Targeting microRNA-125b inhibited the metastasis of Alisertib resistance cells through mediating p53 pathway / 中华肿瘤杂志

Objective: To clarify the mechanisms involvement in Alisertib-resistant colorectal cells and explore a potential target to overcome Alisertib-resistance. Methods: Drug-resistant colon cancer cell line (named as HCT-8-7T cells) was established and transplanted into immunodeficient mice. The metastasis in vivo were observed. Proliferation and migration of HCT-8-7T cells and their parental cells were assessed by colony formation and Transwell assay, respectively. Glycolytic capacity and glutamine metabolism of cells were analyzed by metabolism assays. The protein and mRNA levels of critical factors which are involved in mediating glycolysis and epithelial-mesenchymal transition (EMT) were examined by western blot and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR), respectively. Results: In comparison with the mice transplanted with HCT-8 cells, which were survival with limited metastatic tumor cells in organs, aggressive metastases were observed in liver, lung, kidney and ovary of HCT-8-7T transplanted mice (P<0.05). The levels of ATP [(0.10±0.01) mmol/L], glycolysis [(81.77±8.21) mpH/min] and the capacity of glycolysis [(55.50±3.48) mpH/min] in HCT-8-7T cells were higher than those of HCT-8 cells [(0.04±0.01) mmol/L, (27.77±2.55) mpH/min and(14.00±1.19) mpH/min, respectively, P<0.05]. Meanwhile, the levels of p53 protein and mRNA in HCT-8-7T cells were potently decreased as compared to that in HCT-8 cells (P<0.05). However, the level of miRNA-125b (2.21±0.12) in HCT-8-7T cells was significantly elevated as compared to that in HCT-8 cells (1.00±0.00, P<0.001). In HCT-8-7T cells, forced-expression of p53 reduced the colon number (162.00±24.00) and the migration [(18.53±5.67)%] as compared with those in cells transfected with control vector [274.70±40.50 and (100.00±29.06)%, P<0.05, respectively]. Similarly, miR-125b mimic decreased the glycolysis [(25.28±9.51) mpH/min] in HCT-8-7T cells as compared with that [(54.38±12.70)mpH/min, P=0.003] in HCT-8-7T cells transfected with control. Meanwhile, in comparison with control transfected HCT-8-7T cells, miR-125b mimic also significantly led to an increase in the levels of p53 and β-catenin, in parallel with a decrease in the levels of PFK1 and HK1 in HCT-8-7T cells (P<0.05). Conclusions: Silencing of p53 by miR-125b could be one of the mechanisms that contributes to Alisertib resistance. Targeting miR-125b could be a strategy to overcome Alisertib resistance.

Nicotinamide activates latent HIV-1 ex vivo in ART suppressed individuals, revealing higher potency than the association of two methyltransferase inhibitors, chaetocin and BIX01294

ABSTRACT Background: Latent HIV-1 is a major hurdle in obtaining HIV-1 sustained virological remission (SVR). Here we explored histone deacetylation inhibition property of nicotinamide (NAM; n = 17) for the first time in comparison to a combination of methyltransferase inhibitors (MTIs; Chaetocin and BIX01294; n = 25) to reactivate latent HIV ex vivo in CD8-depleted PBMCs from antiretroviral treated aviremic individuals. Results: NAM reactivated HIV-1 from 13/17 (76.4%) samples compared to 20/25 (80.0%) using MTIs with mean viral load (VLs) of 4.32 and 3.22 log10 RNA copies/mL, respectively (p = 0.004). Mean purging time after NAM and MTIs stimulation was 5.1 and 6.75 days, respectively (p = 0.73). Viral purging in autologous cultures exhibited blunted HIV recovery with fluctuating VLs followed by a complete viral extinction when expanded in allogenic system. Electron microscopy from five supernatants revealed anomalous viral particles, with lack of complete viral genomes when characterized by ultradeep sequencing through metagenomics approach (n = 4). Conclusion: NAM alone was more potent HIV-1 activator than combination of MTIs, with potential of clinical use.

