[Clinical and genetic characteristics of children with dopa-responsive dystonia caused by tyrosine hydroxylase gene variations].

Objective: To explore the clinical and genetic characteristics of children with dopa-responsive dystonia (DRD) caused by tyrosine hydroxylase (TH) gene variations. Methods: Clinical data of 9 children with DRD caused by TH gene variations diagnosed in the Department of Children Rehabilitation, the Third Affiliated Hospital of Zhengzhou University from January 2017 to August 2022 were retrospectively collected and analyzed, including the general conditions, clinical manifestations, laboratory tests, gene variations and follow-up data. Results: Of the 9 children with DRD caused by TH gene variations, 3 were males and 6 were females. The age at diagnosis was 12.0 (8.0, 15.0) months. The initial symptoms of the 8 severe patients were motor delay or degression. Clinical symptoms of the severe patients included motor delay (8 cases), truncal hypotonia (8 cases), limb muscle hypotonia (7 cases), hypokinesia (6 cases), decreased facial expression (4 cases), tremor (3 cases), limb dystonia (3 cases), diurnal fluctuation (2 cases), ptosis (2 cases), limb muscle hypertonia (1 case) and drooling (1 case). The initial symptom of the very severe patient was motor delay. Clinical symptoms of the very severe patient included motor delay, truncal hypotonia, oculogyric crises, status dystonicus, hypokinesia, decreased facial expression, and decreased sleep. Eleven TH gene variants were found, including 5 missense variants, 3 splice site variants, 2 nonsense variants, and 1 insertion variant, as well as 2 novel variants (c.941C>A (p.T314K), c.316_317insCGT (p.F106delinsSF)). Nine patients were followed up for 40 (29, 43) months, and no one was lost to follow-up. Seven of the 8 severe patients were treated by levodopa and benserazide hydrochloride tablets and 1 severe patient was treated by levodopa tablets. All the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets. Although the weight of the patients increased and the drug dosage was not increased, the curative effect remained stable and there was no obvious adverse reaction. One severe patient developed dyskinesia in the early stage of treatment with levodopa and benserazide hydrochloride tablets and it disappeared after oral administration of benzhexol hydrochloride tablets. Until the last follow-up, motor development of 7 severe patients returned to normal and 1 severe patient still had motor delay due to receiving levodopa and benserazide hydrochloride tablets for only 2 months. The very severe patient was extremely sensitive to levodopa and benserazide hydrochloride tablets and no improvement was observed in this patient. Conclusions: Most of the DRD caused by TH gene variations are severe form. The clinical manifestations are varied and easily misdiagnosed. Patients of the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets, and it takes a long time before full effects of treatment become established. Long-term effect is stable without increasing the drug dosage, and no obvious side effect is observed.

[Etiological survey and traceability analysis of five cases of typhoid fever during the same period in Yantai city].

Objective: To analyze the etiological characteristics and molecular epidemiological correlation of five cases of typhoid fever during the same period in yantai city. Methods: Six S. Typhis strains were isolated from 5 typhoid patients and epidemiological samples in Yantai city in 2018. The onset time of the cases were from May 26, 2018 to July 24, 2018, distributed in Shuidao Town of Muping District, Dengzhou Street of Penglai District, Donglai Street of Longkou District, Wenhua Street of Muping District and Fulaishan Street of zhifu District. S. Typhis strains were analyzed by conventional bacterial isolation method and XbaⅠ/BlnⅠ double-enzyme digestion pulse-field gel electrophoresis (PFGE). Meanwhile, ViaB virulence gene detection and 27 common antibiotics sensitivity tests were conducted to study the etiology of S. Typhis. Results: Six strains of S. Typhi were isolated from 5 patients and the domestic egg of one patient, which were divided into 4 PFGE patterns by PFGE-XbaⅠ and PFGE-BlnⅠ and among which 3 strains had the same PFGE patterns.One multi-drug resistant strain (foreign patient), one single-drug resistant strain (patient with a history of provincial retention), and one completely sensitive strain were detected. The three strains of the same PFGE pattern exhibit the same drug-sensitive phenotype which were intermediate against aminoglycosides and quinolones and susceptibility against the other antibiotics.All of the strains carried the ViaB virulence factor except the strain from the foreign patient. Conclusion: Local S. Typhi is susceptibility or intermediate against antibiotics commonly used in clinic.Sporadic cases of typhoid fever and typhoid imported infections still need attention.

