Investigation of anti-Alzheimer's activity of aqueous extract of areca nuts (Areca catechu L): in vitro and in vivo studies / Investigación de la actividad anti-Alzheimer del extracto acuoso de nueces de areca (Areca catechu L): estudios in vitro e in vivo

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Sever cognitive and memory impairments, huge increase in the prevalence of the disease, and lacking definite cure have absorbed worldwide efforts to develop therapeutic approaches. Since many drugs have failed in the clinical trials due to multifactorial nature of AD, symptomatic treatments are still in the center attention and now, nootropic medicinal plants have been found as versatile ameliorators to reverse memory disorders. In this work, anti-Alzheimer's activity of aqueous extract of areca nuts (Areca catechu L.) was investigated via in vitro and in vivo studies. It depicted good amyloid ß (Aß) aggregation inhibitory activity, 82% at 100 µg/mL. In addition, it inhibited beta-secretase 1 (BACE1) with IC50 value of 19.03 µg/mL. Evaluation of neuroprotectivity of the aqueous extract of the plant against H2O2-induced cell death in PC12 neurons revealed 84.5% protection at 1 µg/mL. It should be noted that according to our results obtained from Morris Water Maze (MWM) test, the extract reversed scopolamine-induced memory deficit in rats at concentrations of 1.5 and 3 mg/kg.

La enfermedad de Alzheimer (EA) es un trastorno neurodegenerativo relacionado con la edad. Los severos deterioros cognitivos y de la memoria, el enorme aumento de la prevalencia de la enfermedad y la falta de una cura definitiva han absorbido los esfuerzos mundiales para desarrollar enfoques terapéuticos. Dado que muchos fármacos han fallado en los ensayos clínicos debido a la naturaleza multifactorial de la EA, los tratamientos sintomáticos siguen siendo el centro de atención y ahora, las plantas medicinales nootrópicas se han encontrado como mejoradores versátiles para revertir los trastornos de la memoria. En este trabajo, se investigó la actividad anti-Alzheimer del extracto acuoso de nueces de areca (Areca catechu L.) mediante estudios in vitro e in vivo. Representaba una buena actividad inhibidora de la agregación de amiloide ß (Aß), 82% a 100 µg/mL. Además, inhibió la beta-secretasa 1 (BACE1) con un valor de CI50 de 19,03 µg/mL. La evaluación de la neuroprotección del extracto acuoso de la planta contra la muerte celular inducida por H2O2 en neuronas PC12 reveló una protección del 84,5% a 1 µg/mL. Cabe señalar que, de acuerdo con nuestros resultados obtenidos de la prueba Morris Water Maze (MWM), el extracto revirtió el déficit de memoria inducido por escopolamina en ratas a concentraciones de 1,5 y 3 mg/kg.

Physalin B reduces Aβ secretion through down-regulation of BACE1 expression by activating FoxO1 and inhibiting STAT3 phosphorylation / 中国天然药物

Physalin B (PB), one of the major active steroidal constituents of Solanaceae Physalis plants, has a wide variety of biological activities. We found that PB significantly down-regulated β-amyloid (Aβ) secretion in N2a/APPsw cells. However, the underlying mechanisms are not well understood. In the current study, we investigated the changes in key enzymes involved in β-amyloid precursor protein (APP) metabolism and other APP metabolites by treating N2a/APPsw cells with PB at different concentrations. The results indicated that PB reduced Aβ secretion, which was caused by down-regulation of β-secretase (BACE1) expression, as indicated at both the protein and mRNA levels. Further research revealed that PB regulated BACE1 expression by inducing the activation of forkhead box O1 (FoxO1) and inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). In addition, the effect of PB on BACE1 expression and Aβ secretion was reversed by treatment with FoxO1 siRNA and STAT3 antagonist S3I-201. In conclusion, these data demonstrated that PB can effectively down-regulate the expression of BACE1 to reduce Aβsecretion by activating the expression of FoxO1 and inhibiting the phosphorylation of STAT3.

