Intramolecular chaperone-assisted dual-anchoring activation (ICDA): a suitable preorganization for electrophilic halocyclization.

The halocyclization reaction represents one of the most common methodologies for the synthesis of heterocyclic molecules. Many efforts have been made to balance the relationship between structure, reactivity and selectivity, including the design of new electrophilic halogenation reagents and the utilization of activating strategies. However, discovering universal reagents or activating strategies for electrophilic halocyclization remains challenging due to the case-by-case practice for different substrates or different cyclization models. Here we report an intramolecular chaperone-assisted dual-anchoring activation (ICDA) model for electrophilic halocyclization, taking advantage of the non-covalent dual-anchoring orientation as the driving force. This protocol allows a practical, catalyst-free and rapid approach to access seven types of small-sized, medium-sized, and large-sized heterocyclic units and to realize polyene-like domino halocyclizations, as exemplified by nearly 90 examples, including a risk-reducing flow protocol for gram-scale synthesis. DFT studies verify the crucial role of ICDA in affording a suitable preorganization for transition state stabilization and X+ transfer acceleration. The utilization of the ICDA model allows a spatiotemporal adjustment to straightforwardly obtain fast, selective and high-yielding synthetic transformations.

Differentiation of essential oils in Atractylodes lancea and Atractylodes koreana by gas chromatography with mass spectrometry.

Atractylodes rhizome is a valuable traditional Chinese medicinal herb that comprises complex several species whose essential oils are the primary pharmacologically active component. Essential oils of Atractylodes lancea and Atractylodes koreana were extracted by hydrodistillation, and the yield was determined. The average yield of essential oil obtained from A. lancea (2.91%) was higher than that from A. koreana (2.42%). The volatile components of the essential oils were then identified by a gas chromatography with mass spectrometry method that demonstrated good precision. The method showed clear differences in the numbers and contents of volatile components between the two species. 41 and 45 volatile components were identified in A. lancea and A. koreana, respectively. Atractylon (48.68%) was the primary volatile component in A. lancea, while eudesma-4(14)-en-11-ol (11.81%) was major in A. koreana. However, the most significant difference between A. lancea and A. koreana was the major component of atractylon and atractydin. Principal component analysis was utilized to reveal the correlation between volatile components and species, and the analysis was used to successfully discriminate between A. lancea and A. koreana samples. These results suggest that different species of Atractylodes rhizome may yield essential oils that differ significantly in content and composition.

A self-assembly aptasensor based on thick-shell quantum dots for sensing of ochratoxin A.

A novel self-assembling aptasensor was fabricated by precisely attaching three phosphorothioate-modified capture aptamers onto a single thick-shell quantum dot in a controllable manner for monitoring of ochratoxin A (OTA), a poisonous contaminant widespread in foodstuffs. Herein, CdSe/CdS QDs coated in ten layer CdS shells were synthesized using a continual precursor injection method. Analysis of the prepared CdSe/CdS QDs showed a zinc-blende structure, high photoluminescence quantum yields (>80%), and a photoemission peak with a narrow full-width at half-maximum (about 29 nm), all qualities that render them as a superior choice for optical applications. By adjusting the number of phosphorothioate bases in the anchor domain, the tunable-valency aptasensor was able to self-assemble. In the sensing strategy, the thick-shell quantum dot was provided as an acceptor while OTA itself was used as a donor. In the presence of OTA, the capture aptamers drive the aptasensor function into a measurable signal through a fluorescence resonance energy transfer (FRET) system. The newly developed aptasensor had a detection limit as low as 0.5 ng mL(-1), with a linear concentration in the range of 1 to 30 ng mL(-1), and therefore meets the requirements for rapid, effective, and anti-interference sensors for real-world applications. Moreover, the high quality thick-shell QDs provide an ideal alternative for highly sensitive imaging and intensive illumination in the fields of biotechnology and bioengineering.

