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1.
Int J Pharm ; : 119600, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32629070

RESUMO

A potent antipsychotic drug candidate, 7-(4-(4-(6-fluorobenzo[d]-isoxazol-3-yl)-piperidin-1-yl)butoxy)-4-methyl-8-chloro -2H-chromen-2-one mesylate(CY611), with good in vitro and in vivo antipsychotic effects was investigated for preformulation evaluation by crystallography methods. Three anhydrous polymorphs(Form I-III), a monohydrate(Form IV), and a NMP solvate(Form V) were discovered and characterized by powder X-ray diffraction, thermal analysis, attenuated total reflection-fourier transform infrared spectroscopy and scanning electron microscopy. Form I, monohydrate Form IV, and a NMP solvate Form V of the drug candidate were isolated, and their structures were determined by single crystal X-ray diffraction. IDR and relative stability experiment were performed. Although Form II has the fastest release rate in water, it easy transformed to monohydrate which has the lowest release rate. In vivo pharmacokinetic study showed that the Form III has the highest bioavailability at 35.4%. Considering the balance between the physicochemical properties, bioavailability and manufacturability of the available polymorphs, Form III may be the optimal form candidate for the eventual formulation.

2.
Cell Death Dis ; 11(5): 393, 2020 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-32447342

RESUMO

Prostate cancer (PC) is a prevalent male malignancy with high occurrence rate. Recent studies have showed that small nucleolar host genes (SNHGs) and their homolog small nucleolar RNAs (snoRNAs) elicit regulatory functions in carcinogenesis. Present study aimed to investigate the role of SNHG17 and its homolog SNORA71B in PC. Function of SNHG17 and SNORA71B in PC is detected by CCK-8, colony formation, flow cytometry analysis of apoptosis, and transwell migration assay. The mechanism whereby SNHG17 regulated SNORA71B was detected by RIP, pulldown, ChIP, and luciferase reporter assays. Results depicted that transcript 6 of SNHG17 and SNORA71B were upregulated in PC. Knockdown of SNHG17 or SNORA71B weakened proliferation, invasion, migration, and epithelial-to-mesenchymal transition (EMT) and strengthened apoptosis. Mechanistically, SNHG17 and SNORA71B were transcriptionally activated by signal transducer and activator of transcription 5A (STAT5A). SNHG17 positively regulated SNORA71B in PC cell lines and other cell lines. SNHG17 sponged miR-339-5p to upregulate STAT5A and therefore to cause transactivation of SNORA71B. Rescue experiments delineated that SNORA71B was required for the regulation of SNHG17 on PC. Moreover, SNHG17 silence hindered tumorigenesis of PC in vivo. In conclusion, current study first revealed that lncRNA SNHG17 aggravated prostate cancer progression through regulating its homolog SNORA71B via a positive feedback loop, which might do help to the pursuit of better PC treatment.

3.
J Pharm Sci ; 109(7): 2156-2165, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32240697

RESUMO

Different solid forms possess various physicochemical properties, which can significantly affect the stability, bioavailability, and manufacturability of the final product. DP-VPA, a complex of 1-stearoyl-2-valproyl-sn-glycero-3-phosphatidylcholine (DP-VPA-C18) and 1-palmitoyl-2-valproyl-sn-glycero-3-phosphatidylcholine (DP-VPA-C16), is currently under development as an antiepileptic drug. DP-VPA-C16 and DP-VPA-C18 crystallize together in solid solution forms. The solid forms of DP-VPA solid solution were studied herein. Powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), dynamic vapor sorption (DVS) and optical microscopy were used to characterize the different crystalline forms, known as polymorphs. The physicochemical properties, including hygroscopicity, thermodynamic behavior, and relative stability, of each form were investigated. DVS analysis showed that DP-VPA solid solution reduced the hygroscopicity of DP-VPA-C16. The relative humidity stability study revealed that Forms A and B are relatively stable, while Forms A-1, B-1, C and D are highly unstable under natural humidity. Further analysis revealed that Form A transforms into Form B through milling. Given the physicochemical properties of the available physical forms, Form B may be the optimal form for the formulation and development of antiepileptic drugs.