An integrative metabolomics and network pharmacology method for exploring bioactive components and preliminary pharmacodynamics in medicinal parts of Harrisonia perforata / 中国中药杂志

In this paper,metabolomics and network pharmacology were used to investigate the bioactive components of Harrisonia perforata and their possible mechanisms of action. Metabolites in the flowers,fruits,branches,leaves and stalks of H. perforata were analyzed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Meanwhile,multiple statistical analysis methods including principal component analysis( PCA) and orthogonal partial least squares discriminant analysis( OPLS-DA)were applied to screen and identify differential compounds. With metabolomics method,9 differential compounds were preliminarily identified from leaves and other non-traditional medicinal parts. Subsequently,these compounds were explored by using network pharmacology. With gastrointestinal absorption and drug-likeness as limiting conditions,they were imported into the Swiss ADME,from which 7 compounds with potential medicinal activity were obtained. Then,their targets were predicted by PharmMapper,with Human Protein Targets Only and Normalized Fit Score>0. 9 set as limiting conditions,and 60 standardized potential targets were identified with Uniprot. KEGG( Kyoto encyclopedia of genes and genomes) pathway data was obtained using metascape and the " potential active ingredients-target-pathway" network was constructed with Cytoscape 3. 7. 2. The enrichment analysis of KEGG demonstrated that the 60 targets were enriched in 78 signaling pathways( min overlap: 3,P value cutoff: 0. 01,min enrichment: 1. 5),many of which are related to anti-bacteria,anti-inflammation and anti-virus,such as IL-17 signaling pathway,RIG-I-like receptor signaling pathway and NOD-like receptor signaling pathway. Finally,depending on the clinical activity of H. perforata,the relevant signaling pathways were analyzed through experimental data and literature. Dehydroconiferyl alcohol was reported to have the anti-inflammatory effect and perforamone D to possess the antimycobacterial activity. The KEGG pathway enrichment analysis showed that dehydroconiferyl alcohol could act on the Alzheimer's disease( AD) signaling pathway by targeting CDK5 R1 and BACE1. ACh E inhibitor is the most promising drug to treat AD,while dehydroconiferyl alcohol has been proved to inhibit ACh E according to literature. The experimental results revealed that the extract of leaves of H. perforata can effectively inhibit the growth of Staphylococcus aureus. These are consistent with the enrichment analysis results of KEGG. This study explored the bioactive components and pharmacodynamics of the leaves of the H. perforata,laying a theoretical foundation for its in-depth development and rational application.

Expression and significance of CK5/6, P63, P40, CK7, TTF-1, NapsinA, CD56, Syn and CgA in biopsy specimen of squamous cell carcinoma, adenocarcinoma and small cell lung carcinoma / Expresión y significado de CK5/6, P63, P40, CK7, TTF-1, NapsinA, CD56 Syn y CgA en muestras de biopsia de carcinoma de células escamosas, adenocarcinoma y carcinoma de células pequeñas de pulmón

Nine tumor and various potential biomarkers were measured and combined the information to diagnose disease, all patients accepted fiber bronchoscopy brush liquid based cytologyand histopathology examination in order to reliably detect lung cancer. The samples from 314 Chinese lung cancer patients were obtained and CK5/6, P63, P40, CK7, TTF-1, NapsinA CD56, Syn and CgA were measured with the immunohistochemical SP method and analyzed correlation of the expression of these markers with pathological and clinical features of squamous cell carcinoma, adenocarcinoma, and small cell lung carcinoma. Squamous cell carcinoma, adenocarcinoma and small cell carcinoma were 61 cases, 114 cases and 139 cases,CK5/6 and P63 expression were more frequent in squamous cell carcinoma, with sensitivity and specificity of 77.05 % and 96.44 %, 83.61 % and 88.93 %,and compared with adenocarcinoma and small cell carcinoma difference was statistically significant (P<0.05), The incidences of a positive P40 expression were 100 % in squamous cell carcinoma, with specificity of 98.81 %.CK7, TTF-1 and NapsinA expression were more frequent in adenocarcinoma, with sensitivity and specificity of 85.09 % and 78.69 %, 79.82 % and 93.44 %, 56.14 % and 95.08 %, and compared with squamous cell carcinoma and small cell carcinoma difference was statistically significant (P<0.05). TTF-1, Syn, CgA and CD56 expression were more frequent in adenocarcinoma, with sensitivity and specificity of 86.33 % and 93.44 %, 89.21 % and 98.36 %, 74.10 % and 100 %, 96.40 % and 96.72 %. The combined detection of CK5/6, P63 and P40 were more useful and specific in differentiating squamous cell carcinoma. CK7, TTF-1 and NapsinA were more useful and specific in differentiating lung adenocarcinoma. The impaired CD56, TTF-1, Syn and CgA reflects the progression of small cell lung cancer.