BRD4 interacts with PML/RARα in acute promyelocytic leukemia / 医学前沿

Bromodomain-containing 4 (BRD4) has been considered as an important requirement for disease maintenance and an attractive therapeutic target for cancer therapy. This protein can be targeted by JQ1, a selective small-molecule inhibitor. However, few studies have investigated whether BRD4 influenced acute promyelocytic leukemia (APL), and whether BRD4 had interaction with promyelocytic leukemia-retinoic acid receptor α (PML/RARα) fusion protein to some extent. Results from cell viability assay, cell cycle analysis, and Annexin-V/PI analysis indicated that JQ1 inhibited the growth of NB4 cells, an APL-derived cell line, and induced NB4 cell cycle arrest at G1 and apoptosis. Then, we used co-immunoprecipitation (co-IP) assay and immunoblot to demonstrate the endogenous interaction of BRD4 and PML/RARα in NB4 cells. Moreover, downregulation of PML/RARα at the mRNA and protein levels was observed upon JQ1 treatment. Furthermore, results from the RT-qPCR, ChIP-qPCR, and re-ChIP-qPCR assays showed that BRD4 and PML/RARα co-existed on the same regulatory regions of their target genes. Hence, we showed a new discovery of the interaction of BRD4 and PML/RARα, as well as the decline of PML/RARα expression, under JQ1 treatment.

Effect of JQ1 on expression of autoimmune-related genes in CD4+T cells of systemic lupus erythematosus / 中南大学学报(医学版)

To investigate the effect of bromodomain and extra-terminal (BET) protein inhibitor JQ1 on expression of autoimmune-related genes in CD4+T cells from patients with systemic lupus erythematosus (SLE).
 Methods: Peripheral CD4+T cells were isolated by positive selection with CD4 microbeads. The percentage of CD4+T cells were detected by flow cytometry. CD4+T cells were treated by JQ1 at 100 nm/L for 6, 24, 48 h. The expression of T cell-related genes was measured by quantitative real-time PCR (qPCR). The secretion levels of cytokines in culture supernatant were measured by ELISA at 48 h.
 Results: The percentage of CD4+T cells isolated by CD4 microbeads is 97.2%. Compared with the control group, the mRNA expression levels of IFNG, IL-17F, IL-21, CXCR5 and FOXP3 were down-regulated at 6, 24 and 48 h (P<0.05), and IL-17A mRNA level was decreased at 6 and 24 h (P<0.01); while IL-4 mRNA level was up-regulated at 24, 48 h (P<0.01), and TGF-β1 mRNA level was up-regulated at 6 and 48 h (P<0.05) in SLE CD4+T cells treated with JQ1. The secretion levels of IFN-γ and IL-21 in JQ1-treated group were decreased significantly (P<0.05), while the secretion levels of IL-4 and TGF-β were up-regulated compared with control group (P<0.05).
 Conclusion: JQ1 can reverse the immune dysregulation and improve the immunity homeostasis in CD4+T cells from patients with SLE.

Mechanism of action of BET bromodomain inhibitor JQ1 in treating airway remodeling in asthmatic mice / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of action of BET bromodomain inhibitor JQ1 in treating airway remodeling in asthmatic mice.</p><p><b>METHODS</b>A total of 24 mice were randomly divided into control group, ovalbumin (OVA)-induced asthma group (OVA group), and JQ1 intervention group (JQ1+OVA group), with 8 mice in each group. OVA sensitization/challenge was performed to establish a mouse model of asthma. At 1 hour before challenge, the mice in the JQ1+OVA group were given intraperitoneal injection of JQ1 solution (50 μg/g). Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 24 hours after the last challenge, and the total number of cells and percentage of eosinophils in BALF were calculated. Pathological staining was performed to observe histopathological changes in lung tissue. RT-PCR and Western blot were used to measure the mRNA and protein expression of E-cadherin and vimentin during epithelial-mesenchymal transition (EMT).</p><p><b>RESULTS</b>Compared with the control group, the OVA group had marked infiltration of inflammatory cells in the airway, thickening of the airway wall, increased secretion of mucus, and increases in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the OVA group, the JQ1+OVA group had significantly alleviated airway inflammatory response and significant reductions in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the control group, the OVA group had significant reductions in the mRNA and protein expression of E-cadherin and significant increases in the mRNA and protein expression of vimentin (P<0.01); compared with the OVA group, the JQ1+OVA group had significant increases in the mRNA and protein expression of E-cadherin and significant reductions in the mRNA and protein expression of vimentin (P<0.01); there were no significant differences in these indices between the JQ1+OVA group and the control group (P>0.05).</p><p><b>CONCLUSIONS</b>Mice with OVA-induced asthma have airway remodeling during EMT. BET bromodomain inhibitor JQ1 can reduce airway inflammation, inhibit EMT, and alleviate airway remodeling, which provides a new direction for the treatment of asthma.</p>

