases 1 and 2 (TPH1 and TPH2) play a key role in the synthesis of serotonin (5-HT), a hormone and neurotransmitter, in the peripheral organs and brain, respectively. The main aim ...of this study was to clarify the distribution of mRNA of the
Tph1
and
Tph2
genes in brain structures under normal conditions and after inflammation. The experiments were carried out on young (4 weeks old) male C57BL/6 mice. The animals were divided into three groups: intact, control, injected ip with saline, and injected ip with 2 mg/kg of bacterial lipopolysaccharide (LPS). Markers of inflammation, spleen mass and thymus mass were assayed 5 days after the saline or LPS administration. In the frontal cortex, hippocampus, striatum, hypothalamus, and midbrain the concentrations of 5-HT and its main metabolite, 5-hydroxyindole acetic acid (5-HIAA), and TPH activity were assayed using HPLC, while
Tph1
and
Tph2
mRNA were quantified using quantitative real-time RT-PCR. A dramatic increase of spleen mass and decrease of thymus mass 5 days after LPS administration was shown. A significant increase of 5-HT and 5-HIAA levels in the midbrain as well as decrease of 5-HIAA concentration and TPH activity in hypothalamus in mice treated with LPS and saline compared with intact animals was revealed. The highest concentration of
Tph2
gene mRNA was observed in the midbrain in 5-HT neuron bodies, while this gene mRNA level was lower in 5-HT endings (cortex, hippocampus, striatum, and hypothalamus). Trace amounts of
Tph1
mRNA was found in all studied brain structures in mice of the three groups. Thus,
Tph1
gene expression in the mouse brain is too low to significantly affect 5-HT synthesis in normal conditions and during inflammation.
Tryptophan hydroxylases 1 and 2 (TPH1 and TPH2) play a key role in the synthesis of serotonin (5-HT), a hormone and neurotransmitter, in the peripheral organs and brain, respectively. The main aim of ...this study was to clarify the distribution of mRNA of the Tph1 and Tph2 genes in brain structures under normal conditions and after inflammation. The experiments were carried out on young (4 weeks old) male C57BL/6 mice. The animals were divided into three groups: intact, control, injected ip with saline, and injected ip with 2 mg/kg of bacterial lipopolysaccharide (LPS). Markers of inflammation, spleen mass and thymus mass were assayed 5 days after the saline or LPS administration. In the frontal cortex, hippocampus, striatum, hypothalamus, and midbrain the concentrations of 5-HT and its main metabolite, 5-hydroxyindole acetic acid (5-HIAA), and TPH activity were assayed using HPLC, while Tph1 and Tph2 mRNA were quantified using quantitative real-time RT-PCR. A dramatic increase of spleen mass and decrease of thymus mass 5 days after LPS administration was shown. A significant increase of 5-HT and 5-HIAA levels in the midbrain as well as decrease of 5-HIAA concentration and TPH activity in hypothalamus in mice treated with LPS and saline compared with intact animals was revealed. The highest concentration of Tph2 gene mRNA was observed in the midbrain in 5-HT neuron bodies, while this gene mRNA level was lower in 5-HT endings (cortex, hippocampus, striatum, and hypothalamus). Trace amounts of Tph1 mRNA was found in all studied brain structures in mice of the three groups. Thus, Tph1 gene expression in the mouse brain is too low to significantly affect 5-HT synthesis in normal conditions and during inflammation.
Parkinson's disease (PD) is the most common age-related movement disorder characterized by the progressive loss of nigrostriatal dopaminergic neurons. To date, PD treatment strategies are mostly ...based on dopamine replacement medicines, which can alleviate motor symptoms but do not slow down the progression of neurodegeneration. Thus, there is a need for disease-modifying PD therapies. The aim of this work was to evaluate the neuroprotective effects of the novel compound PA96 on dopamine neurons in vivo and in vitro, assess its ability to alleviate motor deficits in MPTP- and haloperidol-based PD models, as well as PK profile and BBB penetration. PA96 was synthesized from (1
,2
,6
)-3-methyl-6-(prop-1-en-2-yl) cyclohex-3-ene-1,2-diol (Prottremin) using the original three-step stereoselective procedure. We found that PA96: (1) supported the survival of cultured näive dopamine neurons; (2) supported the survival of MPP
-challenged dopamine neurons in vitro and in vivo; (3) had chemically appropriate properties (synthesis, solubility, etc.); (4) alleviated motor deficits in MPTP- and haloperidol-based models of PD; (5) penetrated the blood-brain barrier in vivo; and (6) was eliminated from the bloodstream relative rapidly. In conclusion, the present article demonstrates the identification of PA96 as a lead compound for the future development of this compound into a clinically used drug.
The annual turquoise killifish (
) is a laboratory model organism for neuroscience of aging. In the present study, we investigated for the first time the levels of serotonin and its main metabolite, ...5-hydroxyindoleacetic acid, as well as the activities of the key enzymes of its synthesis, tryptophan hydroxylases, and degradation, monoamine oxidase, in the brains of 2-, 4- and 7-month-old male and female
. The marked effect of age on the body mass and the level of serotonin, as well as the activities of tryptophan hydroxylases and monoamine oxidase in the brain of killifish were revealed. The level of serotonin decreased in the brain of 7-month-old males and females compared with 2-month-old ones. A significant decrease in the tryptophan hydroxylase activity and an increase in the monoamine oxidase activity in the brain of 7-month-old females compared to 2-month-old females was shown. These findings agree with the age-related alterations in expression of the genes encoding tryptophan hydroxylases and monoamine oxidase.
is a suitable model with which to study the fundamental problems of age-related changes of the serotonin system in the brain.
The study investigates the use of protatran compounds as growth stimulators for Bacillus thuringiensis bacteria, which are widely used as producers of biopesticides. Cultivation of the Bacillus ...thuringiensis strain subsp. kurstaki was carried out in a Luria-Bertani (LB) liquid medium. Protatrans (2-Me-C 6 H 4 OCH 2 COO - were added to the NN+(CH2CH2OH)3 (1), 4-Cl-C6H4 -SCH2COO-NN(CHCHOH) (2) and 4-Cl-CSOCHCOO NN+(CH2CH2OH)3 (3) media in concentrations of 1×10 -4 –1×10 -8 wt %. The LB medium without the addition of compounds 1–3 was used as a control. Cultures were incubated at a temperature of 30°C for 24 hours. The number of Bacillus thuringiensis cells was determined by serial dilution. The maximum growth was observed in a medium containing 1×10 -4 wt % of protatran 3. The number of cells was almost 10 times (966.7%) higher than in the control. In media with 1×10 -5 , 1×10 -6 , 1×10 -7 and 1×10 -8 wt % of compound 3, the number of cells was 4–7 times higher than in the control (by 371.7–666.7%). Protatrans 1 and 2 had a positive effect on Bacillus thuringiensis, increasing the number of cells by 83–292% compared to control. Therefore, it was demonstrated for the first time that commercially available non-toxic protatran compounds in microconcentrations are powerful growth stimulators for the entomopathogenic bacteria Bacillus thuringiensis. This indicates the potential for significant improvement and cost reduction of biotechnology for the production of bacterial insecticides based on Bacillus thuringiensis, used in agriculture, forestry and homesteads to control harmful insects.