Se midieron tumores y utilizaron nueve biomarcadores potenciales y se analizó la información para diagnosticar la enfermedad. A todos los pacientes se les realizó citología en líquido con broncoscopía de fibra y examen histopatológico para detectar de manera confiable el cáncer pulmonar. Se obtuvieron muestras de 314 pacientes chinos con cáncer de pulmón y CK5 / 6, P63, P40, CK7, TTF-1, Napsina A, CD56, Syn y CgA se midieron a través de histoquímica SP y analizaron la correlación de la expresión de estos marcadores con características patológicas y clínicas de carcinoma de células escamosas, adenocarcinoma y carcinoma de células pequeñas en el cáncer de pulmón. El carcinoma de células escamosas, el adenocarcinoma y el carcinoma de células pequeñas fueron 61 casos, 114 casos y 139 casos, respectivamente, la expresión de CK5 / 6 y P63 fueron más frecuentes en el carcinoma de células escamosas, con una sensibilidad y especificidad del 77,05 % y 96,44 %, 83,61 % y 88,93 %, y en comparación con el adenocarcinoma y el carcinoma de células pequeñas, la diferencia fue estadísticamente significativa (P <0,05). La incidencia de ap la expresión positiva P40 fue del 100 % en el carcinoma de células escamosas, con una especificidad del 98,81 %. La expresión de CK7, TTF-1 y NapsinA fueron más frecuentes en el adenocarcinoma, con una sensibilidad y especificidad del 85,09 % y 78,69 %, 79,82 % y 93,44 %, 56,14 % y 95,08 %, y en comparación con el carcinoma de células escamosas y la diferencia de carcinoma de células pequeñas fue estadísticamente significativa (P <0,05) .TTF-1, Syn, CgA y la expresión de CD56 fueron más frecuentes en adenocarcinoma, con sensibilidad y especificidad de 86.33 % y 93.44 %, 89.21 % y 98.36 %, 74.10 % y 100 %, 96.40 % y 96.72 %. La detección combinada de CK5 / 6, P63 y P40 fue más útil y específica en la diferenciación del carcinoma de células escamosas. CK7, TTF-1 y NapsinA fueron más útiles y específicos para diferenciar el adenocarcinoma de pulmón. El deterioro de CD56, TTF-1, Syn y CgA refleja la progresión del cáncer de pulmón de células pequeñas.

Partial Characterization of Two Cathepsin D Family Aspartic Peptidases of Clonorchis sinensis

Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel β-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.

Successful application of virtual screening and molecular dynamics simulations against antimalarial molecular targets

The main challenge in the control of malaria has been the emergence of drug-resistant parasites. The presence of drug-resistant Plasmodium sp. has raised the need for new antimalarial drugs. Molecular modelling techniques have been used as tools to develop new drugs. In this study, we employed virtual screening of a pyrazol derivative (Tx001) against four malaria targets: plasmepsin-IV, plasmepsin-II, falcipain-II, and PfATP6. The receiver operating characteristic curves and area under the curve (AUC) were established for each molecular target. The AUC values obtained for plasmepsin-IV, plasmepsin-II, and falcipain-II were 0.64, 0.92, and 0.94, respectively. All docking simulations were carried out using AutoDock Vina software. The ligand Tx001 exhibited a better interaction with PfATP6 than with the reference compound (-12.2 versus -6.8 Kcal/mol). The Tx001-PfATP6 complex was submitted to molecular dynamics simulations in vacuum implemented on an NAMD program. The ligand Tx001 docked at the same binding site as thapsigargin, which is a natural inhibitor of PfATP6. Compound TX001 was evaluated in vitro with a P. falciparum strain (W2) and a human cell line (WI-26VA4). Tx001 was discovered to be active against P. falciparum (IC50 = 8.2 µM) and inactive against WI-26VA4 (IC50 > 200 µM). Further ligand optimisation cycles generated new prospects for docking and biological assays.