BRD4 inhibition suppresses cell growth, migration and invasion of salivary adenoid cystic carcinoma

BACKGROUND: Bromodomain-containing protein 4 (BRD4) inhibition is a new therapeutic strategy for many malignancies. In this study, we aimed to explore the effect of BRD4 inhibition by JQ1 on in vitro cell growth, migration and invasion of salivary adenoid cystic carcinoma (SACC). METHODS: The human normal epithelial cells and SACC cells (ACC-LM and ACC-83) were treated with JQ1 at concentrations of 0, 0.1, 0.5 or 1 µM. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Cell apoptosis and cell cycle distribution was evaluated by Flow cytometry. Immunofluorescence staining was used to examine the expression of BRD4 in SACC cells. The quantitative real-time polymerase chain reaction (qRT-PCR) assay and western blot assay were performed to examine messenger RNA (mRNA) and protein levels in SACC cells. Wound- healing assay and transwell assay were used to evaluate the activities of migration and invasion of SACC cells. RESULTS: JQ1 exhibits no adverse effects on proliferation, cell cycle and cell apoptosis of the normal human epithelial cells, while suppressed proliferation and cell cycle, and induced apoptosis of SACC cells, down-regulated the mRNA and protein levels of BRD4 in SACC cells, meanwhile reduced protein expressions of c-myc and BCL-2, two known target genes of BRD4. Moreover, JQ1 inhibited SACC cell migration and invasion by regulating key epithelial-mesenchymal transition (EMT) characteristics including E-cadherin, Vimentin and Twist. CONCLUSIONS: BRD4 is an important transcription factor in SACC and BRD4 inhibition by JQ1 may be a new strategy for SACC treatment.

Effect of BRD4 Inhibitor JQ1 on Proliferation Inhibition and Apotosis Induction in Jurkat Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect and possible mechanism of bromo-domain inhibitors (JQ1) on proliferation inhibition and inducing apoptosis of acute T lymphocyte leukemia cell line (Jurkat) .</p><p><b>METHODS</b>Jurkat cell line was treated by JQ1 at different concentrations. MTT was used to detect the cell proliferation inhibition rate. The flow cytometry with AnnexinV-FITC/PI fluorescence staining was used to detect the changes of apoptosis rate, and real-time fluorescent quantitative PCR was used to detect c-Myc/Notch1 gene expression levels.</p><p><b>RESULTS</b>With the increasing of drug concentration and prolonging of time, the inhibitory rate of Jurkat cell growth was enhanced in time-dose dependent manner; Jurkat cells was treated by 0.8, 1.6, and 4 µ mol/LJQ1 for 48 h and 72 h, the cell apoptosis rate was enhanced with the increase of drug concentration and prolonging of time, and the difference was statistically different in comparison with the control group(P<0.05); PCR detection indicated that Notch1 and c-Myc mRNA expression was reduced in 48 h after JQ1 treatment, which was statistically different from the control group,(P<0.05) .</p><p><b>CONCLUSION</b>JQ1 can effectively inhibit the growth of Jurkat cell line, and potentially induce apoptosis through Notch1 and c-Myc gene. Hence JQ1 may be one of new methods used to treat T-ALL.</p>

Comparison of clinical bracket point registration with 3D laser scanner and coordinate measuring machine

OBJECTIVE: The aim of the present study was to assess the diagnostic value of a laser scanner developed to determine the coordinates of clinical bracket points and to compare with the results of a coordinate measuring machine (CMM). METHODS: This diagnostic experimental study was conducted on maxillary and mandibular orthodontic study casts of 18 adults with normal Class I occlusion. First, the coordinates of the bracket points were measured on all casts by a CMM. Then, the three-dimensional coordinates (X, Y, Z) of the bracket points were measured on the same casts by a 3D laser scanner designed at Shahid Beheshti University, Tehran, Iran. The validity and reliability of each system were assessed by means of intraclass correlation coefficient (ICC) and Dahlberg's formula. RESULTS: The difference between the mean dimension and the actual value for the CMM was 0.0066 mm. (95% CI: 69.98340, 69.99140). The mean difference for the laser scanner was 0.107 ± 0.133 mm (95% CI: -0.002, 0.24). In each method, differences were not significant. The ICC comparing the two methods was 0.998 for the X coordinate, and 0.996 for the Y coordinate; the mean difference for coordinates recorded in the entire arch and for each tooth was 0.616 mm. CONCLUSION: The accuracy of clinical bracket point coordinates measured by the laser scanner was equal to that of CMM. The mean difference in measurements was within the range of operator errors. .

