Objective. This study aims at establishing and validating an in vitro system to screen drug inducers of CYPs mediated via hPXR, as well as studying transcriptional regulation of CYPs mediated via hPXR by helicid and its two metabolites. Methods. Cloning the nuclear receptor hPXR and the promoters of CYP3A4, CYP2B6, CYP2C9, and inserting the trans-element to the upstream of firefly luciferase reporter gene of the pGL4.17 vectors, then cotransfecting the report vectors and hPXR expression plasmid to HepG2 cell line. After 24 hours, the transfected cells were treated with helicid (0.004, 0.04, and 0.4 μmol/L) and its metabolite I and metabolite II (0.0004, 0.004, and 0.04 μmol/L) for 48 h, while rifampin (10 μmol/L) was included as the positive control and 0.1% DMSO as the negative control group. Cells were lysized and luciferase activity was determined using a dual luciferase reporter assay kit. Results. Helicid and its metabolites did not significantly increase promoter activities of CYP3A4, CYP2B6, and CYP2C9 in HepG2 cells transfected with PXR expression plasmid (P > 0.05). Conclusion. PXR-expressed CYP3A4, CYP2B6, and CYP2C9 dual luciferase reporter gene platforms were successfully established, and helicid and its metabolites I, II do not significantly induce the transcription of CYP3A4, CYP2B6, and CYP2C9.
Fast-spiking, parvalbumin-expressing GABAergic interneurons/basket cells (BCs) play a key role in feedforward and feedback inhibition, gamma oscillations, and complex information processing. For these functions, fast propagation of action potentials (APs) from the soma to the presynaptic terminals is important. However, the functional properties of interneuron axons remain elusive. Here, we examined interneuron axons by confocally targeted subcellular patch-clamp recording in rat hippocampal slices. APs were initiated in the proximal axon ~20 μm from the soma, and propagated to the distal axon with high reliability and speed. Subcellular mapping revealed a stepwise increase of Na+ conductance density from the soma to the proximal axon, followed by a further gradual increase in the distal axon. Active cable modeling and experiments with partial channel block indicated that low axonal Na+ conductance density was sufficient for reliability, but high Na+ density was necessary for both speed of propagation and fast-spiking AP phenotype. Our results suggest that a supercritical density of Na+ channels compensates for the morphological properties of interneuron axons (small segmental diameter, extensive branching, and high bouton density), ensuring fast AP propagation and high-frequency repetitive firing.
A high-fat diet (HFD) is a well-known risk factor for cardio-cerebrovascular disease but the relationship between a HFD and depressive symptoms remains unknown.
Compare changes in behavioral and measures of brain glucose metabolism in rats fed a HFD to those of rats fed a standard diet.
Twenty male Sprague-Dawley rats were randomly assigned to a study group (n=10) that received a high fat diet for 9 weeks or a control group (n=10) that received a standard diet for 9 weeks. At baseline and at the end of the 9-week trial assessments included body weight, serum lipids (total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol), the sucrose preference test, and the open field test. The rate of brain glucose metabolism in different brain regions (assessed using micro-positron emission tomography) at the end of the trial was also compared between the two groups of rats.
Nine weeks of a HFD in rats resulted in the expected increase in weight and changes in serum lipid levels, but it was also associated with a decreased preference for sucrose (which may be due to a loss of interest in pleasurable activities), increased weight-adjusted water intake, and a significant deactivation of the right thalamus and right striatum (based on decreased rates of glucose metabolism). In the HFD group the magnitude of the drop in the sucrose preference was strongly correlated to the magnitude of the deactivation of the right thalamus (r=0.78) and the right striatum (r=0.81).