Amyloid β Protein Aggravates Neuronal Senescence and Cognitive Deficits in 5XFAD Mouse Model of Alzheimer's Disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Amyloid β (Aβ) has been established as a key factor for the pathological changes in the brains of patients with Alzheimer's disease (AD), and cellular senescence is closely associated with aging and cognitive impairment. However, it remains blurred whether, in the AD brains, Aβ accelerates the neuronal senescence and whether this senescence, in turn, impairs the cognitive function. This study aimed to explore the expression of senescence-associated genes in the hippocampal tissue from young to aged 5XFAD mice and their age-matched wild type (WT) mice to determine whether senescent neurons are present in the transgenic AD mouse model.</p><p><b>METHODS</b>The 5XFAD mice and age-matched wild type mice, both raised from 1 to 18 months, were enrolled in the study. The senescence-associated genes in the hippocampus were analyzed and differentially expressed genes (DEGs) were screened by quantitative real-time polymerase chain reaction. Cognitive performance of the mice was evaluated by Y-maze and Morris water maze tests. Oligomeric Aβ (oAβ) (1-42) was applied to culture primary neurons to simulate the in vivo manifestation. Aging-related proteins were detected by Western blotting analysis and immunofluorescence.</p><p><b>RESULTS</b>In 5XFAD mice, of all the DEGs, the senescence-associated marker p16 was most significantly increased, even at the early age. It was mainly localized in neurons, with a marginal expression in astrocytes (labeled as glutamine synthetase), nil expression in activated microglia (labeled as Iba1), and negatively correlated with the spatial cognitive impairments of 5XFAD mice. oAβ (1-42) induced the production of senescence-related protein p16, but not p53 in vitro, which was in line with the in vivo manifestation.</p><p><b>CONCLUSIONS</b>oAβ-accelerated neuronal senescence may be associated with the cognitive impairment in 5XFAD mice. Senescence-associated marker p16 can serve as an indicator to estimate the cognitive prognosis for AD population.</p>

Diagnosis value of TTF-1, Napsin A and Ki-67 index in primary salivary gland-type lung carcinomas / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic characteristics of primary salivary gland-type lung carcinomas, and the immunophenotypic value of TTF-1, Napsin A and Ki-67 in their differential diagnosis.</p><p><b>METHODS</b>Totally 48 special type lung cancer surgical removal specimens were collected in China-Japan Friendship Hospital during September 2009 to December 2014. A panel of immunohistochemical markers (TTF-1, Napsin A, Ki-67, CK5/6, CK7 and p63) were conducted on these specimens.</p><p><b>RESULTS</b>The 48 cases of special type lung cancer included 25 cases of primary salivary gland-type lung carcinoma (18 cases of adenoid cystic carcinoma and 7 cases of mucoepidermoid carcinoma), 5 cases pulmonary adenocarcinoma with mucoepidermoid carcinoma-like or adenoid cystic carcinoma-like structure, and 18 cases of pulmonary adenosquamous carcinoma. Compared with pulmonary adenocarcinoma with mucoepidermoid carcinoma-like or adenoid cystic carcinoma-like structure and pulmonary adenosquamous carcinoma, primary salivary gland-type lung carcinomas have special characteristics in median age, sex, location, tumor size, LN involvement and pleura invasion, with negative TTF-1 and Napsin A expression as well as lower Ki-67 index detected by immunohistochemistry. Primary salivary gland-type lung carcinomas usually have an indolent behavior.</p><p><b>CONCLUSIONS</b>Primary salivary gland-type lung carcinomas are low-aggressive entities. The origins of primary salivary gland-type lung carcinomas were different from that of pulmonary adenocarcinoma with mucoepidermoid carcinoma-like or adenoid cystic carcinoma-like structure and pulmonary adenosquamous carcinoma. Negative TTF-1 and Napsin A expression as well as Ki-67 index lower than 20% have special value for primary salivary gland-type lung carcinomas in their differential diagnosis.</p>

inhibition of Candida albicans secreted aspartyl proteinase by triangular gold nanoparticles

The aim of this study was to synthesize triangular gold nanoparticles, and then to evaluate their capability for inhibition of Candida albicans secreted aspartyl proteinase 2 [Sap2]. To synthesize the nanoparticles, hydrogen tetrachloroaurate and hexadecyl trimethyl ammonium bromide were incubated in presence of Sn[IV] meso-tetra[N-methyl-4-pyridyl] porphine tetratosylate chloride, and then characterized. Next, thirty clinical isolates of Candida albicans were obtained from patients suffering from vaginal candidiasis. Each Candida albicans isolate was first cultured in YCB-BSA medium, incubated for 24 h at 35°C. Then, 100 microL of triangular gold nanoparticles at three concentrations [16, 32, and 64 microg/mL] were added to Candida suspension, and incubated for 24 and 48 h at 35°C. To evaluate Sap activity, 0.1 mL of medium and 0.4 mL of 0.1 M sodium citrate buffer [pH 3.2] containing BSA 1% w/v were added, and incubated 15 minutes at 37°C. Then, the optical density of each tube was read at 280 nm. Enzyme activity was expressed as the amount [microM] of tyrosine equivalents released per min per ml of culture supernatant. This study showed that the size of the nanoparticles was 70 +/- 50 nm. Sap activity evaluation demonstrated triangular gold nanoparticles could inhibit the enzyme, and the higher incubation time and concentration led to more decrease of Sap activity. For the first time, we demonstrated triangular gold nanoparticles as a novel inhibitor of Sap enzyme which may be useful for treatment of candidiasis

