Hyperforin and its analogues inhibit CYP3A4 enzyme activity

Literature indicates that herb-drug interaction of St. John's wort is largely due to increased metabolism of the co-administered drugs that are the substrates of cytochrome P450 (CYP) 3A4 enzyme, alteration of the activity and/or expression of the enzyme. The major St. John's wort constituents, acylphloroglucinols, were evaluated for their effects on CYP3A4 enzyme activity to investigate their roles in herb-drug interaction. Hyperforin and four oxidized analogues were isolated from the plant and fully characterized by mass spectral and NMR analysis. These acylphloroglucinols inhibited activity of CYP3A4 enzyme potently in the fluorometric assay using the recombinant enzyme. Furoadhyperforin (IC(50) 0.072 microM) was found to be the most potent inhibitor of CYP3A4 enzyme activity, followed by furohyperforin isomer 1 (IC(50) 0.079 microM), furohyperforin isomer 2 (IC(50) 0.23 microM), hyperforin (IC(50) 0.63 microM) and furohyperforin (IC(50) 1.3 microM). As the acylphloroglucinols are potent inhibitors of the CYP3A4 enzyme, their modulation of the enzyme activity is unlikely to be involved in increased drug metabolism by St. John's wort.     

Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae

Polyphosphates of different chain lengths (P₃, P₄, P₁₅, P₃₅), (1 μM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p₄A and P₄ (1 μM) was 40 and 60%, respectively, whereas 1 μM Ap₄A was not inhibitory. P₄ and P₁₅ were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA. In (a), P₄ and P₁₅ behaved as competitive inhibitors, with K_i values of 0.5 μM and 0.2 μM, respectively. In addition, P₄ (at 1 μM) and P₁₅ (at 0.3 μM) changed the Hill coefficient (n_H) from 1 (control) to about 1.3 and 1.6, respectively. In (b), the inhibition by P₄ and P₁₅ decreased V and modified only slightly the K_m values of the enzyme towards tRNA.     

A DNA vaccine against cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) prevents tumor growth

Co-stimulatory signaling pathway triggered by the binding of B7.1/B7.2 (CD80/86) of antigen-presenting cells (APCs) to CD28 of T cells is required for optimal T-cell activation. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding with a greater affinity. Ipilimumab, a monoclonal antibody against CTLA-4, has shown positive efficacy in a pivotal clinical trial for the treatment of metastatic melanoma and was approved by FDA. However, the cost of monoclonal antibody-based therapeutics might limit the number of patients treated. To develop a novel therapeutics specifically targeting CTLA-4, we constructed a DNA vaccine by cloning the sequence of CTLA-4 fused with a transmembrane domain sequence of placental alkaline phosphatase (PLAP) into a mammalian expression plasmid, pVAC-1. Immunization with the resulting construct, pVAC-1-hCTLA-4, elicited antibody specific to human CTLA-4 with cross reactivity to murine CTLA-4, which was sufficient for inhibiting B16F10 tumor growth in c57BL/6 mice in the absence of measurable toxicity. Coupling liposome with pVAC-1-mCTLA-4 could break tolerance to self-antigen in BALB/c mice and induce potent immunity against murine CTLA-4, and suppress growth of subcutaneous renal cell carcinoma (Renca).     

The SKP1-Cul1-F-box and leucine-rich repeat protein 4 (SCF-FbxL4) ubiquitin ligase regulates lysine demethylase 4A (KDM4A)/Jumonji domain-containing 2A (JMJD2A) protein

Chromatin-modifying enzymes play a fundamental role in regulating chromatin structure so that DNA replication is spatially and temporally coordinated. For example, the lysine demethylase 4A/Jumonji domain-containing 2A (KDM4A/JMJD2A) is tightly regulated during the cell cycle. Overexpression of JMJD2A leads to altered replication timing and faster S phase progression. In this study, we demonstrate that degradation of JMJD2A is regulated by the proteasome. JMJD2A turnover is coordinated through the SKP1-Cul1-F-box ubiquitin ligase complex that contains cullin 1 and the F-box and leucine-rich repeat protein 4 (FbxL4). This complex interacted with JMJD2A. Ubiquitin overexpression restored turnover and blocked the JMJD2A-dependent faster S phase progression in a cullin 1-dependent manner. Furthermore, increased ubiquitin levels decreased JMJD2A occupancy and BrdU incorporation at target sites. This study highlights a finely tuned mechanism for regulating histone demethylase levels and emphasizes the need to tightly regulate chromatin modifiers so that the cell cycle occurs properly.     