4.
Chin Med J (Engl) ; 133(7): 792-799, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-32149767

RESUMO

BACKGROUND: Regulatory policy (RP) is known as a major factor to improve health care system performance. A significant difference in maternal mortality rates (MMRs) was observed between New York city (NYC) and Shanghai (SH), both first-class international metropolises. This study aims to adopt a quantitative evaluation model to analyze whether RP differences contribute to the different MMRs of the two cities. METHODS: Based on collection of all publicly released policy documents regarding maternal health in the two cities, we assessed and compared the status of their maternal health care RPs from 2006 to 2017 through a series of quantitative indicators as regulatory elements coverage rate (RECR), departmental responsibility clarity rate (DRCR), and accountability mechanism clarity rate (AMCR), based on two characteristics of comprehensiveness and effectiveness of RPs. Pearson correlation analysis, principal component analysis, and linear regression analysis were used to test the relationships between the indicators and MMR in SH and NYC. RESULTS: By 2017, disparities of maternal health care RP are found between SH and NYC, from the indicators of RECR (100% vs. 77.0%), DRCR (38.9% vs. 45.1%), and AMCR (29.2% vs. 22.5%). From 2006 to 2017, RECR, DRCR, and AMCR in SH have shown a higher growth of 8.7%, 53.2%, and 45.2%, compared with growth of 25.0%, 12.5%, and 2.9% in NYC. The three indicators were found all negatively correlated with MMR in SH (Coefficients = -0.831, -0.833, and -0.909, and P < 0.01), while only RECR and DRCR had negative correlation with MMR in NYC (Coefficients = -0.736 and -0.683, and P < 0.05). Linear regression showed that the principal components of the three indicators were found with significant impact on MMRs both in SH (R = 0.914, R = 0.836, P < 0.001) and NYC (R = 0.854, R = 0.357, P = 0.04). CONCLUSION: Compared with NYC, the more comprehensive and effective maternal health care RPs in SH had a stronger impact on MMR control, which contributed to the differences between the two cities' MMRs to some extent. The methods and indicators we adopted for assessment are reasonable and comparable.

5.
J Burn Care Res ; 41(4): 892-899, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32112091

RESUMO

After transplantation, skin grafts contract to different degrees, thus affecting the appearance and function of the skin graft sites. The exact mechanism of contracture after skin grafting remains unclear, and reliable treatment measures are lacking; therefore, new treatment methods must be identified. Many types of centripetal contraction forces affect skin graft operation, thus leading to centripetal contracture. Therefore, antagonizing the centripetal contraction of skin grafts may be a feasible method to intervene in skin contracture. Here, the authors propose the first reported mechanical stretching method to address contracture after skin grafting. A full-thickness skin graft model was established on the backs of SD rats. The skin in the experimental group was stretched unilaterally or bidirectionally with a self-made elastic stretching device, whereas the skin was non-stretched in the control group. The rats were sacrificed 2 weeks after stretching. The area, length, and width of the skin were measured. The grafts were cut and fixed with formalin. Routine paraffin sections were stained with hematoxylin-eosin, picric acid-Sirius red, Victoria blue, and anti-alpha-smooth muscle actin (SMA). Mechanical stretching made the graft lengthen in the direction of the stress and had an important influence on collagen deposition and alpha-SMA expression in the graft. This method warrants further in-depth study to provide a basis for clinical application.

6.
Eur J Med Chem ; 191: 112144, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087465

RESUMO

We designed and synthesized a novel series of piperidine propionamide derivatives as potent sigma-1 (σ1) receptor antagonists and mu (µ) opioid receptor agonists, and measured their affinity for σ1 and µ receptors in vitro through binding assays. The basic scaffold of the new compounds contained a 4-substituted piperidine ring and N-aryl propionamide. Compound 44, N-(2-(4-(4-fluorobenzyl) piperidin-1-yl) ethyl)-N-(4-methoxy-phenyl) propionamide, showed the highest affinity for σ1 receptor (Ki σ1 = 1.86 nM) and µ receptor (Ki µ = 2.1 nM). It exhibited potent analgesic activity in the formalin test (ED50 = 15.1 ± 1.67 mg/kg) and had equivalent analgesic effects to S1RA (σ1 antagonist) in a CCI model. Therefore, Compound 44, which has mixed σ1/µ receptor profiles, may be a potential candidate for treating neuropathic pain.