Haloperidol inhibits Memapsin 2: innovation by docking simulation and in vitro assay

A number of drugs exhibit unexpected pharmacological effects related to their ability to bind more than one receptor in humans. Haloperidol a typical antipsychotic drug appeared in several reports to be used in schizophrenia patients in which the significant of Alzheimer's disease has been reduced. The etiology of the disease is characterized by aggregates of amyloid plaques, largely composed of amyloid- beta peptide formed from the amyloid precursor protein cleaved by Memapsin 2. To investigate if haloperidol can bind to Memapsin 2 active site, an initial molecular docking was performed as a preliminary in-silico screening test followed by in vitro enzyme inhibition assay. Haloperidol was found to fit readily in Memapsin binding site with IC50value 250mM. Haloperidol can be considered as important lead or important target can be modified for more inhibitory activity, with the intention of protection or treatment for Alzheimer's disease

Diagnostic value of combined detection of HNF-1β and Napsin A in the diagnosis of ovarian clear cell carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the diagnostic value of HNF-1β and Napsin A for ovarian clear cell carcinomas, serous carcinomas, endometrioid adenocarcinomas and metastatic Krukenberg tumors.</p><p><b>METHODS</b>Immunohistochemical EnVision method was used to detect the expression of HNF-1β and Napsin A in 38 cases of ovarian clear cell carcinoma, 30 cases of high-grade serous carcinoma, 22 cases of endometrioid adenocarcinoma and 16 cases of metastatic Krukenberg tumor. Expression of HNF-1β and Napsin A were compared, and sensitivity and specificity of clear cell carcinoma of the ovary were analysed.</p><p><b>RESULTS</b>The positive rate of HNF-1β in the ovarian clear cell carcinoma was 100%(38/38), higher than those in high-grade serous carcinoma and endometrioid adenocarcinoma (P<0.05), although significant difference was not observed from that of metastatic Krukenberg tumor (P>0.05). Napsin A expressed in 97.4% (37/38) of ovarian clear cell carcinoma, 6.7% (2/30) of high-grade serous carcinoma, 22.7% (5/22) of endometrioid adenocarcinoma. Napsin A expression in clear cell carcinoma was higher than those in high-grade serous carcinoma and endometrioid adenocarcinoma (P<0.01), and no expression of Napsin A was seen in metastatic Krukenberg tumor (P>0.05). The sensitivity and specificity of HNF-1β in the diagnosis of ovarian clear cell carcinoma were 100% and 52.9%, those of Napsin A were 97.4% and 91.2%, those of both HNF-1β and Napsin A were 97.4% and 91.2%, respectively. The sensitivity and specificity of HNF-1β or Napsin A in the diagnosis of ovarian clear cell carcinoma were 100% and 52.9%, respectively.</p><p><b>CONCLUSIONS</b>HNF-1β is a more sensitive marker for the diagnosis of ovarian clear cell carcinoma, whereas Napsin A is a more specific marker. The combined detection of HNF-1β and Napsin A may be helpful for the diagnosis of clear cell carcinoma of the ovary.</p>