OBJETIVO: o objetivo do presente estudo foi avaliar o valor diagnóstico de um scanner a laser desenvolvido para determinar as coordenadas dos pontos de colagem de braquetes, comparando seus resultados aos resultados obtidos com uma máquina de medição coordenada (MMC). MÉTODOS: esse estudo experimental diagnóstico foi conduzido com modelos ortodônticos obtidos a partir da arcada superior de 18 pacientes adultos, com oclusão normal de Classe I. Inicialmente, as coordenadas dos pontos de colagem de braquetes de todos os modelos foram mensuradas por uma MMC. Em seguida, as coordenadas tridimensionais (X, Y, Z) dos pontos foram mensuradas nos mesmos modelos por um scanner a laser 3D, desenvolvido na Universidade de Shahid Beheshti. A eficácia e confiabilidade dos dois sistemas foram avaliadas pelo Coeficiente de Correlação Intraclasse (CCI) e pela fórmula de Dahlberg. RESULTADOS: a diferença entre a média da dimensão mensurada pela MMC e o valor real obtido foi de 0,0066mm (IC 95%: 69,98340 - 69,99140). A diferença média para o scanner a laser foi de 0,107 ± 0,133 (95% IC: -0,002 - 0,24). Em cada método, as diferenças não foram significativas. Ao comparar os dois métodos, o CCI gerou um valor de 0,998 para a coordenada X e de 0,996 para a coordenada Y. A diferença média para as coordenadas registradas em cada dente da arcada foi de 0,616mm. CONCLUSÃO: a precisão das coordenadas do ponto de colagem dos braquetes foi a mesma no scanner a laser e na MMC. A diferença média entre as medições manteve-se dentro dos limites de erros operacionais. .

Comparing the effects of small molecules BIX-01294, Bay K8644, RG-108 and valproic acid, and their different combinations on induction of pluripotency marker-genes by Oct4 in the mouse brain

Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid [VPA] are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction [PCR] was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell [NSC] markers. Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog [p<0.05]. Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc [p<0.001]. VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc [p<0.01], Pax6 and Sox1 [p<0.001]. These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4

Mechanism of Notch1 Pathway in SUP-B15 Cell Apoptosis Induced by JQ1 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>The study was aimed to investigate the possible mechanism of Notch1 pathway in apoptosis of Ph(+) human ALL Cells(SUP-B15 cells) induced by bromodomain inhibitors JQ1.</p><p><b>METHODS</b>The SUP-B15 cells were treated with different concentrations of JQ1 for different times. The cell proliferation was analyzed with cytotoxicity test(MTT method). Cell cycle was detected by fluorescence microscopy and flow cytometry. The mRNA expression of MIS2, Notch1, Hes1, BCR-ABL in Notch1 pathway was detected by real-time quantitative PCR.</p><p><b>RESULTS</b>JQ1 0-4 µmol/L could significantly inhibit the viability of SUP-B15 cells treated in does-and time-dependent manner. After SUP-B15 cells were treated with 1,2,4 µmol/L JQ1 for 48 h, the JQ1 could induce S cycle arrest in does-dependent manner which was statistical different from the control at the same time (P<0.05). MIS2, Notch1, Hes1, BCR-ABL mRNA expression was down-regulated by JQ1 which was statistical different from the control (P<0.05).</p><p><b>CONCLUSION</b>The JQ1 can effectively inhibit the growth and proliferation of SUP-B15 cells and the Notch1 pathway may be one of the important apoptosis mechanisms in Ph(+) ALL cells induced by JQ1.</p>

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Development and in vitro evaluation of estradiol transdermal film-forming spray / 药学学报

To develop estradiol transdermal film-forming spray (TFS), various polymers were screened using solvent appearance, spray ability, film-forming rate and appearance as indices. The influence of polymer type, plasticizer and penetration enhancer on the transdermal flux were investigated by selecting porcine skin as model, and transdermal flux of TFS was compared with commercial patch and gel. The drug existing state in the formed film was investigated by differential scanning calorimetry (DSC). The solvent appearances, spray abilities, film-forming rates and appearances of eudragit E PO, RL PO, hydroxypropyl cellulose EF, polyvinylpyrrolidone K30, Plasdone S630 and Agrimer VA64 were suitable for the preparation of TFS. TFS prepared by Eudragit RL PO had the biggest transdermal flux of estradiol among all the polymers investigated. Triethyl citrate, the plasticizer, decreased the transdermal flux. Azone increased the transdermal flux, while oleic acid, isopropyl myristate and menthol had opposite effects. TFS had a higher transdermal rate and a higher accumulative penetrated estradiol of 24 h than commercial patch and gel. The DSC result showed that estradiol was spread as molecule in the formed film of TFS. It was indicated that TFS could be expected to be an effective transdermal drug delivery system.

