Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4-S5 linker in activation and slow inactivation.

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

Baohuoside I Inhibits the Proliferation of Hepatocellular Carcinoma Cells via Apoptosis Signaling and NF-kB Pathway.

Baohuoside I is a flavonoid isolated from Epimedium koreanum Nakai and has many pharmacological activities. However, its role in liver cancer remains unclear. This study aimed to investigate the inhibitory effect of Baohuoside I on the Human Hepatocellular Carcinoma (HCC) cell lines QGY7703, and underlying mechanisms. QGY7703 cells were used as the model to assess the function of Baohuoside I in vitro. The effects of Baohuoside I on QGY7703 cells' growth, proliferation, and invasiveness were confirmed by CCK-8, lactate dehydrogenase release, and invasion assays. Cell apoptosis was analyzed by flow cytometry, and the levels of cleaved Caspase-3, Bax, and Bcl-2 were quantified by western blot. Western blot analysis, nuclear translocation of NF-κB, and Q-PCR were used to measure the expression of affected molecules. In QGY7703 cells, Baohuoside I induced the expression of molecules related to NF-κB pathway. The toxicity of Baohuoside I on QGY7703 cells was also confirmed in vivo, in a tumor model. Baohuoside I had a significant toxic effect on QGY7703 cells from a concentration of 10 µM. This compound significantly inhibited the proliferation of QGY7703 cells by inducing apoptosis and downregulating NF-κB signaling pathway. Thus, Baohuoside I is a novel candidate drug and opens new possibilities of clinical strategies for HCC treatment.

[Investigation and analysis of influence of geographical environment factors on spatial distribution of flavonoid of Epimedium koreanum].

The study is aimed to clarify the spatial distribution of Epimedium koreanum( Ek) high-quality production areas. Through visiting and field investigation,collecting the distribution information of Ek samples,and based on the four kinds of flavonoids in Ek,the high-quality production areas and distribution of Ek distribution of the main environmental factors were drawn using GIS technology,the maximum entropy model( MaxEnt),geographical detector statistical analysis method,and the statistical significance of regression equation were obtained. Considering the content of 4 main flavonoids in Ek,the results of this study showed that the main environmental factors,such as precipitation,annual precipitation variation coefficient,annual average temperature and clay content exhibited the greatest influence on the growth suitability of Ek. Ek materials quality concentrated distribution in southeastern Jilin province Changbai mountain hinterland and northeastern Liaoning province. Ek with high content of epimedine A and epimedine C are mainly distributed in the southeastern Jilin province and northeastern Liaoning province,Ek with high epimedine B is distributed in eastern Liaoning province; high icariin Ek was found in most area of northeastern Liaoning province,a small amount distributed in the southeast of Jilin province. This study predicted the climate suitability distribution of Ek,and provided reference for the rational planning and establishment of the standardized cultivation base of Ek.

Actin-like 6A predicts poor prognosis of hepatocellular carcinoma and promotes metastasis and epithelial-mesenchymal transition.

UNLABELLED: Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide because of metastasis. Epithelial-mesenchymal transition (EMT) is widely considered to be crucial to the invasion-metastasis cascade during cancer progression. Actin-like 6A (ACTL6A) is initially verified important for cell proliferation, differentiation, and migration. In this study, we find that ACTL6A plays an essential role in metastasis and EMT of HCC. ACTL6A expression is up-regulated in HCC cells and tissues. A high level of ACTL6A in HCCs is correlated with aggressive clinicopathological features and is an independent poor prognostic factor for overall and disease-free survival of HCC patients. Ectopic expression of ACTL6A markedly promotes HCC cells migration, invasion, as well as EMT in vitro and promotes tumor growth and metastasis in the mouse xenograft model. Opposite results are observed when ACTL6A is knocked down. Mechanistically, ACTL6A promotes metastasis and EMT through activating Notch signaling. ACTL6A knockdown has the equal blockage effect as the Notch signaling inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester, in HCC cells. Further studies indicate that ACTL6A might manipulate SRY (sex determining region Y)-box 2 (SOX2) expression and then activate Notch1 signaling. CONCLUSIONS: ACTL6A promotes metastasis and EMT by SOX2/Notch1 signaling, indicating a prognostic biomarker candidate and a potential therapeutic target for HCC.