7.
Int Immunopharmacol ; 80: 106189, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31931374

RESUMO

INTRODUCTION: Liver injury induced by burn plus delayed resuscitation (B + DR) is life threatening in clinical settings. Mitochondrial damage and oxidative stress may account for the liver injury. MitoQ is a mitochondria-targeted antioxidant. We aimed to evaluate whether MitoQ protects against B + DR-induced liver injury. METHODS: Rats were randomly divided into three groups: (1) the sham group; (2) the B + DR group, which was characterized by third-degree burn of 30% of the total body surface area plus delayed resuscitation, and (3) the treatment group, in which rats from the B + DR model received the target treatment. MitoQ was injected intraperitoneally (i.p) at 15 min before resuscitation and shortly after resuscitation. In the vitro experiments, Kupffer cells (KCs) were subjected to hypoxia/reoxygenation (H/R) injury to simulate the B + DR model. Mitochondrial characteristics, oxidative stress, liver function, KCs apoptosis and activation of the NLRP3 inflammasome in KCs were measured. RESULTS: B + DR caused liver injury and oxidative stress. Excessive ROS lead to liver injury by damaging mitochondrial integrity and activating the mitochondrial DNA (mtDNA)-NLRP3 axis in KCs. The oxidized mtDNA, which was released into the cytosol during KCs apoptosis, directly bound and activated the NLRP3 inflammasome. MitoQ protected against liver injury by scavenging intracellular and mitochondrial ROS, preserving mitochondrial integrity and function, reducing KCs apoptosis, inhibiting the release of mtDNA, and suppressing the mtDNA-NLRP3 axis in KCs. CONCLUSION: MitoQ protected against B + DR-induced liver injury by suppressing the mtDNA-NLRP3 axis.

8.
Cell Signal ; 56: 1-14, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30465826

RESUMO

As the most commonly occurring form of primary renal tumor, renal cell carcinoma (RCC) is a malignancy accompanied by a high mortality rate. 3-phosphoinositide-dependent protein kinase 1 (PDK1) has been established as a protein target and generated considerable interest in both the pharmaceutical and academia industry. The aim of the current study was to investigate the effect of si-PDK1 on the RCC cell apoptosis, proliferation, migration, invasion and epithelial mesenchymal transition (EMT) in connection with the PI3K-PDK1-Akt pathway. Microarray analysis from the GEO database was adopted to identify differentially expressed genes (DEGs) related to RCC, after which the positive expression of the PDK1 protein in tissue was determined accordingly. The optimal silencing si-RNA was subsequently selected and RCC cell lines 786-O and A498 were selected and transfected with either a si-PDK1 or activator of the PI3K-PDK1-Akt pathway for grouping purposes. The mRNA and protein expressions of PDK1, the PI3K-PDK1-Akt pathway-, EMT- and apoptosis-related genes were then evaluated. The effect of si-PDK1 on cell proliferation, apoptosis, invasion and migration was then analyzed. Through microarray analysis of GSE6344, GSE53757, GSE14762 and GSE781, PDK1 was examined. PDK1 was determined to be highly expressed in RCC tissues. Si-PDK1 exhibited marked reductions in relation to the mRNA and protein expression of PDK1, PI3K, AKT as well as Vimentin while elevated mRNA and protein expressions of E-cadherin were detected, which ultimately suggested that cell migration, proliferation and invasion had been inhibited coupled with enhanced levels of cell apoptosis. While a notable observation was made highlighting that the PI3K-PDK1-Akt pathway antagonized the effect of PDK1 silencing. Taken together, the key observations of this study provide evidence suggesting that high expressions of PDK1 are found in RCC, while highlighting that silencing PDK1 could inhibit RCC cell proliferation, migration, invasion and EMT by repressing the PI3K-PDK1-Akt pathway.


Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Carcinoma de Células Renais/patologia , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Renais/patologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/biossíntese , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Adulto , Idoso , Antígenos CD/metabolismo , Apoptose/genética , Caderinas/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Vimentina/metabolismo
9.
Nat Prod Res ; : 1-6, 2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30580623

RESUMO

A new indole alkaloid, 17-oxo-19-(Z)-naucline, and six known alkaloids 2-7 were isolated from the branches of Nauclea officinalis. The structure of the new compound 1 was characterised mainly by analysing its physical data including IR, 1 D, 2 D NMR, and HR-ESI-MS. Other compounds were identified by comparisons their data with those reported in the literature. Compound1, 4, 5, 6, 7 showed in vitro anti-inflammatory activity decrease the LPS-stimulated production of nitric oxide in RAW264.7 cell, while all compounds exhibited weak cytotoxicity against human tumour cell lines (LOVO, A549 and HepG2).

10.
J Agric Food Chem ; 65(18): 3702-3710, 2017 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-28436658

RESUMO

The essential oils (EOs) derived from aromatic plants such as Piper species are considered to play a role in alleviating neuronal ailments that are associated with inhibition of acetylcholinesterase (AChE). The chemical compositions of 23 EOs prepared from 16 Piper spp. were analyzed by both gas chromatography with a flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 76 compounds were identified in the EOs from the leaves and stems of 19 samples, while 30 compounds were detected in the EOs from the fruits of four samples. Sesquiterpenes and phenylpropanoids were found to be rich in these EOs, of which asaricin, caryophyllene, caryophyllene oxide, isospathulenol, (+)-spathulenol, and ß-bisabolene are the major constituents. The EOs from the leaves and stems of Piper austrosinense, P. puberulum, P. flaviflorum, P. betle, and P. hispidimervium showed strong AChE inhibitory activity with IC50 values in the range of 1.51 to 13.9 mg/mL. A thin-layer chromatography (TLC) bioautography assay was employed to identify active compound(s) in the most active EO from P. hispidimervium. The active compound was isolated and identified as asaricin, which gave an IC50 value of 0.44 ± 0.02 mg/mL against AChE, comparable to galantamine with an IC50 0.15 ± 0.01 mg/mL.


Assuntos
Acetilcolinesterase/química , Inibidores da Colinesterase/química , Proteínas de Peixes/antagonistas & inibidores , Óleos Voláteis/química , Piper/química , Extratos Vegetais/química , Animais , Cromatografia em Camada Delgada , Enguias , Proteínas de Peixes/química , Cromatografia Gasosa-Espectrometria de Massas , Folhas de Planta/química , Óleos Vegetais/química , Caules de Planta/química
11.
J Cell Sci ; 130(5): 1003-1015, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28082423

RESUMO

GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe, we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale.


Assuntos
Técnicas Citológicas/métodos , Proteínas de Fluorescência Verde/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Bases , Genes Reporter , Vetores Genéticos/metabolismo , Luciferases/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Corpos Polares do Fuso/metabolismo
12.
Int J Nanomedicine ; 11: 5971-5987, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27877042

RESUMO

Nanoemulsions (NEs) are used as transdermal drug delivery systems for systematic therapeutic purposes. We hypothesized that the skin permeation profile of an NE could be modulated by incorporating it into a hydrogel containing differing proportions of thickening agent. The objectives of this study were as follows: 1) to determine the stability and skin irritability of NE gels (NGs) containing 1%, 2%, and 3% (w/w) Carbopol® 934 (CP934) (termed NG1, NG2, and NG3, respectively); 2) to compare the skin permeation profiles and drug deposition patterns of the NGs; and 3) to visualize the drug delivery routes of the NGs. Terbinafine and citral were incorporated into the NGs as model drugs. Ex vivo skin permeation tests indicated that the percutaneous flux rates of terbinafine decreased in the order NE (215 µg/cm2) > NG1 (213 µg/cm2) > NG2 (123 µg/cm2) > NG3 (74.3 µg/cm2). The flux rates of citral decreased in the order NE (1,026 µg/cm2) > NG1 (1,021 µg/cm2) > NG2 (541 µg/cm2) > NG3 (353 µg/cm2). The NGs accumulated greater amounts of the drugs in the stratum corneum and less in the epidermis/dermis than did the NE (P<0.05) over a period of 12 h. Laser scanning confocal microscopy indicated that the NGs altered the main drug delivery routes from skin appendages to intercellular paths. Histological images suggested that perturbations to the skin structure, specifically the size of the epidermal intercellular spaces and the separation distance of dermal collagen bundles, could be significantly minimized by increasing the proportion of CP934. These results suggest that adjustments of the CP934 proportions can be used to modulate the skin permeation profiles of NGs for specific therapeutic purposes.