Combined detection of thyroid transcription factor 1 and napsin A in pleural fluid cell blocks facilitates cytopathologic diagnosis of pulmonary adenocarcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the value of detecting thyroid transcription factor 1 (TTF-1) and Noval aspartic proteinase of pepsin family A (napsin A) in pleural fluid cell blocks in cytopathologic diagnosis of pulmonary adenocarcinoma.</p><p><b>METHODS</b>Conventional cell smears of pleural effusions were obtained from 48 patients with a history of lung adenocarcinoma for cytological analysis. The cell blocks were prepared using the cytological specimens and examined with immunohistochemistry for TTF-1 and napsin A. The rates of a positive diagnosis of pulmonary adenocarcinoma were compared between the two methods, and the diagnositic value of TTF-1 and napsin A in pleural fluid cell blocks was evaluated.</p><p><b>RESULTS</b>Immuno- histochemistry of the cell block sections yielded a significantly higher positive rate of diagnosis than cytological analysis of conventional cell smear (84.44% vs 55.56%, P<0.05). Most of the pleural fluid cell blocks showed positive expressions of TTF-1 (36/38, 94.74%) and napsin A (30/38, 78.95%), and none of samples showed TTF-1 or napsin A expression in the mesothelial cells (P<0.05). The combination detection of TTF-1 and napsin A in pleural fluid cell blocks had a high diagnosis value with a diagnostic sensitivity of 97.37% and a specificity of 100% for pulmonary adenocarcinoma.</p><p><b>CONCLUSIONS</b>The combined detection of TTF-1 and napsin A in pleural fluid cell blocks facilitates cytopathologic diagnosis of pulmonary adenocarcinoma.</p>

effect of EFG1 gene silencing on down-regulation of SAP5 gene, by use of RNAi technology

Efg1 transcription factor is believed to be the main regulator of hyphal formation under many different conditions. In addition, it is responsible for positive regulation of the expression of several hyphalspecific genes. SAP5, which encodes secreted aspartic proteinase, is one of the mentioned genes and is crucial for pathogenicity properties. In the present work we have established the experimental conditions for the use of siRNA in the diploid yeast Candida albicans in order to knock-down the EFG1 gene expression as well as the Efg1-dependent gene, SAP5. The 19-nucleotide siRNA was designed according to cDNA sequence of EFG1 gene in C. albicans and modified-PEG/LiAc method was applied for yeast transfection. To quantify the level of both EFG1 and SAP5 gene expression, the cognate mRNAs were measured in C. albicans by quantitative real-time RT-PCR and data was consequently analyzed by use of REST[registered mark] software. Images taken by fluorescent microscopy method indicated the effectiveness of transfection. According to REST[registered mark] software data analysis, expression of EFG1 gene decreased about 2.5-fold using 500 nM of siRNA. A 7-fold decrease in EFG1 gene expression was observed when applying 1 micro M of siRNA [P<0.05]. Consequently, the expression of SAP5 was significantly down-regulated both in yeast treated with 500 and 1000 nM of siRNA [P<0.05]. In conclusion, post-transcriptional gene silencing [PTGS] is likely to be considered as a promising approach to discover new gene targets so as to design fungal-specific antifungal agents, and it is strongly possible that we are taking the right way to battle with C. albicans-associated infections

Immunoprotection by Sap2 and dendritic cells against systemic Candida albicans infection in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of combined immunization with recombinant Sap2 and dendritic cells (DCs) against systemic Canddida albicans infection.</p><p><b>METHODS</b>We constructed a prokaryotic expression vector carrying Sap2 of Candida albicans to obtain Sap2 protein. Murine DCs were sensitized by pulsing with Candida albicans spores and rope. Five groups of mice were immunized with recombinant Sap2 protein and sensitized DCs, sensitized DCs, naive DCs, Sap2, or PBS alone for 3 times, and the effect of the immunization against systemic Candida albicans infection were assessed by observing the survival of the mice, detecting the percentages of CD4⁺ and CD8⁺ cells, CFU in the kidney homogenate, and examining renal pathologies.</p><p><b>RESULTS</b>Immunization with Sap2 and sensitized DCs and with DCs or Sap2 alone all prolonged the mouse survival and produced obvious effect in renal protection and immune enhancement, but such effects were more obvious with the combined immunization.</p><p><b>CONCLUSION</b>Combined immunization with Sap2 protein and DCs offers strong immunoprotection against systemic Candida albicans infection in mice, which provides experimental evidence for the development of new combined vaccines for immunoprotection.</p>