Inhibition of PKR ameliorates lipopolysaccharide-induced acute lung injury by suppressing NF-κB pathway in mice.

Acute lung injury (ALI) is characterized by dramatic lung inflammation and alveolar epithelial cell death. Although protein kinase R (PKR) (double-stranded RNA-activated serine/threonine kinase) has been implicated in inflammatory response to bacterial cell wall components, whether it plays roles in lipopolysaccharide (LPS)-induced ALI remains unclear. This study was aimed to reveal whether and how PKR was involved in LPS-induced ALI pathology and the potential effects of its specific inhibitor, C16 (C13H8N4OS). During the experiment, mice received C16 (100 or 500 ug/kg) intraperitoneally 1 h before intratracheal LPS instillation. Then, whole lung lavage was collected for analysis of total protein levels and proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6. The lungs were tested for Western blot, transferase-mediated dUTP nick-end labeling (TUNEL) stain and immunohistochemistry. Results showed that PKR phosphorylation increased significantly after LPS instillation. Furthermore, PKR specific inhibition attenuated LPS-induced lung injury (hematoxylin and eosin stain), reduced lung protein permeability (total protein levels in whole lung lavage) and suppressed proinflammatory cytokines (TNF-α, IL-1ß and IL-6) and lung apoptosis (TUNEL stain and caspase3 activation). Moreover, mechanism-study showed that C16 significantly suppressed I kappa B kinase (IKK)/I kappa B alpha (IκBα)/NF-κB signaling pathway after LPS challenge. These findings suggested that PKR inhibition ameliorated LPS-induced lung inflammation and apoptosis in mice by suppressing NF-κB signaling pathway.

[Regionalization study of Dioscorea nipponica in Jilin province based on MaxEnt and ArcGIS].

At the urgent practical issue of resource protection and artificial cultivation area selection of Dioscorea nipponica, the dominant environmental factors affecting the distribution of D. nipponicain Jilin province were selected by field investigation and using the maximum information entropy model and geographic information technology. MaxEnt model study found that the standard deviation of seasonal variation of temperature, precipitation in October and other six environmental factors on the growth of D. nipponica are the greatest impacting factors. The range of suitability for the growth of D. nipponica was 4.612 08×10-6-0.544 31, and the regionalization study was divided into four parts: high fitness area, middle fitness area, low fitness area and unfavorable area. The high fitness area is concentrated in the central and southern areas of Jilin Province, using ArcGIS statistical environment factors in the appropriate area of the numerical situation. The results showed that the regionalization study of D. nipponica was basically the same as the actual situation. It is clear that the natural environment suitable for the growth of D. nipponica is also the basis for the protection of the resources and the selection of cultivated area.

[Quality of Schisandrae Chinensis Fructus regionalization based on GIS technology].

This paper is aims to clarify the spatial distribution of high quality medicinal materials Schisandrae Chinensis Fructus. Based on investigation and field investigation, the samples and distribution information of Schisandrae Chinensis Fructus were collected. Based on the data of four kinds of lignin chemical constituents, ecological environment factors and spatial distribution data of Schisandrae Chinensis Fructus, using GIS technology, maximum information entropy model and SPSS statistical analysis method for regionalizing the quality of Schisandrae Chinensis Fructus. The results showed that Schisandrae Chinensis Fructus was mainly distributed in the northeast of Liaoning, east of Jilin, east of Heilongjiang. The content of schisandrin was higher in the samples from northeastern part of Jilin province and the northeastern part of Liaoning province, The content of deoxyschizandrin was higher in the samples from middle of Jilin province and northeastern Hebei province, where the content of schisandrin B was higher in the samples from Jilin area, The higher schisantherin A sample were from southeast of Jilin and northeast of Liaoning. Considering the content of four components in Schisandrae Chinensis Fructus, the quality of Schisandrae Chinensis Fructus was concentrated in the southeast of Jilin and the northeastern part of Liaoning.