Clonal overgrowth of esophageal smooth muscle cells in diffuse leiomyomatosis-Alport syndrome caused by partial deletion in COL4A5 and COL4A6 genes

This is a study of a patient who manifests all of the features of a diffuse leiomyomatosis-Alport syndrome (DL-ATS), and her two-year-old son who has already been diagnosed with Alport syndrome. Fourteen years ago, the patient underwent a partial esophageal resection followed by a replacement with jejunum. Recently, she underwent a surgical resection of the esophagus due to esophageal dysfunction. Genetic analyses of COL4A5 and COL4A6 on the X-chromosome were efficiently performed using the genomic DNA of her son. We have identified a novel deletion of 194-kb in length, encompassing COL4A5-COL4A6 promoters as well as nearly the entire large intron 1 of COL4A5 and intron 2 of COL4A6. To uncover the relationship of the esophagus-specific occurrence of the tumor and the expression of those genes, immunohistochemical analyses of type IV collagen α chains were conducted in the non-affected individuals. The esophageal smooth muscle-specific expression of α5(IV) and α6(IV) chains in the gastrointestinal tract was observed. Moreover, CAG repeat analysis of the androgen receptor gene and an immunohistochemical analysis in the leiomyoma revealed clonal overgrowth of the cells which received X-inactivation on the non-affected allele. These results may suggest that the dominant effect was caused by the partial deletion of the esophageal smooth muscle-specific genes, COL4A5 and COL4A6.     

Peppermint (Mentha piperita L.) extract effectively inhibits cytochrome P450 3A4 (CYP3A4) mRNA induction in rifampicin-treated HepG2 cells

Interaction between foods and drugs is an important consideration in pharmaceutical therapy. Therefore, here, we examined the suppressive effects of the extracts from seven edible herbs on the induction of CYP3A4 gene expression in rifampicin-treated HepG2 cells. We evaluated the structure and suppressive activity of the most effective active compound isolated from dried peppermint (Mentha piperita L.). The structure of the compound was identified as that of pheophorbide a based on spectroscopic data. It suppressed the induction of CYP3A4 mRNA expression by rifampicin in a dose-dependent manner. Quantitative high-performance liquid chromatography showed that 2 g of dry leaves 0.43 mg in one cup of peppermint tea. These findings demonstrate that pheophorbide a suppresses the induction of CYP3A4 mRNA expression in rifampicin-treated HepG2 cells. Pheophorbide is known to cause photosensitivity. However, the effective dose of pheophorbide a that had a suppressive effect was very low, indicating a high safety margin.

Abbreviations: DAD: diode array detector; DMEM: Dulbecco’s modified Eagle's medium; ELISA: enzyme-linked immunosorbent assay; HPLC: high-performance liquid chromatography; PCR: polymerase chain reaction; PXR: pregnane X receptor; CAR: constitutive androstane receptor; AHR: aryl hydrocarbon receptor; TLC: thin-layer chromatography     