Ultra-fast liquid chromatography with tandem mass spectrometry determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up.

A rapid, selective, and sensitive ultra-fast liquid chromatography with tandem mass spectrometry method was developed for the determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up. Through optimizing the sample pretreatment procedures and chromatographic conditions, good linearity (r(2) ≥ 0.9993), low limit of detection (0.5-0.8 µg/kg), and satisfactory recovery (83.54-94.44%) expressed the good reliability and applicability of the established method in various traditional Chinese medicines. Moreover, the aptamer-affinity column, prepared in-house, showed an excellent feasibility owing to its specific identification of ochratoxin A in various kinds of selected traditional Chinese medicines. The maximum adsorption amount and applicability value were 188.96 ± 10.56 ng and 72.3%, respectively. The matrix effects were effectively eliminated, especially for m/z 404.2→358.0 of ochratoxin A. The application of the developed method for screening the natural contamination levels of ochratoxin A in 25 random traditional Chinese medicines on the market in China indicated that only eight samples were contaminated with low levels below the legal limit (5.0 µg/kg) set by the European Union. This study provided a preferred choice for the rapid and accurate monitoring of ochratoxin A in complex matrices.

Aptamer-affinity column clean-up coupled with ultra high performance liquid chromatography and fluorescence detection for the rapid determination of ochratoxin A in ginger powder.

Aptamers are single-stranded oligonucleotides with high affinity and specificity and are widely used in targets separation and enrichment. Here, an aptamer-affinity column (AAC) was firstly prepared in-house through a covalent immobilization strategy. Then, ochratoxin A (OTA) in ginger powder was absorbed and enriched using the new aptamer-based clean-up technology for the first time, and was further analyzed by ultra high performance liquid chromatography with fluorescence detection. After optimization, the average recoveries for blank samples spiked with OTA at 5, 15, and 45 µg/kg ranged from 85.36 to 96.83%. Furthermore, the AAC exhibited a similar accuracy as an immunoaffinity column to clean up OTA in ginger powder. Above all, it exhibited better reusability, twice that of the immunoaffinity column, had lower toxicity and cost, and took less time. Of 25 contaminated ginger powder samples, OTA contamination levels ranged from 1.51 to 4.31 µg/kg, which were lower than the European Union (EU) regulatory limits. All the positive samples were further confirmed by ultra-fast LC with MS/MS. In conclusion, the method of clean-up based on the AAC coupled to ultra-HPLC with fluorescence detection was rapid, specific, and sensitive for the quantitative analysis of OTA in a complex matrix.

GC-MS combined with chemometric techniques for the quality control and original discrimination of Curcumae longae rhizome: analysis of essential oils.

Curcumae longae rhizome is a widely used traditional herb in many countries. Various geographical origins of this herb might lead to diversity or instability of the herbal quality. The objective of this work was to establish the chemical fingerprints for quality control and find the chemical markers for discriminating these herbs from different origins. First, chemical fingerprints of essential oil of 24 C. longae rhizome from four different geographical origins in China were determined by GC-MS. Then, pattern recognition techniques were introduced to analyze these abundant chemical data in depth; hierarchical cluster analysis was used to sort samples into groups by measuring their similarities, and principal component analysis and partial least-squares discriminate analysis were applied to find the main chemical markers for discriminating these samples. Curcumae longae rhizome from Guangxi province had the highest essential oil yield (4.32 ± 1.45%). A total of 46 volatile compounds were identified in total. Consistent results were obtained to show that C. longae rhizome samples could be successfully grouped according to their origins, and turmerone, ar-turmerone, and zingiberene were the characteristic components for discriminating these samples of various geographical origins and for quality control. This finding revealed that fingerprinting analysis based on GC-MS coupled with chemometric techniques could provide a reliable platform to discriminate herbs from different origins, which is a benefit for quality control.

[Rapid detection of extrinsic harmful contaminants using quantum dots-coated probes and their application prospects in traditional Chinese medicine].