The Specific Protein Kinase R (PKR) Inhibitor C16 Protects Neonatal Hypoxia-Ischemia Brain Damages by Inhibiting Neuroinflammation in a Neonatal Rat Model.

BACKGROUND Brain injuries induced by hypoxia-ischemia in neonates contribute to increased mortality and lifelong neurological dysfunction. The specific PKR inhibitor C16 has been previously demonstrated to exert a neuroprotective role in adult brain injuries. However, there is no recent study available concerning its protective role in hypoxia-ischemia-induced immature brain damage. Therefore, we investigated whether C16 protects against neonatal hypoxia-ischemia injuries in a neonatal rat model. MATERIAL AND METHODS Postnatal day 7 (P7) rats were used to establish classical hypoxia-ischemia animal models, and C16 postconditioning with 100 ug/kg was performed immediately after hypoxia. Western blot analysis was performed to quantify the phosphorylation of the PKR at 0 h, 3 h, 6 h, 12 h, 24 h, and phosphorylation of NF-κB 24h after hypoxia exposure. The TTC stain for infarction area and TUNEL stain for apoptotic cells were assayed 24 h after the brain hypoxia. Gene expression of IL-1ß, IL-6, and TNF-α was performed at 3 h, 6 h, 12 h, and 24 h. RESULTS The level of PKR autophosphorylation was increased dramatically, especially at 3 h (C16 group vs. HI group, P<0.01). Intraperitoneal C16 administration reduced the infarct volume and apoptosis ratio after this insult (C16 group vs. HI group<0.01), and C16 reduced proinflammatory cytokines mRNA expression, partly through inhibiting NF-κB activation (C16 group vs. HI group<0.05). CONCLUSIONS C16 can protect immature rats against hypoxia-ischemia-induced brain damage by modulating neuroinflammation.

Comparative analysis of codon usage patterns in chloroplast genomes of ten Epimedium species.

BACKGROUND: The Phenomenon of codon usage bias exists in the genomes of prokaryotes and eukaryotes. The codon usage pattern is affected by environmental factors, base mutation, gene flow and gene expression level, among which natural selection and mutation pressure are the main factors. The study of codon preference is an effective method to analyze the source of evolutionary driving forces in organisms. Epimedium species are perennial herbs with ornamental and medicinal value distributed worldwide. The chloroplast genome is self-replicating and maternally inherited which is usually used to study species evolution, gene expression and genetic transformation. RESULTS: The results suggested that chloroplast genomes of Epimedium species preferred to use codons ending with A/U. 17 common high-frequency codons and 2-6 optimal codons were found in the chloroplast genomes of Epimedium species, respectively. According to the ENc-plot, PR2-plot and neutrality-plot, the formation of codon preference in Epimedium was affected by multiple factors, and natural selection was the dominant factor. By comparing the codon usage frequency with 4 common model organisms, it was found that Arabidopsis thaliana, Populus trichocarpa, and Saccharomyces cerevisiae were suitable exogenous expression receptors. CONCLUSION: The evolutionary driving force in the chloroplast genomes of 10 Epimedium species probably comes from mutation pressure. Our results provide an important theoretical basis for evolutionary analysis and transgenic research of chloroplast genes.

[On origin of Oviductus Ranae in Chinese Pharmacopoeia].

OBJECTIVE: To discuss the problem about the origin of Oviduetus Ranae in the Chinese Pharmacopoeia according to historical documents, the researches reported recently and the author research. METHOD: Through comprehensive analysis of the documents and materials reported, the original animal sources of Oviduetus Ranae was discussed in terms of historical records, morphology, karyotype, Ag-Belt and isoenzyme electrophoresis, gene levels and so on. RESULT AND CONCLUSION: The original animal sources of Oviduetus Ranae is Rana dybowskii,its order element is an effective species in China. In order to avoid the problem of species confusion about the origin of Oviduetus Ranae, author suggests that R. dybowskii should be the original animal of Oviduetus Ranae.