γ-Aminobutyric acid-mediated neurotransmission in cerebellar-hypothalamic circuit attenuates gastric mucosal injury induced by ischemia-reperfusion.

BACKGROUND: Excessive greater splanchnic nerve (GSN) activation contributes to the progression of gastric ischemia-reperfusion (GI-R) injury. This study was designed to investigate the protective mechanism of cerebellar fastigial nucleus (FN) stimulation against GI-R injury. METHODS: The GI-R injury model was induced in rats by clamping the celiac artery for 30 min, and then reperfusion for 30 min, 1, 3, 6, or 24 h, respectively. KEY RESULTS: Microinjection of L-Glu (3, 6, 12 µg) into the FN dose-dependently attenuated GI-R injury and GSN activity. In addition, there was an enhancement of gastric mucosal blood flow in GI-R rats. Pretreatment with the glutamic acid decarboxylase antagonist into the FN, the GABAA receptor antagonist into the lateral hypothalamic area or lesion of superior cerebellar peduncle all reversed the protective effects of the FN stimulation. Furthermore, the FN stimulation reduced the TUNEL-positive gastric mucosal cell and Bax-positive gastric mucosal cell in GI-R rats. CONCLUSIONS & INFERENCES: These results indicate that the protective effects of the FN stimulation against GI-R injury may be mediated by attenuation of the excessive GSN activation, gastric mucosal cell apoptosis, and Bax expression in GI-R rats.

Prediction of human absorption of natural compounds by the non-everted rat intestinal sac model.

A major concern in natural drug research is that many substances with potent biological activity in vitro are unable to generate good activity in vivo owing to their poor water-solubility, poor permeability and/or poor stability. The permeability of drug candidates across the intestinal mucosa is one of the most important factors in defining drug bioavailability and biological activity. In order to screen promising compounds for further investigation, a non-everted rat intestinal sac model has been developed successfully to assay the permeability of natural compounds and to predict their human absorption. In this system, the drug solution was placed in non-everted intestinal sacs (NEIS), which were placed in an acceptor solution and the permeability of drug across intestine walls was determined. The feasibility of this method has been validated and demonstrated for 11 model compounds chosen from currently marketed drugs whose human fraction absorbed (Fa) data have been reported. The results of the studies indicate that a good relationship exists between the permeability of the model drugs and their corresponding Fa data. The permeability of 13 natural compounds was evaluated using this system. Only fraxinellone and vitexin-7-glucoside exhibited high intestinal permeability, and predictive of excellent human absorption, which awaits confirmation from further investigation in vivo. This model provides an alternative method to everted intestinal sacs for the evaluation of in vitro permeability in rats, and for estimating human absorption of drugs. It may therefore hold great promise for oral absorption screening of new drug candidates.

Effects of microinjection of melatonin and its receptor antagonists into anterior hypothalamic area on blood pressure and heart rate in rats.

AIM: To examine the effects of microinjection of melatonin and its receptor antagonists into the anterior hypothalamic area (AHA) on blood pressure (BP) and heart rate (HR) in normotensive and stress-induced hypertensive rats. METHODS: Melatonin and its receptor antagonists were microinjected into the AHA, then BP, mean arterial pressure (MAP), and HR were synchronously recorded. RESULTS: Microinjection of melatonin produced a fall in MAP. Prazosin, an antagonist of melatonin ML2 receptor, could not antagonize the depressive response induced by melatonin. While luzindole, a competitive antagonist of melatonin ML1 receptor, was able to almost completely prevented the depressive response induced by injection of melatonin. CONCLUSION: Melatonin acts as a hypotensive factor and the effects are mainly due to activation of ML1 receptors in rat brain, and the AHA may be one of the important central areas where melatonin can exert modulatory effects on BP and HR.

[Role of angiotensin-(1-7) in amino-acid-neurotransmitter-mediated blood pressure regulation in rat rostral ventrolateral medulla].

The present study was undertaken to investigate the linkage between angiotensin-(1-7) [Ang-(1-7)] and the release of amino acid neurotransmitters in the the rostral ventrolateral medulla (RVLM) by techniques of microinjection, microdialysis combined with high performance liquid chromatography (HPLC)-fluorescent detection. Unilateral microinjection of Ang-(1-7) into the RVLM of anesthetized rats produced an increase in mean arterial pressure (MAP) accompanied by an increased release of glutamate (Glu). In contrast, microinjection of Ang779, a selective antagonist of Ang-(1-7) receptor, caused a decrease in MAP with a decreased release of Glu and an increased release of glycine, taurine and gamma-aminobutyric acid. The pressor effect of Ang-(1-7) and the depressor effect of Ang779 were in part blocked by corresponding antagonists of amino acid receptors. These results suggest that the pressor effect of Ang-(1-7) in the RVLM may be partially due to an increased release of Glu, whereas the depressor effect of Ang779 may be partially attributed to a decreased release of Glu and an increased release of inhibitory amino acid neurotransmitters.