The contamination of extrinsic harmful contaminants including mycotoxins, heavy metals and pesticides, etc, brings serious risks to traditional Chinese medicines (TCMs), further to human health. Due to their unique photoluminescence, chemiluminescence, electrochemical and electrochemiluminescence properties, semiconductor quantum dots (QDs) nanoparticles are widely used to immobilize bioprobes and biosensors, etc. In this review, the luminescence characteristics and specific ligands of QDs probles which are used to determine contaminants were summed up. Then, the applications of QDs-coated novel probes in the determination of mycotoxins, heavy metals and pesticides were discussed in detail. In addition, the contamination levels and characteristics of extrinsic harmful residues in TCMs were investigated. Further, the maximum levels of those contaminants in TCMs were compared with those set by various countries. Finally, the future development trends and problems of QDs-coated probes in the determination of those extrinsic residues in TCMs were prospected.

The HER4 cytoplasmic domain, but not its C terminus, inhibits mammary cell proliferation.

Unlike the proliferative action of other epidermal growth factor (EGF) receptor family members, HER4/ErbB4 is often associated with growth-inhibitory and differentiation signaling. These actions may involve HER4 two-step proteolytic processing by intramembraneous gamma-secretase, releasing the soluble, intracellular 80-kDa HER4 cytoplasmic domain, s80HER4. We demonstrate that pharmacological inhibition of either gamma-secretase activity or HER4 tyrosine kinase activity blocked heregulin-dependent growth inhibition of SUM44 breast cancer cells. We next generated breast cell lines stably expressing GFP-s80HER4 [green fluorescent protein (GFP) fused to the N terminus of the HER4 cytoplasmic domain, residues 676-1308], GFP-CT(HER4) (GFP fused to N terminus of the HER4 C-terminus distal to the tyrosine kinase domain, residues 989-1308), or GFP alone. Both GFP-s80HER4 and GFP-CTHER4 were found in the nucleus, but GFP-s80HER4 accumulated to a greater extent and sustained its nuclear localization. s80HER4 was constitutively tyrosine phosphorylated, and treatment of cells with a specific HER family tyrosine kinase inhibitor 1) blocked tyrosine phosphorylation; 2) markedly diminished GFP-s80HER4 nuclear localization; and 3) reduced signal transducer and activator of transcription (STAT)5A tyrosine phosphorylation and nuclear localization as well as GFP-s80HER4:STAT5A interaction. Multiple normal mammary and breast cancer cell lines, stably expressing GFP-s80HER4 (SUM44, MDA-MB-453, MCF10A, SUM102, and HC11) were growth inhibited compared with the same cell line expressing GFP-CTHER4 or GFP alone. The s80HER4-induced cell number reduction was due to slower growth because rates of apoptosis were equivalent in GFP-, GFP-CTHER4-, and GFP-s80HER4-expressing cells. Lastly, GFP-s80HER4 enhanced differentiation signaling as indicated by increased basal and prolactin-dependent beta-casein expression. These results indicate that surface HER4 tyrosine phosphorylation and ligand-dependent release of s80HER4 are necessary, and s80HER4 signaling is sufficient for HER4-dependent growth inhibition.

Effect of focal adhesion kinase (FAK) downregulation with FAK antisense oligonucleotides and 5-fluorouracil on the viability of melanoma cell lines.