These findings support hypotheses about the role of a HFD in the causal pathway for depressive symptoms. Further work is needed to clarify the underling mechanism, but it appears that the interaction between the content of the diet and the limbic system-striatum-thalamus circuit plays a role in both eating behavior and depressive symptoms.
high-fat diet; depression; animal model; glucose metabolism; micro-PET
Huoxue Rongluo Tablet was made of tall gastrodis tuber, dahurian angelica root, honeysuckle stem, grassleaf sweetflag rhizome, common flowering quince fruit, figwort root, red peony root and peach seed at a ratio of 3:2:6:2:3:3:3:3. Huoxue Rongluo Tablet is a well-established and common pre-scription for the treatment of cerebral infarction. In this study, a rat model of cerebral ischemia was established and the animals were intragastrically administered Huoxue Rongluo Tablet. This treat-ment reduced infarct volume, decreased matrix metalloproteinase-9 expression, and improved neurological function. Moreover, the effects of Huoxue Rongluo Tablet were better than those of buflomedil pyridoxal phosphate. These results indicate that Huoxue Rongluo Tablet is effective in treating cerebral infarction by regulating matrix metalloproteinase-9 protein expression.
neural regeneration; traditional Chinese medicine; Huoxue Rongluo Tablet; cerebral infarction; neuroprotection; matrix metalloproteinase-9; buflomedil pyridoxal phosphate; grants-supported paper; neuroregeneration
Objective and design
This study is aimed at exploring the role of neurokinin-1 receptor (NK-1R) in the development of allergic rhinitis (AR) in rats.
Sprague–Dawley rats were sensitized and challenged with ovalbumin to induce AR. The rats were treated intranasally with saline, control, or NK-1R-specific small interfering RNA (siRNA) before and during the challenge period. The numbers of sneezes and nose rubs and amount of nasal secretion in individual rats were measured. The levels of NK-1R expression in the nasal mucosal tissues after the last challenge were determined. The numbers of eosinophils in the collected nasal lavage fluid and the levels of serum interleukin (IL)-5 in individual rats were determined.
The levels of NK-1R expression in the nasal mucosal tissues of the AR rats that had been treated with saline or control siRNA were significantly higher than those in the healthy controls and the rats treated with NK-1R-specific siRNA, demonstrating NK-1R silencing. Furthermore, knockdown of NK-1R expression significantly reduced the amounts of sneezing, nose rubbing, and nasal secretions in AR rats. Knockdown of NK-1R expression also significantly eliminated eosinophil infiltration in the nasal tissues and reduced the levels of serum IL-5 in rats.
Knockdown of NK-1R expression decreased allergic inflammation in nasal mucosal tissues and alleviated the allergic rhinitis symptoms, suggesting that NK-1R may be a critical mediator of the development of AR.
Allergic rhinitis; NK-1R; siRNA; Rats
Chronic unpredictable stress (CUS) can cause behavioral and physiological abnormalities that are important to the prediction of symptoms of depression that may be associated with cerebral glucose metabolic abnormalities. Curcumin showed potential antidepressant effects, but whether or not it can reverse cerebral functional abnormalities and so ameliorate depression remains unknown.
To investigate the effects of curcumin on brain activity in CUS rats, rats were subjected to 3 weeks of CUS and then treated with curcumin orally at a dose of 40 mg/kg/day for one month. 18 F fluorodeoxyglucose (18 F-FDG)-micro positron emission tomography (micro-PET) neuroimaging was used to detect changes in cerebral metabolism. Body weight, sucrose preference, and open field tests were used to record depressive behaviors during CUS and after curcumin treatment.
Three weeks of CUS significantly decreased body weight, sucrose preference, sucrose consumption, total distance travelling, and the number of rearing events. It also induced metabolic alterations in several parts of the brain, showing increased glucose metabolism in the right hemisphere. After curcumin treatment for one month, sucrose preference, sucrose consumption, total distance travelling, and the number of rearing events returned to normal levels. Curcumin treatment also induced strong deactivation of the left primary auditory cortex and activation of amygdalohippocampal cortex.
Curcumin was found to ameliorate the abnormalities in the behavior and brain glucose metabolism caused by CUS, which may account for its antidepressive effects.