Effect of exogenous hydrogen sulfide on BACE-1 enzyme expression and β-amyloid peptide metabolism in high-glucose primary neuronal culture / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of exogenous hydrogen sulfide (H2S) on β-site APP cleaving enzyme 1 (BACE-1) and β-amyloid peptide (Aβ) metabolism in primary culture of neurons under high-glucose condition.</p><p><b>METHODS</b>The cortical neurons in primary culture under normal and high glucose (60 mmol/L) conditions for 24 h were exposed to 25, 50 and 100 µmol/L NaHS. Aβ1-42 concentration in the cell culture was measured by ELISA, and BACE-1 mRNA and protein levels were detected by fluorescent quantitative real-time PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Compared with the neurons cultured in normal glucose, the neurons exposed to high glucose showed significantly increased Aβ1-42 concentration and BACE-1 mRNA and protein expressions (P<0.05). Exposure to 25, 50 and 100 µmol/L NaHS significantly decreased Aβ1-42 concentration and BACE-1 mRNA and protein expressions in the high-glucose cell culture (P<0.05).</p><p><b>CONCLUSION</b>Neurons exposed to high glucose exhibit increased Aβ1-42 levels and BACE-1 mRNA and protein expressions, which can be concentration-dependently decreased by NaHS.</p>

The role of Toll-like receptor 4 on inflammation and Aβ formation in cortex astrocytes / 生理学报

To investigate the role and possible molecular mechanism of astrocytes in inflammation and amyloid β-protein (Aβ) formation, in this research, by using LPS to stimulate cultured rat astrocytes in vitro with or without anti-Toll-like receptor 4 (TLR4) antibody pretreatment, we first detected the TLR4, TNF-α, IL-1β, β-amyloid precursor protein (β-APP) and β-site APP clearing enzyme 1 (BACE1) mRNA with real-time PCR, and TLR4, NF-κB/P65 protein in cultured astrocytes by Western blot, and then further probed the translocation of NF-κB/P65 using immunofluorescence and the contents of TNF-α, IL-1β and Aβ in culture supernatant through ELISA. We found that all of these indexes increased at different degrees after LPS-stimulation. However, if pretreatment with anti- TLR4 antibody, such stimulating effects of LPS on the nuclear translocation of NF-κB/P65 and TNF-α, IL-1β, Aβ contents in astrocytic culture supernatant were reduced significantly or disappeared in comparison with the group with only LPS-administration. Our results suggest that TLR4 in astrocytes might play an important role in the inflammation and Aβ formation through the TLR4/NF-κB signaling pathway, thus providing new knowledge and understanding of the inflammatory hypothesis of AD pathogenesis.

Effect of PNS on the activity and content of BACE1 in the brain of SAMP8 mice with Alzheimer's disease / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the effect of Panax notoginseng saponin (PNS) on the activity and content of beta-secretase in the brain of senescence accelerated mouse-prone 8 (SAMP8) mice with Alzheimer's disease.</p><p><b>METHODS</b>Totally 32 SAMP8 mice were randomly divided into the normal control group, the high dose PNS group (200 mg/kg), the low dose group (100 mg/kg), and the huperzine A group (0.3 mg/kg), 8 in each group. Equal volume of double distilled water was given to those in the normal control group. All medication was given by gastrogavage, once daily for two successive months. The activity of BACE1 was assayed by direct immunofluorescent method (DIF). The content of BACE1 protein was detected by Western blot.</p><p><b>RESULTS</b>The relative fluorescence units (RFU/microg) was 2.008 +/- 0.031 in the high dose PNS group, 2.221 +/- 0.029 in the low dose PNS group, and 2.267 +/- 0.076 in the huperzine A group, all lower than that in the normal control group (2.403 +/- 0.058; all P < 0.01). The content of BACE1 protein was 0.900 +/- 0.028 in the high dose PNS group, 1.000 +/- 0.032 in the low dose PNS group, and 0.837 +/- 0.080 in the huperzine A group, all lower than that in the normal control group (2.210 +/- 0.074, all P < 0.01).</p><p><b>CONCLUSION</b>PNS higher than 100 mg/kg could decrease the activity of BACE1 and down-regulate the content of BACE1 protein in the brain of SAMP8 mice.</p>