In vitro transdermal delivery of the active fraction of xiangfusiwu decoction based on principal component analysis / 药学学报

The objective of the present study was to establish a method based on principal component analysis (PCA) for the study of transdermal delivery of multiple components in Chinese medicine, and to choose the best penetration enhancers for the active fraction of Xiangfusiwu decoction (BW) with this method. Improved Franz diffusion cells with isolated rat abdomen skins were carried out to experiment on the transdermal delivery of six active components, including ferulic acid, paeoniflorin, albiflorin, protopine, tetrahydropalmatine and tetrahydrocolumbamine. The concentrations of these components were determined by LC-MS/MS, then the total factor scores of the concentrations at different times were calculated using PCA and were employed instead of the concentrations to compute the cumulative amounts and steady fluxes, the latter of which were considered as the indexes for optimizing penetration enhancers. The results showed that compared to the control group, the steady fluxes of the other groups increased significantly and furthermore, 4% azone with 1% propylene glycol manifested the best effect. The six components could penetrate through skin well under the action of penetration enhancers. The method established in this study has been proved to be suitable for the study of transdermal delivery of multiple components, and it provided a scientific basis for preparation research of Xiangfusiwu decoction and moreover, it could be a reference for Chinese medicine research.

Progress in the ligands and their complex structures of farnesoid X receptor / 药学学报

Farnesoid X receptor (FXR) belongs to the nuclear receptor superfamily. It is highly related to the formation of metabolic syndrome and the glucose homeostasis, and therefore represents an important drug target against metabolic diseases and diabetes. In recent years, great progress has been made in the agonists, antagonists, and crystal structures of FXR. The diverse FXR ligands and their structure-activity relationship are reviewed in this article. The advances in the crystal structures of FXR in complex with different ligands are also introduced.

Alzheimer's Disease: Clinical Trials and Future Perspectives / 대한정신약물학회지

Alzheimer's disease is increasingly common in elderly population with a large socioeconomic burden. Current available drugs for Alzheimer's disease are acetylcholinesterase inhibitors and N-methyl-D-aspartate receptor antagonist. Much effort is directed towards not just symptomatic treatments but disease-modifying treatments. Several drugs with differing targets and mechanisms of action are under development for the treatment of Alzheimer's disease. Phase III trials of dimebon, Ginkgo biloba, non-steroidal anti-inflammatory drugs, phenserine, statins, semagacestat, tarenflurbil, tramiprosate, valproate, xaliproden have been completed without demonstrating adequate efficacy. Encouraging results would be expected from ongoing phase III trials of bapineuzumab and solanezumab. The clinical trials for the disease-modifying treatment of Alzheimer's disease have resulted in both promise and disappointment.

Influence of penetration enhancers on in vitro transdermal permeation of L-tethrahydropalmatine / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of different penetration enhancers on the in vitro transdermal permeation of 1-tethrahydropalmatine (L-THP) through rat skin.</p><p><b>METHOD</b>Both natural and chemical synthesis penetration enhancers were applied singly or jointly to investigate the skin permeation rates of l-THP. The skin permeation profiles were evaluated by Valian-Chien permeation cells with isolated rat skin. HPLC-UV method was established to determine the concentration of l-THP in samples.</p><p><b>RESULT</b>As chemical synthesis penetration enhancer was used alone, 8% azone was observed to be the optimal penetration enhancer with the maximal penetration rate of 21.153 microg x cm(-20 x h(-1). Although 2% menthol crystal or 5% eucalyptus oil was effective as a natural penetration enhancer when used alone, the average penetration rate reached only half of that of 8% azone. The penetration potency of either menthol oil or menthol crystal combined with 8% azone was more effective than that of azone alone (P < 0.05).</p><p><b>CONCLUSION</b>Either menthol oil or menthol crystal combined with 8% azone is effective on transdermal penetration of l-THP in vitro. There is significant synergistic effect when natural penetration enhancers combined with chemical synthesis penetration enhancers.</p>

Corneal permeability assay of topical eye drop solutions in rabbits by MRI / 华中科技大学学报（医学）（英德文版）