CA3 pyramidal neurons are important for memory formation and pattern completion in the hippocampal network. It is generally thought that proximal synapses from the mossy fibers activate these neurons most efficiently, whereas distal inputs from the perforant path have a weaker modulatory influence. We used confocally targeted patch-clamp recording from dendrites and axons to map the activation of rat CA3 pyramidal neurons at the subcellular level. Our results reveal two distinct dendritic domains. In the proximal domain, action potentials initiated in the axon backpropagate actively with large amplitude and fast time course. In the distal domain, Na+ channel–mediated dendritic spikes are efficiently initiated by waveforms mimicking synaptic events. CA3 pyramidal neuron dendrites showed a high Na+-to-K+ conductance density ratio, providing ideal conditions for active backpropagation and dendritic spike initiation. Dendritic spikes may enhance the computational power of CA3 pyramidal neurons in the hippocampal network.
Objective: To investigate the incidence of aneuploidy in fetuses with congenital heart defects (CHDs) and to further identify submicroscopic changes and global DNA methylation levels as potential biomarkers in complex CHD cases.
Methods: Fetuses at high risk for birth defects or with obvious sonographic anomalies were recruited at the Prenatal Diagnosis Center and Ultrasonic Diagnosis Center. Elective fetal karyotyping and DNA copy number and promoter methylation analyses were carried out following parental consent. G-banded karyotyping was performed to detect fetal aneuploidy. Copy number variations (CNVs) were detected using the Affymetrix SNP Array 6.0 and validated by real time PCR. Global DNA methylation analyses were conducted using a Roche NimbleGen Human DNA Methylation 3x720K Array, and DNA methylation differences were assayed by a Sequenom MassARRAY EpiTYPER.
Results: Conventional karyotyping identified 30 cases with aneuploidy in 179 CHD fetuses. Various CNVs were found in two aneuploid fetuses and in five euploid CHD fetuses. Verified segmental deletion or duplications were not directly associated with cardiovascular malformations except in DAAM1 and GATA6. Verifiable aberrant DNA methylation could not be identified in three complex CHD fetuses.
Conclusions: In this study, Trisomy 18, Trisomy 21 and 45,XO were the most common aneuploidies identified in CHD fetuses. In the affected samples, only DAAM1 deletion and GATA6 amplification could be associated with cardiovascular biological processes.
Congenital heart defect; Karyotyping; Copy number variants; Methylation level.
With an increasing incidence of congenital heart defects (CHDs) in recent years, genotype-phenotype correlation and array-based methods have contributed to the genome-wide analysis and understanding of genetic variations in the CHD population. Here, we report a copy number deletion of chromosomal 14q23.1 in a female fetus with complex congenital heart defects. This is the first description of DAAM1 gene deletion associated with congenital heart anomalies.
Compared with the control population, one CHD fetus showed a unique copy number deletion of 14q23.1, a region that harbored DAAM1 and KIAA0666 genes.
Results suggest that the copy number deletion on chromosome 14q23.1 may be critical for cardiogenesis. However, the exact relationship and mechanism of how DAAM1 and KIAA0666 deletion contributes to the onset of CHD is yet to be determined.
Congenital heart defect; Copy number deletion; DAAM1 gene
Objective and design
To determine whether the neurokinin-1 receptor (NK1R) plays a role in the activation of RBL-2H3 mast cells after FcεRΙ aggregation.
Materials and methods
NK1R expression in RBL-2H3 cells was inhibited by small hairpin RNA (shRNA) against NK1R, and determined by western blotting. For activation, both NK1R knockdown and control RBL-2H3 cells were sensitized by dinitrophenol (DNP)-specific IgE and stimulated with the antigen DNP-bovine serum albumin (BSA). Following the activation of RBL-2H3 cells, monocyte chemoattractant protein (MCP-1) production and intracellular calcium flux were monitored by ELISA and confocal microscopy assay, respectively. For investigation of the signaling mechanism, phosphorylation of mitogen-activated protein kinases (MAPKs) after RBL-2H3 cell activation was assessed by western blotting.
shRNA-NK1R mediated an effective inhibition of NK1R expression in RBL-2H3 cells. Protein production of MCP-1 was reduced by more than 55 % in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells. In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression.
NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.
Electronic supplementary material
The online version of this article (doi:10.1007/s00011-012-0523-x) contains supplementary material, which is available to authorized users.