Assuntos
Acrilatos/química , Acrilatos/metabolismo , Nanomedicina , Pele/metabolismo , Administração Cutânea , Administração Tópica , Animais , Emulsões , Géis , Cobaias , Masculino , Permeabilidade , Pele/citologia , Absorção Cutânea
13.
Exp Ther Med ; 12(1): 451-456, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27347077

RESUMO

The present study investigated the H19 gene methylation status in male infertility. Between March 2013 and June 2014, semen samples were collected from 15 normal fertile males and 15 males experiencing infertility, and routine analysis and sperm morphological assessment were performed. The semen samples were subjected to density gradient centrifugation to separate the sperm fraction, and genomic DNA from the sperms was extracted and treated for bisulfite modification. Following in vitro amplification by polymerase chain reaction (PCR), the purified PCR products were cloned into pMD®18-T vectors and successful cloning was confirmed by restriction enzyme digestion. Positive clones were sequenced and the DNA methylation status was analyzed. The overall methylation rate in the normal fertile group was 100% (270/270), whereas in the infertile group the methylation rate was lower at 94.1% (525/558), revealing a statistically significant decrease in overall methylation rate in the infertile patients compared with the control group (χ2=15.12; P<0.001). The average methylation rates of CpG 1, 3 and 6 in the infertile group were statistically different from those in the normal control group (all P<0.05). The abnormal methylation of imprinted gene H19 is associated with male infertility, suggesting that H19 may serve as a biomarker for the detection of defects in human spermiogenesis.

14.
J Asian Nat Prod Res ; 18(8): 730-6, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26982483

RESUMO

Two new compounds, (Z,R)-1-phenylethylcinnamate (1) and (1R,2R,3R,6S)-pipoxide (2) were isolated from the aerial part of Piper hainanense, along with 12 known compounds, including nine benzene derivatives (4-11), one isobutylamide (12), and two polyoxygenated cyclohexene derivatives (13-14). Their structures were elucidated on the basis of the HRESIMS, 1D and 2D NMR spectroscopic analyses, and ECD in cases of 2 and 3. The absolute configuration of ellipeiopsol B (3) was determined for the first time. All these compounds 1-14 were reported from the titled plant for the first time. Most of the isolates were tested for their cytotoxicities against five human cancer cell lines. Four of which, 2, 3, 9, 14 showed moderate bioactivities. Among them, the new compound 2 showed potential cytotoxicity against SMMC-7721, MCF-7, and SW-480 with IC50 values of 9.7, 15.0, and 13.2 µM, respectively.


Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Cinamatos/isolamento & purificação , Cinamatos/farmacologia , Cicloexenos/isolamento & purificação , Cicloexenos/farmacologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Piper/química , Antineoplásicos Fitogênicos/química , Cinamatos/química , Cicloexenos/química , Ensaios de Seleção de Medicamentos Antitumorais , Medicamentos de Ervas Chinesas/química , Humanos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Triterpenos/química
15.
Nat Prod Res ; 30(9): 1068-74, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26539898

RESUMO

Two new triterpenoids (1-2) were isolated and elucidated from the roots of Gypsophila oldhamiana, together with four known triterpenoids (3-6). Their structures were identified to be 3ß-hydroxyolean-13(18)-ene-23, 28-dioic acid (1), 3ß, 12α-dihydroxy-23-carboxyolean-28, 13ß-olide (2), 3ß, 16α-dihydroxy-23-oxoolean-13(18)-en-28-oic acid (3), gypsogenin (4), quillaic acid (5) and gypsogenic acid (6) by spectral methods. All compounds were tested for their cytotoxicities against human tumour cell lines (lung cancer H460 and gastric cancer SGC-7901) and for their antiangiogenic effects using a zebra fish model. All compounds showed interesting antiangiogenic activities and the significant cytotoxicities against H460.


