Epilepsy ; genetics ; metabolism ; Glutamic Acid ; metabolism ; Humans ; Ion Channels ; genetics ; Receptors, GABA-A ; genetics

Due to the negative autopsy and without cardiac structural abnormalities, unexpected sudden cardiac death （USCD） is always a tough issue for forensic pathological expertise. USCD may be associated with parts of fatal arrhythmic diseases. These arrhythmic diseases may be caused by disorders of cardiac ion channels or channel-related proteins. Caveolin can combine with multiple myocardial ion channel proteins through its scaffolding regions and plays an important role in maintaining the depolarization and repolarization of cardiac action potential. When the structure and function of caveolin are affected by gene mutations or abnormal protein expression, the functions of the regulated ion channels are correspondingly impaired, which leads to the occurrence of multiple channelopathies, arrhythmia or even sudden cardiac death. It is important to study the effects of caveolin on the functions of ion channels for exploring the mechanisms of malignant arrhythmia and sudden cardiac death.
Arrhythmias, Cardiac/physiopathology* ; Autopsy ; Caveolins/metabolism* ; Channelopathies/genetics* ; Death, Sudden, Cardiac/pathology* ; Forensic Pathology ; Humans ; Ion Channels/metabolism* ; Mutation ; Myocardium

OBJECTIVETo observe the effects of chronic lead exposure on mRNA and protein expression of ASIC1a, ASIC2a, ASIC2b in hippocampus of baby-rats.

METHODSThe Wistar pregnant rats were randomly divided into 3 groups fed with distilled water or lead contained water (0.2% and 1.0% lead acetate) respectively, 5 rats in each group. The lead-exposure ranged from the 0 day of pregnancy to the offspring weaned. Then the baby-rats were fed with lead water like their mothers and killed at postnatal day 8 or 50. Atomic absorption spectrometry was used to determine lead content in the brain. RT-PCR and Western blotting were used to observe mRNA and protein expression of ASIC1a, ASIC2a and ASIC2b in their hippocampus respectively.

RESULTSThe brain lead content of test groups was higher than that of the control group (P < 0.01), and the lead content of the postnatal day 50 was higher than that in postnatal day 8 (P < 0.01). Compared with the control group, ASIC1a mRNA expression of 1.0% lead exposure in the hippocampus was uptrend (P < 0.01), ASIC1a protein expression of each test group was downtrend (P < 0.05), while for ASIC2a and ASIC2b mRNA and protein, there was no significant differences observed (P > 0.05).

CONCLUSIONASIC1a expression in hippocampus can be changed by chronic lead exposure.

Acid Sensing Ion Channels ; Animals ; Female ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Nerve Tissue Proteins ; genetics ; metabolism ; Pregnancy ; Prenatal Exposure Delayed Effects ; genetics ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Sodium Channels ; genetics ; metabolism

OBJECTIVETo investigate the ion channel genes related with the genesis and development of heart failure after myocardial infarction through analyzing the differential gene expressions in non-infarcted myocardial tissues of post-myocardial infarction heart failure (PIHF) rat model, and that of normal rat, and the bio-informatics Stc-GO analysis on them.

METHODSRat model of PIHF was established by left anterior descending coronary artery ligation in six SD rats, and a control group with six sham-operative rats was set. Myocardial samples were taken in two batches from them (three rats in each group) at the heart failure formation stage and the stable stage (10 days and 8 weeks after operation) respectively. Total RNA extraction, probe preparation with reverse transcription, hybridization with double-channel cDNA microarray of rat's ion channel genes, and computerized differential gene expression screening were conducted, and Stc-GO functional clustering analysis was performed on the outcomes obtained to search out the GO sort of significance in them.

RESULTSAt 10 days after operation, 319 common differential expressed genes were found from the 746 target genes, all of them were up-regulated. At 8 weeks after operation, in the 276 differential expressed genes, 274 were up-regulated while the other two down-regulated. The up-regulated genes were those concerning receptors of various hormones, cytokines, neuro-hormones, growth factors and nuclear receptor, protein phosphorylase, G protein, various ion channels mediated by ligand or voltage, transport protein, receptor interfering protein, etc. The down-regulated genes concerning the potassium channel and transport protein, etc. Stc-GO analysis found that the six genes concerning adrenergic receptor kinase beta 1 (betaARK1), amiloride-sensitive cation channel 2 neuronal (Accn2), voltage-dependent calcium ion channel gamma subunit 1 (Cacng1), cyclic nucleotide gated channel alpha 1 (Cnga1), Glutamate receptor ionotropic kainite 2 (Grik 2) and neurotrophic tyrosine kinase receptor type 2 (Ntrk 2), were all the significantly up-regulated differential genes of the model group related with the sham-operative group, and all showed a down-regulating trend as time goes on, and four genes in them were validated by the RT-PCR test.