Neurokinin-1 receptor; Mast cell; FcεRΙ; Signaling pathway
In the title compound, C15H13N3O3, the dihedral angle between the benzene rings is 1.01 (3)° and that between the nitro group and its attached ring is 5.99 (15)°. In the crystal, molecules are linked by N—H⋯O hydrogen bonds generating C(4) chains along .
In the title compound, C15H13FN2O, the dihedral angle between the benzene rings is 16.9 (2)°. The F atom and the O atom are in a syn conformation. In the crystal, molecules are linked by N—H⋯O hydrogen bonds to generate C(4) chains propagating along the b-axis direction.
This study aimed to investigate the prevalence and risk factors for anxiety and depression symptoms in outpatient migraineurs in mainland China. In addition, we evaluated whether the Hospital Anxiety and Depression Scale (HADS) provided sufficient validity to screen depression and anxiety. A cross-sectional study was conducted consecutively at our headache clinic. Migraine was diagnosed according to International Classification of Headache Disorders, 2nd edition (ICHD-II). Demographic characteristics and clinical features were collected by headache questionnaire. Anxiety and depression symptoms about migraineurs were assessed using HADS. Several questionnaires were simultaneously used to evaluate patients with depressive disorder including the Hamilton Depression Rating Scale-17 (HAMD), Hamilton Anxiety Rating Scale (HAMA) and HADS. Pearson correlation analysis was applied to test the validity of HADS. 176 outpatients with migraine (81.8 % female) were included. Overall, 17.6 and 38.1 % participants had depression and anxiety, respectively. Possible risk factors for depression in migraineurs included headache intensity of first onset of migraine, migraine with presymptom, migraine with family history and migraine disability. The possible risk factors for anxiety included fixed attack time of headache in one day and poor sleeping, and age represented a protective factor for anxiety. The correlation coefficient of HADS-A and HADS-D with HAMA and HAMD was 0.666 and 0.508, respectively (P < 0.01). This study demonstrates that depression and anxiety comorbidity in our mainland Chinese migraineurs are also common, and several risk factors were identified that may provide predictive value. These findings can help clinicians to identify and treat anxiety and depression in order to improve migraine management.
Anxiety; Cross-sectional study; Depression; Migraine; Risk factor
After gene mutation, the pcDNA3.1/APP595/596 plasmid was transfected into HEK293 cells to establish a cell model of Alzheimer's disease. The cell model was treated with donepezil or compound Danshen tablets after culture for 72 hours. Reverse transcription-PCR showed that the mRNA expression of amyloid protein precursor decreased in all groups following culture for 24 hours, and that there was no significant difference in the amount of decrease between donepezil and compound Danshen tablets. Our results suggest that compound Danshen tablets can reduce expression of the mRNA for amyloid protein precursor in a transgenic cell model of Alzheimer's disease, with similar effects to donepezil.
amyloid protein precursor; Alzheimer's disease; transgenic cell model; compound Danshen tablets; Chinese medicine; neural regeneration
For patients with pregnancy-induced thalassemia, fetal cord blood or amniotic fluid is invasively collected in the traditional diagnosis and prediction of thalassemia. However, there is no specific molecular target in the diagnosis of thalassemia using fetal DNA from the plasma of pregnant women.
The promoter of cell surface adhesion molecule (IGSF4) gene was found to be down-regulated in patients with homozygous thalassemia, and the expression of IGSF4 was closely associated with the methylation of its promoter. In the present study, mass spectrometric sequencing of methylation was performed using MassARRAY to detect the 12 CpG sites in the promoter of IGSF4 gene.
The methylation degree of these 12 CpG sites was significantly higher than that in healthy subjects (P<0.05). Hierarchical clustering was done in 23 patients with thalassemia and 5 healthy individuals. Results revealed the promoter of IGSF4 gene was highly methylated in thalassemia patients, which was dramatically different from that in healthy subjects (P<0.05). Methylation-specific PCR (MSP) was employed to confirm the methylation of the promoter of IGSF4 gene and results were consistence with those obtained in sequencing with MassARRAY. Real-time PCR showed, when compared with heterozygous subjects, the expression of IGSF4 was significantly down-regulated in thalassemia patients (ratio=0.18).