Assuntos
Caryophyllaceae/química , Triterpenos/análise , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Embrião não Mamífero , Humanos , Espectroscopia de Ressonância Magnética , Ácido Oleanólico/análogos & derivados , Extratos Vegetais/química , Raízes de Plantas/química , Espectrometria de Massas por Ionização por Electrospray , Peixe-Zebra
16.
Mol Microbiol ; 98(4): 770-86, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26256689

RESUMO

Members of Cdc14 phosphatases are common in animals and fungi, but absent in plants. Although its orthologs are conserved in plant pathogenic fungi, their functions during infection are not clear. In this study, we showed that the CDC14 ortholog is important for pathogenesis and morphogenesis in Fusarium graminearum. FgCDC14 is required for normal cell division and septum formation and FgCdc14 possesses phosphatase activity with specificity for a subset of Cdk-type phosphorylation sites. The Fgcdc14 mutant was reduced in growth, conidiation, and ascospore formation. It was defective in ascosporogenesis and pathogenesis. Septation in Fgcdc14 was reduced and hyphal compartments contained multiple nuclei, indicating defects in the coordination between nuclear division and cytokinesis. Interestingly, foot cells of mutant conidia often differentiated into conidiogenous cells, resulting in the production of inter-connected conidia. In the interphase, FgCdc14-GFP localized to the nucleus and spindle-pole-body. Taken together, our results indicate that Cdc14 phosphatase functions in cell division and septum formation in F. graminearum, likely by counteracting Cdk phosphorylation, and is required for plant infection.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Fusarium/patogenicidade , Regulação Fúngica da Expressão Gênica , Monoéster Fosfórico Hidrolases/metabolismo , Núcleo Celular/química , Citocinese , Proteínas Fúngicas/genética , Fusarium/crescimento & desenvolvimento , Deleção de Genes , Hifas/crescimento & desenvolvimento , Morfogênese , Mutação , Monoéster Fosfórico Hidrolases/genética , Doenças das Plantas/microbiologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Triticum/microbiologia
17.
Theor Biol Med Model ; 12: 14, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26246000

RESUMO

BACKGROUND: A single-chain bispecific antibody (scBsAb; an engineered antibody), has promising clinical applications. Nonetheless, the effect of different interchain linkers on its activity is poorly understood. METHODS: Gene synthesis was used to splice the anti-γ-seminoprotein single-chain antibody (anti-γ-Sm scFv) gene with the anti-CD3 single-chain antibody (anti-CD3 scFv) gene via different interchain peptide linkers. The Phyre2 software was used to predict spatial configuration of different scBsAbs. Eukaryotic expression vectors carrying scBsAbs were constructed by molecular cloning techniques and these plasmids were transfected into HeLa cells with liposomes. scBsAbs were purified by Ni(2+)-NTA agarose and analysed for antigen binding by an enzyme-linked immunosorbent assay (ELISA). Blood pharmacokinetics and inhibition of prostate tumour growth in nude mice were analysed in in vivo experiments. RESULTS: Bioinformatics analysis and prediction showed that none of the three linkers, Fc, 205C', and HSA, had a significant effect on protein folding of anti-γ-Sm scFv or anti-CD3 scFv. Nevertheless, the spatial structures of the three linkers were noticeably different. Anti-γ-Sm × anti-CD3 scBsAb with an Fc, 205C', or HSA linker was successfully constructed, and these antibodies had similar protein expression levels. ELISA showed that all the three scBsAbs bound to Jurkat cells and the LNCaP membrane antigen, although binding of (205C')scBsAb was weaker than that of the two parental scFvs (P < 0.05). In contrast, binding strength of (HSA)scBsAb and (Fc)scBsAb was close to that of the parental scFvs (P > 0.05). Pharmacokinetic analysis showed that the half-clearance time of the elimination phase (T(1/2ß)) for (HSA)scBsAb was the longest: up to 4.4 h. Compared with γ-Sm ScFv, the three scBsAbs all had a much stronger inhibitory effect on the growth of prostate cancer (P < 0.05), but there were no significant differences among the three scBsAbs (P > 0.05). CONCLUSIONS: HSA is the optimal linker for the anti-γ-Sm × anti-CD3 scBsAb and may improve antigen-binding affinity of antibodies and prolong physiological retention time. Interchain linkers affect the function of scBsAbs; these effects may have important implications for construction of antibodies.