A Potential Mechanism of Sodium Channel Mediating the General Anesthesia Induced by Propofol.

General anesthesia has revolutionized healthcare over the past 200 years and continues to show advancements. However, many phenomena induced by general anesthetics including paradoxical excitation are still poorly understood. Voltage-gated sodium channels (Na V ) were believed to be one of the proteins targeted during general anesthesia. Based on electrophysiological measurements before and after propofol treatments of different concentrations, we mathematically modified the Hodgkin-Huxley sodium channel formulations and constructed a thalamocortical model to investigate the potential roles of Na V . The ion channels of individual neurons were modeled using the Hodgkin-Huxley type equations. The enhancement of propofol-induced GABAa current was simulated by increasing the maximal conductance and the time-constant of decay. Electroencephalogram (EEG) was evaluated as the post-synaptic potential from pyramidal (PY) cells. We found that a left shift in activation of Na V was induced primarily by a low concentration of propofol (0.3-10 µM), while a left shift in inactivation of Na V was induced by an increasing concentration (0.3-30 µM). Mathematical simulation indicated that a left shift of Na V activation produced a Hopf bifurcation, leading to cell oscillations. Left shift of Na V activation around a value of 5.5 mV in the thalamocortical models suppressed normal bursting of thalamocortical (TC) cells by triggering its chaotic oscillations. This led to irregular spiking of PY cells and an increased frequency in EEG readings. This observation suggests a mechanism leading to paradoxical excitation during general anesthesia. While a left shift in inactivation led to light hyperpolarization in individual cells, it inhibited the activity of the thalamocortical model after a certain depth of anesthesia. This finding implies that high doses of propofol inhibit the network partly by accelerating Na V toward inactivation. Additionally, this result explains why the application of sodium channel blockers decreases the requirement for general anesthetics. Our study provides an insight into the roles that Na V plays in the mechanism of general anesthesia. Since the activation and inactivation of Na V are structurally independent, it should be possible to avoid side effects by state-dependent binding to the Na V to achieve precision medicine in the future.

[Quality evaluation on eighteen samples of forest frog's oviduct from different habitat of northeast China].

OBJECTIVE: To compare eighteen samples of Forest frog's oviduct from different regions of northeast China, in order to fomulate the quality evaluation standard. METHODS: According to the documents, comparing the target constituent of Forest frog's oviduct, including the mositure, ash, protein, lipid and expansibility were analysed. RESULTS: It was similar to the chemical constituent in Forest frog's oviduct from different habitiat of northeast China. CONCLUSION: The germplasm of this species is stable.

Docosahexaenoic Acid Rescues Synaptogenesis Impairment and Long-Term Memory Deficits Caused by Postnatal Multiple Sevoflurane Exposures.

Sevoflurane exposures were demonstrated to induce neurotoxicity in the developing brain in both human and animal studies. However, there is no effective approach to reverse it. The present study aimed to evaluate the feasibility of utilizing docosahexaenoic acid (DHA) to prevent sevoflurane-induced neurotoxicity. P6 (postnatal 6 days) mice were administrated DHA after exposure to 3% sevoflurane for two hours daily in three consecutive days. Molecular expressions of synaptic makers (PSD95, synaptophysin) and synaptic morphological changes were investigated by Western blot analysis and transmission electron microscopy, respectively. Meanwhile, Morris water maze test was used to assess spatial memory of mice at P31 (postnatal 31 days). DHA restored sevoflurane-induced decreased level of PSD95 and synaptophysin expressions and increased PSD areas and also improved long-term spatial memory. These results suggest that DHA could rescue synaptogenesis impairment and long-term memory deficits in postnatal caused by multiple sevoflurane exposures.