4E-BP1、4E-BP1-4A 重组慢病毒载体的构建及对胃癌细胞 HGC27生长的影响

目的:构建4E-BP1及其 T37A、T46A、S65A、T70A 突变体4E-BP1-4A 基表达的重组慢病毒载体,研究其对胃癌 HGC27细胞生长的影响.方法:PCR 扩增4E-BP1基及其突变体4E-BP1-4A 基并克隆到 pCDH 载体,构建成 pCDH-4E-BP1、pCDH-4E-BP1-4A,将其与包装载体共转染293T 细胞,包装成 Lenti-4E-BP1及 Lenti-4E-BP1-4A重组慢病毒载体,将此慢病毒感染胃癌 HGC27细胞,Western 印迹鉴定病毒载体介导的4E-BP1、4E-BP1-4A 蛋白的表达,MTT、克隆形成和软琼脂方法研究过量表达4E-BP1、4E-BP1-4A 对胃癌 HGC27细胞生长的影响.结果:包装成 Lenti-4E-BP1及 Lenti-4E-BP1-4A 重组慢病毒载体,并将此慢病毒载体感染胃癌 HGC27细胞;MTT、克隆形成、软琼脂实验表明过量表达4E-BP1可抑制胃癌 HGC27细胞的生长,过量表达4E-BP1-4A 时抑制效果更明显.结论:构建了4E-BP1、4E-BP1-4A 的重组慢病毒表达载体,在胃癌 HGC27细胞中过量表达4E-BP1可抑制细胞生长,过量表达4E-BP1-4A 的抑制效果更明显.     

The porcine proteasome subunit A4 (PSMA4) gene: isolation of a partial cDNA, linkage and physical mapping

A partial cDNA clone encoding the porcine proteasome subunit A4 ( PSMA4 or proteasome subunit C9) has been isolated from a porcine muscle cDNA library and sequenced. A biallelic Taq I RFLP was identified in Large White, Landrace and Duroc breeds. Moreover, the 3'-untranslated region of the gene showed a triallelic SSCP. By linkage analysis the PSMA4 locus was assigned to pig chromosome 7 and by radioactive in situ hybridization this locus was mapped to the region 7q13–q14.     

Activin A induces neuronal differentiation and survival via ALK4 in a SMAD-independent manner in a subpopulation of human neuroblastomas

Activin A is a multifunctional homo-dimeric protein that belongs to the transforming growth factor (TGF)-β superfamily. In neurons, activin has neuroprotective effects both in vitro and in vivo, but it inhibits neuronal differentiation in some cell lines. Here we report that activin A can promote neuronal differentiation in particular cases. We examined activin A-induced neuronal differentiation and survival in a selected subpopulation of a human neuroblastoma cell line, SK-N-SH, grown in low-serum (differentiation-inducing) conditions. Activin A caused dramatic neurite outgrowth, and increased the expression of neuronal markers and the transactivation of dopamine β-hydroxylase. We demonstrated that the activin A signal is transduced through the activin A type 1 receptor, ALK4, and transactivates several TGF-β target genes in a SMAD-independent manner. That is, activin A did not induce the phosphorylation of SMAD2/3, the interaction of SMAD2/3 with SMAD4, the binding of SMAD2/3 to the promoter of TGF-β target genes, or the accumulation of SMAD2/3 in the nucleus. These results suggest that, in particular cases, activin A can induce neuronal differentiation and support neuronal survival in vitro. These findings may reflect previously unknown functions of activin A in neuronal cells in vivo.     

Identification and evaluation of bioactive natural products as potential inhibitors of human microtubule affinity-regulating kinase 4 (MARK4)

Microtubule affinity-regulating kinase 4 (MARK4) has recently been identified as a potential drug target for several complex diseases including cancer, diabetes and neurodegenerative disorders. Inhibition of MARK4 activity is an appealing therapeutic option to treat such diseases. Here, we have performed structure-based virtual high-throughput screening of 100,000 naturally occurring compounds from ZINC database against MARK4 to find its potential inhibitors. The resulted hits were selected, based on the binding affinities, docking scores and selectivity. Further, binding energy calculation, Lipinski filtration and ADMET prediction were carried out to find safe and better hits against MARK4. Best 10 compounds bearing high specificity and binding efficiency were selected, and their binding pattern to MARK4 was analyzed in detail. Finally, 100 ns molecular dynamics simulation was performed to evaluate; the dynamics stability of MARK4-compound complex. In conclusion, these selected natural compounds from ZINC database might be potential leads against MARK4, and can further be exploited in drug design and development for associated diseases.     

Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis

Abnormal BRAF and p16INK4A co-exist in 60% of melanomas. BRAF mutation also occurs in 80% of benign nevi where it turns-on p16INK4A resulting in proliferative senescence; loss of p16INK4A removes the inhibitory block leading to melanoma development. Since only melanomas with wild-type BRAF have amplified CDK4 and cyclin D1 genes, p16INK4A-CDK4/6-cyclin D pathway is viewed as linearly downstream of BRAF. Thus, co-occurrence of aberrant BRAF and INK4A may be remnant of changes during melanoma formation without functional significance. To explore this notion, we simultaneously knocked down BRAF (via siRNA) and expressed INK4A cDNA in melanoma cells and observed enhanced growth inhibition. Notably, although each alone had no statistically significant effect on apoptosis, co-expression of BRAF siRNA and INK4A cDNA caused potent apoptosis, which was associated with up-regulation of BIM and down-regulation of BCL2. Our results suggest that aberrant BRAF and INK4A cooperate to promote proliferation and survival of melanoma cells.     

Cytoplasmic Poly（A） Binding Protein 4 Is Highly Expressed in Human Colorectal Cancer and Correlates with Better Prognosis

Cytoplasmic poly(A) binding protein 4(PABPC4) is an RNA-processing protein that plays an important role in the regulation of gene expression.The aim of this study was to investigate the expression pattern and identify the potential clinical significance of PABPC4 in colorectal cancer.Immunohistochemical analysis revealed that 26.7%(27/101 patients) of primary colorectal tumors and 60.5%(23/38 patients) of corresponding adjacent,normal tissues showed high cytoplasmic expression of PABPC4,whereas expression was absent in 98%(43/44 patients) of distant,normal tissues.Using Kaplan-Meier analysis,we observed that the expression of PABPC4 was significantly correlated with disease-free survival and overall survival in patients with stageⅡand stageⅢcolorectal cancer(P = 0.022 and P = 0.020,respectively).PABPC4 expression was positively associated with survival outcome,and may have predictive value in the prognosis of patients with colorectal cancer.Taken together,our findings indicate that PABPC4 may play a role in the pathogenesis of colorectal cancer.     

Plant growth and phosphorus accumulation of wild type and two root hair mutants of Arabidopsis thaliana (Brassicaceae)

Arabidopsis thaliana root hairs grow longer and denser in response to low-phosphorus availability. We tested the hypothesis that wild-type Arabidopsis would acquire more phosphorus under phosphorus-limiting conditions than mutants that do not have the root hair response. The growth and phosphorus acquisition of wild-type Arabidopsis (WS) were compared to two root hair mutants (rhd6 and rhd2) under eight phosphorus treatments ranging from 0.4 mmol/m to 54 mmol/m phosphorus. At the lowest phosphorus treatment, all plants were small and showed severe phosphorus stress symptoms. At 1.5 mmol/m phosphorus, WS plants had greater shoot biomass, absolute growth rate, total phosphorus, and specific phosphorus absorption than the two root hair mutants. At the highest phosphorus treatment, there was no difference between genotypes in any of the parameters measured. We conclude that the response of increased root hair growth under low phosphorus availability in Arabidopsis is important in increasing phosphorus acquisition under phosphorus-limiting conditions.     

Insulin-like growth factor-I and Bcl-X(L) inhibit c-jun N-terminal kinase activation and rescue Schwann cells from apoptosis