CONCLUSIONIon channel genes concerning Accn2, Grik2, Ntrk2 and Cacng1 were up-regulated in PIHF, and its mechanism is waiting for further study.

Animals ; Heart Failure ; etiology ; genetics ; metabolism ; Ion Channels ; metabolism ; Male ; Myocardial Infarction ; complications ; genetics ; metabolism ; Oligonucleotide Array Sequence Analysis ; Rats ; Rats, Sprague-Dawley ; Transcriptome

In Candida albicans, adaptation to environmental pH is relevant to its pathogenicity. Calcium signaling pathway involves in many stress responses and often accompany with Ca2+ fluctuation. We constructed CCH1 and MID1 mutant strains and studied their effect on calcium influx and further investigated the regulation by Crz1p transcription factor. We used PCR-directed gene disruption to construct cch1delta/delta and mid1delta/delta null mutant. By using a flow cytometry-based method we monitored the free cytosolic Ca2+ levels under alkaline stress. Moreover, we constructed pPHO89-LacZ plasmids and by beta-Galactosidase assays, we analyzed the changes of LacZ activities after gene disruption. The results showed that alkaline stress induced calcium burst reduced obviously in cch1delta/delta and mid1delta/delta mutant strains, also for LacZ activities, and fully abolished in crz1delta/delta mutant strain. Finally, by realtime PCR, we confirmed the regulation role of Crz1p in CCH1 and MID1 genes but in a calcineurin independent way. Studies on the effect of calcium pathway on response to alkaline stress will provide an important theoretical basis for Candida albicans infection-oriented treatment and new drug targets.
Calcium Channels ; metabolism ; Candida albicans ; genetics ; metabolism ; physiology ; Fungal Proteins ; genetics ; physiology ; Gene Expression Regulation, Fungal ; Hydrogen-Ion Concentration ; Signal Transduction ; Stress, Physiological ; Transcription Factors ; metabolism ; physiology

This paper was designed in middle cerebral artery occlusion (MCAO) model of rats, to explore the role of transient receptor potential channel 4 (TRPC4) as Ca(2+) selective channel by detecting the changes of the expression of TRPC4 in different parts of cerebral tissues under the condition of focal cerebral ischemia. The rats were sacrificed after MCAO surviving time 6 h, 12 h, 1 d, 3 d. As determined by Western blot, the expressions of TRPC4 in striatum and hippocampus of 12 h, 1 d, 3 d groups were significant higher than that in the control group (P<0.05). Immunohistochemical staining showed that the TRPC4 immunoreactive substances were present in the membrane of neurons. Compared with the control group, immunostaining positive cells increased in hippocampus and striatum of cerebral ischemia groups. The TRPC4 immunostaining positive cells increased significantly in 1d-group and 3d-group (P<0.05). It suggests that as a Ca(2+) selective channel, the variance of the expression of TRPC4 may play a role in acute and delayed neuronal injury in focal cerebral ischemia.
Animals ; Cation Transport Proteins ; biosynthesis ; genetics ; Corpus Striatum ; metabolism ; Hippocampus ; metabolism ; Infarction, Middle Cerebral Artery ; metabolism ; Ion Channels ; biosynthesis ; genetics ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; TRPV Cation Channels

The present study was carried out to determine the functional properties of Kv4.2 expressed in mammalian cells in comparison with native transient potassium outward current (I(A)) in the hippocampal neurons. Transient transfection, cell culture and whole cell voltage clamp techniques were used. The results showed that I(A) in cultured rat hippocampal neurons and Kv4.2 expressed in HEK293 cells both displayed "A"-type current properties. The activation curves of I(A) and Kv4.2 were better fitted by simple Boltzmann function with V(1/2) 10.0+/-3.3 mV, k 13.9+/-2.6 mV for I(A) and V1/2 -9.7+/-4.1 mV, k 15.8+/-5.7 mV for Kv4.2, respectively. The steady-state inactivation curves of I(A) had a midpoint of -93.0+/-11.4 mV and a slope of 9.0+/-1.5 mV. The voltage-dependence of inactivation for Kv4.2 exhibited midpoint and slope values of -59.4+/-12.2 mV and 8.0+/-3.1 mV, respectively. The time constants (tau) of recovery from inactivation of I(A) and Kv4.2 were 27.9+/-14.1 ms and 172.8+/-10.0 ms, respectively. These results suggest that Kv4.2 is probably a major isoform contributing to I(A) in hippocampus neurons.
Animals ; Animals, Newborn ; Cells, Cultured ; Female ; Gene Transfer Techniques ; Hippocampus ; metabolism ; physiology ; Ion Transport ; Male ; Neurons ; metabolism ; physiology ; Patch-Clamp Techniques ; Potassium Channel Blockers ; Potassium Channels ; genetics ; physiology ; Potassium Channels, Voltage-Gated ; Rats ; Rats, Wistar ; Shal Potassium Channels

OBJECTIVETo investigate the correlation between uncoupling protein 2 (UCP2) expression and myocardial mitochondria injury in rats with sepsis induced by lipopolysaccharide (LPS).