The expression of IGSF4 was closely related to the methylation of its promoter, suggesting the methylation of IGSF4 gene is tissue-specific for thalassemia. These findings provide evidence for the non-invasive prenatal diagnosis of thalassemia in terms of epigenetics.
thalassemia; DNA methylation; IGSF4; MassARRAY; noninvasive prenatal diagnosis
The title compound, C20H19NO, is a substructure of CP-640186, a potent inhibitor of mammalian acetyl-coenzyme A carboxylases. In the crystal structure, the amide group forms a dihedral angle of 87.0 (1)° with the plane of the anthracene unit and the piperidine ring adopts a chair conformation. Molecules are arranged into layers parallel to (100) and adjacent anthracene units within layers form dihedral angles of 13.2 (1)°. C—H⋯O interactions from the piperidine rings to the C=O group of the amide are observed between layers.
AIM: To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice.
METHODS: The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 μg. Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected.
RESULTS: After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23 (3.1±0.49) was higher than that in the control group (0.787±0.12, P<0.01). None in the control group had a detectable level of anti-HEV IgG.
CONCLUSION: DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice.
Objective: To determine the protective effect of monosialoganglionside (GM1) and evaluate the influence of GM1 on expression of N-methyl-D-aspartate receptor subunit 1 (NMDAR1) in Sprague-Dawley (SD) rats with focal cerebral ischemia-reperfusion (I/R). Methods: Left middle cerebral artery (MCA) was occluded by an intraluminal suture for 1 h and the brain was reperfused for 72 h in SD rats when infarct volume was measured, GM1 (10 mg/kg) was given ip (intraperitoneally) at 5 min (group A), 1 h (group B) and 2 h (group C) after MCA occlusion (MCAo). Expression of NMDAR1 was detected by Western blot at various time after reperfusion (4 h, 6 h, 24 h, 48 h and 72 h) in ischemic hemispheres of the rats with or without GM1 administered. Results: (1) Adjusted relative infarct volumes of groups A and B were significantly smaller than that of group C and the control group (P<0.01 and P<0.05, respectively). (2) Expression level of NMDAR1 was temporally high at 6 h after reperfusion, and dipped below the normal level at 72 h after reperfusion. GM1 at 5 min after MCAo significantly suppressed the expression of NMDAR1 at 6 h after reperfusion (P<0.05 vs the control). At 72 h after reperfusion, the NMDAR1 expression level of rats treated with GM1 administered (at 5 min or 2 h after MCAo) was significantly higher than that of the control (P<0.05). Conclusion: GM1 can time-dependently reduce infarct volume in rats with focal cerebral I/R partly through stabilizing the expression of NMDAR1.
G(M1) ganglioside; Middle cerebral artery occlusion; Reperfusion; N-methyl-D-aspartate receptors; Rats
Objective: To characterize the expression of aquaporin-4 (AQP4), one of the aquaporins (AQPs), in human brain specimens from patients with traumatic brain injury or brain tumors. Methods: Nineteen human brain specimens were obtained from the patients with traumatic brain injury, brain tumors, benign meningioma or early stage hemorrhagic stroke. MRI or CT imaging was used to assess brain edema. Hematoxylin and eosin staining were used to evaluate cell damage. Immunohistochemistry was used to detect the AQP4 expression. Results: AQP4 expression was increased from 15 h to at least 8 d after injury. AQP4 immunoreactivity was strong around astrocytomas, ganglioglioma and metastatic adenocarcinoma. However, AQP4 immunoreactivity was only found in the centers of astrocytomas and ganglioglioma, but not in metastatic adenocarcinoma derived from lung. Conclusion: AQP4 expression increases in human brains after traumatic brain injury, within brain-derived tumors, and around brain tumors.
Aquaporin-4 (AQP4); Traumatic brain injury; Astrocytoma; Ganglioglioma; Metastatic adenocarcinoma; Brain edema