Inhibition of focal adhesion kinase (FAK), a non-receptor tyrosine kinase linked to tumour cell survival, causes cell rounding, loss of adhesion and apoptosis in human cancer cell lines. In this study, we tested antisense oligonucleotide inhibitors of FAK, in combination with 5-fluorouracil (5-FU), to increase its sensitivity in human melanoma cell lines. Antisense oligonucleotides directed to the 5' mRNA sequence of FAK and missense control oligonucleotides were used. In BL melanoma cells, treatment with FAK antisense oligonucleotide was associated with a 2.5-fold increase in cell death compared with treatment with control oligonucleotide (33+/-2% vs. 13+/-3%, P<0.0001). 5-FU alone had no effect on BL cells (4.4% cell death, P=0.15). The addition of 5-FU after antisense oligonucleotide resulted in a significant synergistic increase in cell death equal to 69+/-2% compared with treatments with antisense oligonucleotide alone, 5-FU alone and control oligonucleotide (P<0.0001). Similar results were found in the C8161 melanoma cell line. In both cell lines, reduction in cell viability was accompanied by an increased loss of adhesion and increased apoptosis that was proportional to the decrease in viability. Treatment with antisense oligonucleotide plus 5-FU resulted in significantly decreased p125FAK expression in both C8161 and BL melanoma cell lines, demonstrated by Western blot analyses. These data show that the downregulation of FAK by antisense oligonucleotide combined with 5-FU chemotherapy results in a greater loss of adhesion and greater apoptosis in melanoma cells than treatment with either agent alone, suggesting that the combination may be a potential therapeutic agent for human melanoma in vivo.

Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein.

Tumor cells resist the apoptotic stimuli associated with invasion and metastasis by activating survival signals that suppress apoptosis. Focal adhesion kinase (FAK), a tyrosine kinase that is overexpressed in a variety of human tumors, mediates one of these survival signals. Attenuation of FAK expression in tumor cells results in apoptosis that is mediated by caspase 8- and FADD-dependent pathways, suggesting that death receptor pathways are involved in the process. Here, we report a functional link between FAK and death receptors. We have demonstrated that FAK binds to the death domain kinase receptor-interacting protein (RIP). RIP is a major component of the death receptor complex and has been shown to interact with Fas and tumor necrosis factor receptor 1 through its binding to adapter proteins. We have shown that RIP provides proapoptotic signals that are suppressed by its binding to FAK. We thus propose that FAK overexpression in human tumors provides a survival signal function by binding to RIP and inhibiting its interaction with the death receptor complex.

Activated Src increases adhesion, survival and alpha2-integrin expression in human breast cancer cells.

Focal adhesion kinase (FAK) is an intracellular kinase that localizes to focal adhesions. FAK is overexpressed in human tumours, and FAK regulates both cellular adhesion and anti-apoptotic survival signalling. Disruption of FAK function by overexpression of the FAK C-terminal domain [FAK-CD, analogous to the FRNK (FAK-related non-kinase) protein] leads to loss of adhesion and apoptosis in tumour cells. We have shown that overexpression of an activated form of the Src tyrosine kinase suppressed the loss of adhesion induced by dominant-negative; adenoviral FAK-CD and decreased the apoptotic response in BT474 and MCF-7 breast cancer cell lines. This adhesion-dependent apoptosis was increased by the Src-family kinase inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. We have also shown that expression of activated Src in breast cancer cells increased the expression of alpha2-integrin and that overexpression of alpha2-integrin suppressed FAK-CD-mediated loss of adhesion. Our results suggest a model in which Src regulates adhesion and survival through enhanced expression of the alpha2-integrin. This provides a mechanism through which Src promotes cellular adhesion and alters the adhesive function of FAK.

Overexpression of the receptor tyrosine kinase Tie-1 intracellular domain in breast cancer.