[Angiotensin II in rostral ventrolateral medulla mediates amino acids release from spinally projecting nerve terminals in the spinal cord].

Microdialysis in the intermediolateral column (IML) was employed to examine amino acids release induced by angiotensin II (ANG II) applied into the rostral ventrolateral medulla (RVLM). Microinjection of ANG II (100 pmol, n = 11) into the RVLM significantly increased (P < 0.01) the release of aspartate (from 4.75 +/- 1.01 to 8.90 +/- 2.28 pmol/20 microliters) and glutamate (from 18.99 +/- 8.64 to 73.88 +/- 29.26 pmol/20 microliters) in the spinal cord. The increase of glutamate release was significantly attenuated (P < 0.05) by pretreatment with losartan (10 nmol, n = 8) at the same RVLM site. Immunofluorescence double labeling combined with confocal microscopic observation demonstrated that 62%-91% of the glutamatergic neurons in the RVLM were double-labeled with AT1 receptors, supporting the view that ANG II-induced glutamate release in the spinal cord may arise from the AT1 receptor-containing glutamatergic spinally projecting neurons in the RVLM.

Central amino acids mediate cardiovascular response to angiotensin II in the rat.

To elucidate the role of the rostral ventrolateral medulla (RVLM) in cardiovascular control through the release of central amino acid neurotransmitters, experiments were performed in Sprague-Dawley (normotensive) rats and spontaneously hypertensive rats (SHR) anesthetized with urethane by using microdialysis sampling from the RVLM for determination of amino acid neurotransmitters. The baseline release of the excitatory amino acid neurotransmitter, glutamate (GLU) from the RVLM in SHR was higher and those of the inhibitory amino acid neurotransmitters, glycine (GLY), taurine (TAU), and gamma-aminobutyric acid (GABA), were lower than in normotensive rats. Microinjection of angiotensin II (ANG II) into the RVLM caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR), accompanied by increased release of GLU in the RVLM. In contrast, microinjection of the ANG II type 1 receptor (AT1) antagonist CV 11974 into the RVLM reduced MAP and HR, accompanied by increased release of GLY, TAU and GABA. These changes in MAP and HR after administration of ANG II or AT1 antagonist were partially blocked by the use of the corresponding antagonist of each amino acid neurotransmitter. Furthermore, these effects were more prominently seen in SHR than in normotensive rats. These results suggest that the release of amino acid neurotransmitters mediate the cardiovascular effects of the angiotensin system in the RVLM, which may be involved in the generation of hypertension in SHR.

The cardiovascular response to medullary cholinergic and corticoid stimulation is calcium channel dependent in rats.

Experiments were performed on anaesthetized Wistar or Sprague-Dawley rats of both sexes. Microinjection of an acetylcholinesterase inhibitor physostigmine (0.4 microgram/0.1 microliter/site) or acetylcholine (ACh, 25 ng/0.1 microliter/site) into the rostral ventrolateral medulla (rVLM) caused an increase in blood pressure (BP), heart rate (HR) and the pressor response produced by stimulation of the dorsal periaqueductal grey (dPAG) in the midbrain. Prior microinjection of the calcium channel blocker verapamil (0.25 microgram/0.1 microliter/site) into the same sites blocked the cardiovascular effect in response to the respective microinjection of the drugs mentioned above. Moreover, verapamil pretreatment blocked the pressor and tachycardiac effect induced by respective microinjection of corticosterone (40 ng/0.1 microliter/site) or aldosterone (40 ng/0.1 microliter/site) into the rVLM, as well as the enhancement of the pressor response to stimulation of the dPAG induced by microinjection of corticoids into the rVLM. These results suggest that the enhancement of cardiovascular activities mediated by cholinergic mechanisms may be due to the activation of postsynaptic calcium channels of neurons in the rVLM. The corticosteroid effect seems to be mediated by similar mechanisms.