Assuntos
Anticorpos Antineoplásicos/imunologia , Especificidade de Anticorpos , Antígeno Prostático Específico/imunologia , Animais , Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Antineoplásicos/metabolismo , Biologia Computacional , Engenharia Genética , Vetores Genéticos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Masculino , Camundongos , Camundongos Nus , Modelos Biológicos , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia
18.
Int J Clin Exp Pathol ; 8(4): 3987-93, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26097585

RESUMO

Epidemiological and histopathological studies have indicated that proliferative inflammatory atrophy (PIA) of the prostate is closely associated with the onset and development of prostate cancer (PCa). However, accurate isolation of PIA still remains a difficult matter, as well as high-quality RNA extraction from isolated PIA. These issues generated a lack of molecular evidence to support the mechanistic explanation proposed for the progression of PIA to PCa. Therefore, the isolation of PIA and the extraction of high-quality RNA from isolated PIA are of great importance to further demonstrate the correlation between PIA and the development of PCa at a molecular level. In this study, clinical samples from radical prostatectomy were stored in liquid nitrogen, PIA was identified by H&E staining of cryosections, PIA clusters were isolated by manual microdissection, total RNA was extracted from the PIA clusters by Trizol, and RNA quality was determined using the Agilent 2100 Bioanalyzer. Our results showed that PIA might be isolated by manual microdissection of cryosections stored in liquid nitrogen from clinical radical prostatectomy and used for extracting high-quality RNA (RIN > 7.5) by Trizol. Therefore, the present study established a valid method to discover molecular evidence in support of the correlation between PIA and the development of PCa.


Assuntos
Atrofia/patologia , Próstata/patologia , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/patologia , Prostatite/patologia , RNA/isolamento & purificação , Atrofia/genética , Atrofia/metabolismo , Progressão da Doença , Humanos , Masculino , Próstata/metabolismo , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Prostatite/genética , Prostatite/metabolismo
19.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 31(3): 328-32, 2015 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-25744837

RESUMO

OBJECTIVE: To establish 5-fluorouracil (5-FU)-resistant human colon cancer HT-29 cell line (HT-29/5-FU) in vitro and observe its biological properties. METHODS: The HT-29/5-FU cell line was established by continuously exposing the HT-29 cells to ascending doses of 5-FU. The morphology and colony formation rate were detected. The growth curve and the chemosensitivity of HT-29/5-FU cell line were determined by MTT assay. Western blotting was used to analyze the expressions of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD). Cell cycle and apoptosis were measured by flow cytometry. RESULTS: The resistance of HT-29/5-FU cells to 5-FU was 3.59-fold greater than that of HT-29 cells. The morphology of HT-29/5-FU cells differed from that of HT-29 cells. Compared with HT-29 cells, HT-29/5-FU cells showed remarkable reduction of cell proliferation and colony formation, higher expressions of TS and DPD, higher percentage of cells in the S phase, and stronger ability of resistance to apoptosis induced by 5-FU. CONCLUSION: The biological characters of HT-29/5-FU cell line may play an important role in 5-FU-resistant mechanisms, which may also contribute to elucidate the potential mechanisms of tumor drug resistance as well as resistance reversal.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Células HT29/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/fisiopatologia , Di-Hidrouracila Desidrogenase (NADP)/genética , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Células HT29/citologia , Células HT29/enzimologia , Humanos , Timidilato Sintase/genética , Timidilato Sintase/metabolismo
20.
Aust J Rural Health ; 2015 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-25819064

RESUMO

OBJECTIVE: To reveal the challenges of village doctors' survival and training in economically developed areas in eastern China. DESIGN: A field survey was used to assess the challenges of village doctors. SETTING: The study was conducted in Changzhou, Jiangsu province, which is an economically developed region in eastern China. PARTICIPANTS: The participants included 844 village doctors, 15 township hospital staff members and 6 health bureau leaders. RESULTS: The main challenges in Changzhou include an insufficient amount of village doctors, difficulties in obtaining professional qualification for village doctors, low salaries and benefits, and difficulties in recruitment. CONCLUSION: With increasing urbanisation in China, the gap between actual and expected income and social security has been increasing. Changes to training have influenced the stability of village doctor teams. Declining attachment of young people to their hometown village has contributed to recruitment difficulties.

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