OBJECTIVE: Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie-1) is a receptor tyrosine kinase that regulates angiogenesis and antiapoptotic survival signaling. Tie-1 expression is generally associated with endothelial cells and neovascularization. We previously identified Tie-1 in human breast tumor samples using a PCR-based screen for protein kinases expressed in breast tumors. The purpose of this study was to determine the cell types expressing Tie-1, whether Tie-1 is expressed in tumor cells, and to examine the regulation of Tie-1 in breast cancer. METHODS: Tie-1 expression was analyzed by Western blot and immunohistochemistry using an antibody to the carboxy terminus of Tie-1. Tie-1 expression was determined in a variety of cancer cell lines, clinical breast and colon tumor samples, and in corresponding benign tissue from the same patient. Tie-1 expression and distribution in breast tumors was scored by immunohistochemistry. RESULTS: Tie-1 was overexpressed in 14/23 breast tumors compared with 0/9 corresponding normal tissues from the same patients. Immunohistochemistry revealed that Tie-1 was overexpressed in epithelial breast cancer cells and ductal carcinoma in situ. In all breast tumor samples, Tie-1 was expressed as a truncated 40- to 43-kD doublet consisting of the intracellular portion of the protein, which contains the tyrosine kinase catalytic domain. The 40- to 43-kD Tie-1 doublet was expressed in a broad variety of cell lines. CONCLUSIONS: We have shown that breast cancer cells overexpress a cleaved form of the Tie-1 protein. Our results implicate the intracellular domain of Tie-1, which includes the catalytic kinase domain, in breast cancer progression.

Focal adhesion kinase N-terminus in breast carcinoma cells induces rounding, detachment and apoptosis.

Focal adhesion kinase (FAK) has a central role in adhesion-mediated cell signalling. The N-terminus of FAK is thought to function as a docking site for a number of proteins, including the Src-family tyrosine kinases. In the present study, we disrupted FAK signalling by expressing the N-terminal domain of FAK (FAK-NT) in human breast carcinoma cells, BT474 and MCF-7 lines, and non-malignant epithelial cells, MCF-10A line. Expression of FAK-NT led to rounding, detachment and apoptosis in human breast cancer cells. Apoptosis was accompanied by dephosphorylation of FAK Tyr(397), degradation of the endogenous FAK protein and activation of caspase-3. Over-expression of FAK rescued FAK-NT-mediated cellular rounding. Expression of FAK-NT in non-malignant breast epithelial cells did not lead to rounding, loss of FAK phosphorylation or apoptosis. Thus FAK-NT contributes to cellular adhesion and survival pathways in breast cancer cells which are not required for survival in non-malignant breast epithelial cells.

Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyses.

PURPOSE: Focal adhesion kinase (FAK), a protein tyrosine kinase that functions in signaling events between cells and their extracellular matrix, is overexpressed in a variety of human solid tumors. To determine whether FAK expression is up-regulated in colorectal cancer, we analyzed FAK mRNA and protein levels in primary colorectal tumors and colorectal liver metastases. EXPERIMENTAL DESIGN: p125(FAK) expression in formalin-fixed paraffin-embedded (FFPE) tissue was studied using immunohistochemical assays on 24 matched primary colorectal carcinomas and colorectal liver metastases as well as 18 different colorectal liver metastases using monoclonal anti-FAK 4.47. FAK mRNA expression was quantitated by real-time PCR on 39 matched normal colorectal mucosa and primary colorectal carcinomas as well as on 17 separate liver metastases. RESULTS: Elevated levels of p125(FAK) expression were demonstrated in both primary colorectal tumors and colorectal liver metastases compared with normal colorectal mucosa. Immunohistochemistry experiments demonstrated equivalent FAK expression in matched samples of colorectal primary tumors and liver metastases. Using real-time PCR in 39 matched samples, FAK mRNA copy number was significantly higher in primary colorectal tumors compared with normal colorectal mucosa. FAK expression was analyzed by both real-time PCR and immunohistochemistry in a separate set of colorectal liver metastases. Immunohistochemistry demonstrated high levels of FAK expression in 89% of samples. Furthermore, FAK mRNA copies in these unmatched liver metastases were significantly higher than the primary tumor FAK mRNA copies. CONCLUSION: These experiments have shown that both primary colorectal cancers and colorectal liver metastases express high levels of FAK mRNA and p125(FAK) protein. Furthermore, the majority of colorectal liver metastases demonstrated robust FAK expression equivalent to or greater than that in the primary colorectal tumor.