[The central mechanism of corticoids in the incidence of stress-induced hypertension].

We observed that in rats during the incidence of hypertension induced by stress, an increase in the plasma concentrations of catecholamines, corticosterone, glucose and lipids is parallel with an elevation of blood pressure and heart rate. Microinjection of corticoids into the rVLM elicits an effect of pressor, tachycardia and enhancement of pressor response to stimulation of the defense area, this may be one of the important causes in the incidence of stress-induced hypertension. This effect is taken place by a rapid membrane effect of the corticoids on the cardiovascular neurons in the rVLM, which is related to an abnormal enhancement of cholinergic activity and activation of calcium channel. Whereas NO plays an inhibitory effect on the cardiovascular neurons in the rVLM.

Effect of central muscarine receptor blockade with DKJ-21 on the blood pressure and heart rate in stress-induced hypertensive rats.

The experiments were performed on Wistar or Sprague-Dawley rats of both sexes divided at random into stress and control groups. The rats in the stress groups were put into cages and subjected to electric foot-shocks and noises for 9-15 days, which caused an increase in blood pressure (BP) and heart rate (HR). In hypertensive rats DKJ-21 (4 mg/1 ml) was injected intravenously (i.v.), and 0.5-1.0 h after administration the BP and HR dropped from the high level to normotensive level. In normotensive rats, however, administration of DKJ-21 had no effect on BP or HR. In separate groups of normotensive rats, pretreatment of DKJ-21 (4 mg/1 ml, i.v.) blocked the pressor and tachycardiac effect induced by microinjection of physostigmine (0.4 microgram/0.1 microliter/site), corticosterone (40 ng/0.1 microliter/site) or aldosterone (40 ng/0.1 microliter/site) into the rostral ventrolateral medulla (rVLM). Furthermore, DKJ-21 also attenuated the enhancement of the pressor response to stimulation of the defense area in the midbrain, which was induced by microinjection of drugs (mentioned above) into the rVLM. These results indicate that i.v. DKJ-21 can selectively block the muscarinic receptors in the rVLM in stress-induced hypertensive rats, which suggests that abnormal enhancement of cholinergic mechanism in the rVLM may be related to hypertensive effects of corticoids in this area.

Role of acetylcholine, corticoids and opioids in the rostral ventrolateral medulla in stress-induced hypertensive rats.

Hypertension was induced in Sprague-Dawley and Wistar rats by irregular foot shocks combined with a buzzing noise for 2 h twice a day for 1-2 weeks. The plasma catecholamine, corticosterone, angiotensin II, glucose and lipids were found to increase in parallel. The acetylcholine (ACh) and choline acetyltransferase in rostral ventrolateral medulla (rVLM) increased markedly). Microinjection of ACh or cholinergic agonists into rVLM induced a pressor effect, and microinjection of M receptor blockers had a depressor effect. Electrophysiological studies showed that the stress-induced hypertension was closely related to the activation of a cholinergic system in rVLM. Microinjection of corticoids into rVLM had led to a pressor response which could be blocked by Ru38486, spironolactone, cholinergic blockers or verapamil. Microinjection of morphine and mu- or delta-receptor agonists into rVLM caused bradycardia and a reduction of arterial pressure that could be blocked by naloxone.

Cardiovascular effects of microinjection of corticoids and antagonists into the rostral ventrolateral medulla in rats.

Experiments were performed on Wistar or Sprague-Dawley rats of both sexes. Microinjection of corticosterone (10 or 40 ng/0.1 microliter/site) into the rostral ventrolateral medulla (rVLM) caused an increase in systolic blood pressure (SBP), heart rate (HR) and pressor response induced by stimulation of the dorsal periaqueductal grey (dPAG) in the midbrain. Microinjection of aldosterone (10 or 40 ng/0.1 microliter/site) into the rVLM had similar effects showing a higher level and longer period than that of corticosterone. All these effects were dose-dependent. Microinjection of glucocorticoid antagonist RU 38486 (40 ng/0.1 microliter/site) or mineralocorticoid antagonist spironolactone (40 ng/0.1 microliter/site) caused a decrease in SBP, HR and the pressor response induced by stimulation of the dPAG. The inhibitory effects of spironolactone were more apparent. These results suggest that both corticoids could exert central modulatory effects on the resting cardiovascular activities and facilitate the pressor response during a defense reaction, and the rVLM is an essential area for the location of the central modulation. These effects may play an important role in the incidence and development of hypertension induced by stress.