This study examined the corneal permeability of topical eye drop solutions added with various corneal penetrating accelerators and gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) by nuclear magnetic resonance imaging (MRI). Twenty-four New Zealand rabbits were randomly divided into 3 groups according to the random digits table: Gd-DTPA group, in which the rabbits received 23.45% Gd-DTPA; hyaluronic acid group, in which 23.45% Gd-DTPA plus 0.2% hyaluronic acid was administered; azone group, in which 23.45% Gd-DTPA with 0.2% azone was given. Fifty microliters of the eye drops was instilled into the conjunctive sac every 5 min, for a total of 6 applications in each group. Contrast medium signals in the cornea, anterior chamber, posterior chamber, and vitreous body were scanned successively by MRI. The morphology and cell density of the corneal endothelium were examined before and 24 h after the treatment. The results showed that the residence time of Gd-DTPA in the conjunctival sac in the hyaluronic acid and azone groups was longer than that in the Gd-DTPA group. The signals in the anterior chamber of the Gd-DTPA and hyaluronic acid groups were increased slightly, and those in the azone group strengthened sharply. The signal intensity continuously rose over 80 min before reaching plateau. The strengthening rate of signals in the anterior chamber was 19.63% in the Gd-DTPA group, 53.42% in the sodium hyaluronate group, and 226.94% in the azone group. No signal was detected in the posterior chamber or vitreous body in all the 3 groups. Corneal morphology and cell density did not show any significant changes after the treatment in all the 3 groups. It was concluded that azone can significantly improve the corneal permeability of drugs that are similar to Gd-DTPA in molecular weight and molecular size, and MRI is a noninvasive technique that can dynamically detect eye drop metabolism in real time.

MLCK and PKC Involvements via Gi and Rho A Protein in Contraction by the Electrical Field Stimulation in Feline Esophageal Smooth Muscle

We have shown that myosin light chain kinase (MLCK) was required for the off-contraction in response to the electrical field stimulation (EFS) of feline esophageal smooth muscle. In this study, we investigated whether protein kinase C (PKC) may require the on-contraction in response to EFS using feline esophageal smooth muscle. The contractions were recorded using an isometric force transducer. On-contraction occurred in the presence of NG-nitro-L-arginine methyl ester (L-NAME), suggesting that nitric oxide acts as an inhibitory mediator in smooth muscle. The excitatory composition of both contractions was cholinergic dependent which was blocked by tetrodotoxin or atropine. The on-contraction was abolished in Ca2+-free buffer but reappeared in normal Ca2+-containing buffer indicating that the contraction was Ca2+ dependent. 4-aminopyridine (4-AP), voltage-dependent K+ channel blocker, significantly enhanced on-contraction. Aluminum fluoride (a G-protein activator) increased on-contraction. Pertussis toxin (a Gi inactivator) and C3 exoenzyme (a rhoA inactivator) significantly decreased on-contraction suggesting that Gi or rhoA protein may be related with Ca2+ and K+ channel. ML-9, a MLCK inhibitor, significantly inhibited on-contraction, and chelerythrine (PKC inhibitor) affected on the contraction. These results suggest that endogenous cholinergic contractions activated directly by low-frequency EFS may be mediated by Ca2+, and G proteins, such as Gi and rhoA, which resulted in the activation of MLCK, and PKC to produce the contraction in feline distal esophageal smooth muscle.

Calcium Sensitization Induced by Sodium Fluoride in Permeabilized Rat Mesenteric Arteries

It was hypothesized that NaF induces calcium sensitization in Ca2+-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with beta-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to Ca2+ (decreased EC50 and increased E(max)). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in Ca2+-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and GTPgammaS-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a Ca2+-dependent manner in beta-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.

Antifungal Activity of Securinine against Some Plant Pathogenic Fungi

The alkaloid securinine was assessed against spore germination of some plant pathogenic and saprophytic fungi (Alternaria alternata, Alternaria brassicae, Alternaria brassicicola, Curvularia lunata, Curvularia maculans, Curvularia pallenscens, Colletotrichum musae, Colletotrichum sp., Erysiphe pisi, Helminthosporium echinoclova, Helminthosporium spiciferum, Heterosporium sp.). Spore germinations of all the tested fungi were inhibited. Alternaria brassicicola, C. lunata, C. pallenscens and H. spiciferum were highly sensitive as complete inhibition of spore germination was observed at very low concentrations (200 ppm).