We previously reported that Schwann cells undergo apoptosis after serum withdrawal. Insulin-like growth factor-I, via phosphatidylinositol-3 kinase, inhibits caspase activation and rescues Schwann cells from serum withdrawal-induced apoptosis. In this study, we examined the role of c-jun N-terminal protein kinase (JNK) in Schwann cell apoptosis induced by serum withdrawal. Activation of both JNK1 and JNK2 was detected 1 h after serum withdrawal with the maximal level detected at 2 h. A dominant negative JNK mutant, JNK (APF), blocked JNK activation induced by serum withdrawal and Schwann cell apoptosis, suggesting JNK activation participates in Schwann cell apoptosis. Serum withdrawal-induced JNK activity was caspase dependent and inhibited by a caspase 3 inhibitor, Ac-DEVD-CHO. Because insulin-like growth factor-I and Bcl-X(L) are both Schwann cell survival factors, we tested their effects on JNK activation during apoptosis. Insulin-like growth factor-I treatment decreased both JNK1 and JNK2 activity induced by serum withdrawal. LY294002, a phosphatidylinositol-3 kinase inhibitor, blocked insulin-like growth factor-I inhibition on JNK activation, suggesting that phosphatidylinositol-3 kinase mediates the effects of insulin-like growth factor-I. Overexpression of Bcl-X(L) also resulted in less Schwann cell death and inhibition of JNK activation after serum withdrawal. Collectively, these results suggest JNK activation is involved in Schwann cell apoptosis induced by serum withdrawal. Insulin-like growth factor-I and Bcl family proteins rescue Schwann cells, at least in part, by inhibition of JNK activity.     

Modelling growth kinetics of Streptomyces coelicolor A3(2) in a pressurised membrane gradostat reactor (MGR)

Limited information is available with regards to the microbial growth kinetics of Streptomyces coelicolor A3(2) immobilised in pressurised membrane gradostat reactors (MGR). The purpose of this study was to quantify the growth kinetics of the filamentous bacterium immobilised on the external surface of ceramic membranes. The single fibre MGR's (SFMGR) were operated using a pneumatic system to supply humidified pressurised air to the extra capillary space (ECS). The nutrient growth medium was supplied to the lumen of the ceramic membrane in the dead-end mode. The growth curve that was obtained for S. coelicolor A3(2) showed the presence of two growth cycles (biphasic growth) from ±66 to 162 h and ±162 to 354 h, respectively, with no noticeable intermediate lag phase. A faster specific growth rate of 0.049 h⁻¹ was obtained for the first growth cycle, with a lower specific growth rate in the range of ±0.013 to 0.019 h⁻¹ obtained for the second growth cycle. The growth kinetics of S. coelicolor A3(2) within a pressurised MGR has not been reported previously.     

Mapping of equine potassium chloride co-transporter (SLC12A4) and amino acid transporter (SLC7A10) and preliminary studies on associations between SNPs from SLC12A4, SLC7A10 and SLC7A9 and osmotic fragility of erythrocytes

Consensus DNA sequences from human, mouse and/or rat were used to design oligonucleotide primers for equine homologues of exons 16, 17 and 20-23 of potassium chloride co-transporter (SLC12A4) and exons 10, 11 and 3, 4, respectively, for two amino acid transporters (SLC7A10 and SLC7A9). DNA sequences of the PCR products showed high sequence identity to these regions. Equine BAC clones were obtained for SLC12A4 and SLC7A10 and mapped to equine chromosomes ECA3p13 and ECA10p15, respectively, by fluorescence in situ hybridization (FISH). Several single nucleotide polymorphisms (SNP) were found. Substitutions of A/G were found within exon 17 of SLC12A4, within intron 11 of SLC7A10 and within intron 3 of SLC7A9. The SNP associated with SLC7A10 and SLC7A9 were sufficiently polymorphic to investigate associations with erythrocyte fragility among a group of 20 thoroughbred horses. A non-parametric rank-sum test showed a weak association between erythrocyte fragility and the SNP associated with SLC7A10 (P < 0.05).     

大豆11S球蛋白Gy5(A3B4)的基因克隆和序列分析

大豆11Ｓ球蛋白(Ｇｌｙｃｉｎｉｎ)是大豆种子的主要贮藏蛋白,分子量为360ｋＤ,由6对相同的蛋白亚基(每对亚基的分子量约60ｋＤ)构成。每对亚基又是由一个酸性Ａ肽(35～45ｋＤ)和一个碱性Ｂ肽(22ｋＤ)通过二硫键连接而成。Ａ肽和Ｂ肽源自同一个基因,即首先由一个大的ｍＲ?..