METHODSThe rat model of sepsis was established through an intraperitoneal injection of LPS. Forty male Sprague-Dawley rats were randomly and equally divided into control group (an intraperitoneal injection of normal saline), sepsis 6 h group (LPS-6 h group), sepsis 12 h group (LPS-12 h group), sepsis 24 h group (LPS-24 h group), and sepsis 48 h group (LPS-48 h group). The serum and heart tissues were harvested at corresponding time points and myocardial mitochondria was extracted. The microplate reader was applied to measure creatine kinase (CK), creatine kinase-MB (CK-MB), and reactive oxygen species (ROS). Flow cytometry was applied to measure the degree of mitochondrial swelling and mitochondrial membrane potential (MMP). Western blot was used to measure the expression level of UCP2. Electron microscopy was applied to observe the morphological changes in heart tissues and myocardial mitochondria.

RESULTSCompared with the control group, the LPS groups had significantly increased serum levels of CK, CK-MB, and myocardial ROS, as well as a significantly increased degree of mitochondrial swelling (P<0.05), and these values reached their peaks at 24 hours after LPS injection. The LPS groups had a significant decrease in MMP (P<0.05), which reached the lowest level at 24 hours after LPS injection. Western blot showed that the LPS groups had a significant increase in the expression level of myocardial UCP2 compared with the control group (P<0.05), which reached its peak at 24 hours after LPS injection. The results of electron microscopy showed mitochondrial swelling, partial rupture of the mitochondrial membrane, and cavity formation in rats in the LPS groups. The most severe lesions occurred in the LPS-24 h group. In rats with LPS, the ROS level in the myocardial mitochondria and the degree of mitochondrial swelling were positively correlated with the expression level of UCP2 (r=0.796 and 0.893, respectively; P<0.05), while MMP was negatively correlated with the expression level of UCP2 (r=-0.903, P<0.05).

CONCLUSIONSIn the rat model of sepsis, the myocardium and myocardial mitochondria have obvious injuries, and the expression level of UCP2 is closely correlated with mitochondrial injury. Therefore, UCP2 might play an important role in myocardial mitochondrial injury in sepsis.

Animals ; Cardiomyopathies ; genetics ; metabolism ; Disease Models, Animal ; Humans ; Ion Channels ; genetics ; metabolism ; Lipopolysaccharides ; adverse effects ; Male ; Mitochondria, Heart ; metabolism ; Mitochondrial Proteins ; genetics ; metabolism ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sepsis ; genetics ; metabolism ; Uncoupling Protein 2

Animals ; Fatty Liver ; metabolism ; Humans ; Ion Channels ; biosynthesis ; genetics ; physiology ; Mitochondrial Proteins ; biosynthesis ; genetics ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Uncoupling Protein 2

OBJECTIVEThis study sought to isolate and identify the proteins that interact with ataxin-3, to confirm the interacted domain, and to provide new clues for exploring the function of ataxin-3 and the pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD).

METHODSYeast two-hybrid screen (MATCHMAKER GAL4 Two-Hybrid System 3) and regular molecular biologic techniques were undertaken to screen human brain cDNA library with mutant ataxin-3 bait. Two baits from both normal and mutant C-terminus of ataxin-3 were created by subcloned methods to determine which domain of ataxin-3 interacts with the putative associated proteins and to find out optimal candidate proteins that interact with C-terminus of ataxin-3. Confocal microscope was used to observe whether ataxin-3 co-localized with the obtained interacting proteins in mammalian cells.

RESULTSFive novel ataxin-3 interacting proteins were obtained, among which were three known proteins, namely human rhodopsin guanosine diphosphate dissociation inhibitor alpha, small ubiquitin-like modifier 1, and human neuronal amiloride-sensitive cation channel 2; the other two were unknown. Interacting domain analysis revealed that an unknown protein interacted with the C-terminus near the polyglutamine tract of ataxin-3, the other four all interacted with the N-terminus. In the nucleus of SH-SY5Y cell, small ubiquitin-like modifier 1 co-localized with the wild-type ataxin-3 and with the intranuclear aggregates formed by the mutant ataxin-3.

CONCLUSIONAn unknown protein probably interacting with C-terminus of ataxin-3 is firstly discovered, and the initiative findings suggest first that the interaction of small ubiquitin-like modifier 1 with N-terminus of ataxin-3 and the relevant sumoylation probably participate in the post-translation modifying of ataxin-3 and in the pathogenesis of SCA3/MJD.

Acid Sensing Ion Channels ; Ataxin-3 ; Cell Line, Tumor ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Confocal ; Mutation ; Nerve Tissue Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Protein Binding ; Recombinant Fusion Proteins ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; SUMO-1 Protein ; genetics ; metabolism ; Sodium Channels ; genetics ; metabolism ; Transfection ; Two-Hybrid System Techniques