Effects of huannao yicong recipe extract on the learning and memory and related factors of Abeta generation in the brain of APP transgenic mice / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effects of Huannao Yicong Recipe (HNYCR)extract on the learning and memory ability, and the expressions of amyloid precursor protein (APP), beta-site APP-cleaving enzyme 1 (BACE1), presenilin-1 (PS-1), and beta amyloid protein (Abeta)in hippocampus CA1 area of APP transgenic mice, and to explore its mechanisms for treating Alzheimer's disease (AD).</p><p><b>METHODS</b>Totally 3-month-old APP695V7171 transgenic mice were used to establish the AD model in this research. They were randomly divided into the model group, the Donepezil group, the large dose HNYCR extract group, the small dose HNYCR extract group, and the normal control group (C57BL/6J mice), 15 in each group. These animals were gavaged for 4 continuous months. Relevant indicators were detected: Morris water maze test was used to measure the spatial learning and memory ability. The immunohistochemical assay was used to detect the expressions of APP, BACE1, PS-1, and Abeta.</p><p><b>RESULTS</b>The times of crossing the original platform and the swimming time and distance in the fourth quadrant of the 7-month-old APP transgenic mice were significantly reduced in Morris water maze test, when compared with the normal control group (P < 0.01). The times of crossing original platform and the swimming time and distance in the fourth quadrant of all treatment groups significantly increased in Morris water maze test, when compared with the model group (P < 0.05). The expressions of APP, BACE1, PS-1, and Abeta in hippocampus CA1 area of 7-month-old model mice increased significantly (P < 0.01), when compared with the normal control group. The expressions of APP, BACE1, PS-1, and Abeta in each 7-month-old intervention groups were significantly reduced, when compared with the model group (P < 0.01).</p><p><b>CONCLUSION</b>Early application of HNYCR extract can obviously improve the learning and memory ability of APP transgenic mice that has declined, reduce the expressions of APP, BACE1, PS-1, and Abeta in the hippocampal CA1 area, reduce the production of Abeta, and slow down the pathological process of brains in APP transgenic mice.</p>

Fragment-based binding efficiency indices in bioactive molecular design: a computational approach to BACE-1 inhibitors

One of the most important targets in Alzheimer disease is Beta site amyloid precursor protein cleaving enzyme-1 [BACE-1]. It is a membrane associated protein and is one of the main enzymes responsible for amyloid beta [A beta] production. Up to now, a considerable number of peptidic and non-peptidic inhibitors of BACE-1 have been developed. Recently, small molecule BACE-1 inhibitors have attracted the attention of scientists, because peptidic inhibitors have many pharmacokinetic problems. In the present study, several small molecule BACE-1 inhibitors were extracted from Brookhaven Protein Databank [PDB] and subjected to dissection analysis to achieve constructing fragments. Atom type, hybridization, and bond order were considered for generated constitutional fragments [simplified structures]. AutoDock version 4.2 was applied to dock various chemical fragments into BACE-1 active site. The benefits of such studies have been well revealed in previous reports. On the basis of obtained binding affinities, fragment-based ligand efficiency [LE] indices were estimated. These theoretical binding efficiencies were applied to further elucidate the key structural features of BACE-1 inhibitors. Typical results of the study were elucidated and we suggested the ways these findings might be beneficial to guide rational bioactive molecular developments. Our study confirmed that the evaluation of ligand-receptor interactions in terms of ligand efficiency indices [binding energy per atom and pK[i] per MW] could be a helpful strategy in structure-based drug discovery [SBDD] strategies

Identification of sulfamoylbenzamide derivatives as selective cathepsin D inhibitors

Aspartic proteases play very important role in post translational processing of proteins and several of them are essential for organism's viability. Here we present the enzyme inhibition activities of different Sulfamoylbenzamide derivatives against two aspartic proteases cathepsin D and plasmepsin II. Cathepsin D is an aspartic protease that degrades proteins at acidic pH in the lysosomes, or extracellular matrix. It is overexpressed by epithelial breast cancer cells and hence hyper-secreted. On the other hand plasmepsin II is an essential enzyme of Plasmodium falciperum. Cathepsin D and Plasmepsin II are pivotal drug targets for treatment of breast cancer and malaria respectively. Virtual screening of Sulfamoylbenzamide compounds followed by enzyme inhibition assays revealed these compounds as selective Cathepsin D inhibitors while inactive against Plasmepsin-II. IC[50] values of five Sulfamoylbenzamide compounds tested are in range of 1.25-2.0 microM. N-[3-chlorophenyl]-2-sulfamoylbenzamide is identified as the most potent of all tested Sulfamoylbenzamide compounds with IC[50] 1.25 microM. It was also noted that the docking score of theses compounds was better in case of Cathepsin D as compared to Plasmepsin-II. Docking score ranges from -29.9 +/- 1.16 to -35.1 +/- 0.13 in case of Cathepsin D, while from -24.0 +/- 0.10 to -29.5 +/- 0.10 in case of Plasmepsin-II