Complete digestion of edible bird's nest releases free N-acetylneuraminic acid and small peptides: an efficient method to improve functional properties.

Edible bird's nest (EBN), an Asian health food, contains insoluble proteins and conjugated N-acetylneuraminic acid (NANA) that are difficult to be absorbed by humans. In order to increase the nutritional value of EBN, we developed methods to digest EBN targeting the release of proteins and NANA. By using simulated gastric fluid under acidic conditions, the complex proteins were fully digested into smaller peptides, and in parallel, NANA was fully released from the conjugated form. The completely digested EBN showed better nutraceutical properties. In a skin whitening test, the EBN digest showed stronger inhibition of melanogenesis of cultured B16 cells and enzymatic activity of tyrosinase, as compared to that of undigested EBN. In addition, the EBN digest exhibited stronger osteogenic activity in cultured osteoblasts. Thus, the complete digestion of EBN could be applied to the development of a new generation of EBN health food products, including EBN drinks and skincare products.

Origin of Red Color in Edible Bird's Nests Directed by the Binding of Fe Ions to Acidic Mammalian Chitinase-like Protein.

The red color of edible bird's nests (EBNs) has remained a mystery for hundreds of years. Here, different analytical methods were employed to identify the color origin of EBNs. The treatment of white EBNs with NaNO2/HCl turned them red. In a simulated-gastric-fluid (SGF)-digested EBN, the HPLC chromatogram, NMR spectrum, circular-dichroism spectrum, and Raman spectrum of a NaNO2-treated white EBN closely resembled those of an authentic red EBN. From the HPLC chromatogram of the SGF-digested EBN, the peptides associated with red color were identified in a red EBN and NaNO2-treated white EBN. Several lines of evidence indicated that the color-containing peptide could be derived from the acidic mammalian chitinase-like (AMCase-like) protein of EBNs. Additionally, there was a noticeable increase in Fe-O-bonding intensity after the color change. On the basis of the findings, we proposed that the oxidation of Fe ions in AMCase-like proteins contributed significantly to the color change of EBNs.

Quercetin Potentiates the NGF-Induced Effects in Cultured PC 12 Cells: Identification by HerboChips Showing a Binding with NGF.

Dementia is a persistent disorder of the mental processes and is strongly related to depression. However, the performance of current antidepression medicine is far from satisfactory. Herbal extract provides an excellent source to identify compounds for possible drug development against depression. Here, HerboChips were employed to search herbal compounds that could bind nerve growth factor (NGF). By screening over 500 types of herbal extracts, the water extract of Ginkgo Folium, the leaf of Ginkgo biloba, showed a strong binding to NGF. The herbal fractions showing NGF binding were further isolated and enriched. By using LC-MS/MS analysis, one of the NGF binding fractions was enriched, which was further identified as quercetin, a major flavonoid in Ginkgo Folium. Quercetin, similar to Ginkgo Folium extract, could enhance the effect of NGF in cultured PC 12 cells, including potentiation of neurite outgrowth and phosphorylation of Erk-1/2. This is the first report of discovering an NGF binding compound by using HerboChips from herbal extracts, which could be further developed for antidepression application.

Ginkgetin induces autophagic cell death through p62/SQSTM1-mediated autolysosome formation and redox setting in non-small cell lung cancer.

Promoting cell death by autophagy could be a novel treatment for cancer. The major player in autophagy, p62, serves as a good therapeutic target. Ginkgetin, a biflavonoid from Ginkgo biloba leaves, exhibited promising anticancer activity in non-small cell lung cancer cell lines, with an IC50 lower than that of cisplatin. This anticancer effect of ginkgetin was illustrated in a xenograft nude mouse model. Ginkgetin induced autophagic cell death in A549 cells, and this effect was markedly reversed by chemical and genetic approaches. Ginkgetin showed potential binding affinity to p62. Upregulation of p62 through chemical and genetic means decreased cell death, lysosome acidification, and autophagosome formation, which consequently disrupted autolysosome formation. In addition, the decreased autophagy induced by p62 overexpression increased Nrf2/ARE activity and the oxygen consumption rate and decreased on formation of reactive oxygen species. These phenomena were exhibited in a reciprocal manner when p62 was knocked down. Thus, p62 may be a potential target in ginkgetin-induced autophagic cell death, and ginkgetin could be developed as a novel anticancer drug.

Yu Ping Feng San reverses cisplatin-induced multi-drug resistance in lung cancer cells via regulating drug transporters and p62/TRAF6 signalling.

Yu Ping Feng San (YPFS), an ancient Chinese herbal decoction composed of Astragali Radix, Atractylodis Macrocephalae Rhizoma and Saposhnikoviae Radix, has been used in the clinic for treating immune deficiency. In cancer therapy, YPFS is being combined with chemotherapy drugs to achieve improved efficacy; however, scientific evidence to illustrate this combination effect is lacking. The present study aims to demonstrate the anti-drug resistance of YPFS in cisplatin (DDP)-resistant non-small cell lung cancer cells (A549/DDP). The application of YPFS exhibited a synergistic enhancement of DDP-induced cytotoxicity as well as of the apoptotic signalling molecules. DDP-induced expression of the multi-drug-resistance efflux transporters was markedly reduced in the presence of YPFS, resulting in a higher intracellular concentration of DDP. In addition, the application of YPFS increased DDP-induced ROS accumulation and MMP depletion, decreased p62/TRAF6 signalling in DDP-treated A549/DDP cells. The co-treatment of DDP and YPFS in tumour-bearing mice reduced the tumour size robustly (by more than 80%), which was much better than the effect of DDP alone. These results indicate that YPFS can notably improve the DDP-suppressed cancer effect, which may be a consequence of the elevation of intracellular DDP via the drug transporters as well as the down regulation of p62/TRAF6 signalling.

Clivorine, an otonecine pyrrolizidine alkaloid from Ligularia species, impairs neuronal differentiation via NGF-induced signaling pathway in cultured PC12 cells.

BACKGROUND: Pyrrolizidine alkaloids (PAs) are commonly found in many plants including those used in medical therapeutics. The hepatotoxicities of PAs have been demonstrated both in vivo and in vitro; however, the neurotoxicities of PAs are rarely mentioned. PURPOSE: In this study, we aimed to investigate in vitro neurotoxicities of clivorine, one of the PAs found in various Ligularia species, in cultured PC12 cells. STUDY DESIGN: PC12 cell line was employed to first elucidate the neurotoxicity and the underlying mechanism of clivorine, including cell viability and morphology change, neuronal differentiation marker and signaling pathway. METHODS: PC12 cells were challenged with series concentrations of clivorine and/or nerve growth factor (NGF). The cell lysates were collected for MTT assay, trypan blue staining, immunocytofluorescent staining, qRT-PCR and western blotting. RESULTS: Clivorine inhibited cell proliferation and neuronal differentiation evidenced by MTT assay and dose-dependently reducing neurite outgrowth, respectively. In addition, clivorine decreased the level of mRNAs encoding for neuronal differentiation markers, e.g. neurofilaments and TrkA (NGF receptor). Furthermore, clivorine reduced the NGF-induced the phosphorylations of TrkA, protein kinase B and cAMP response element-binding protein in cultured PC12 cells. CONCLUSION: Taken together, our results suggest that clivorine might possess neurotoxicities in PC12 cells via down-regulating the NGF/TrkA/Akt signaling pathway. PAs not only damage the liver, but also possess neurotoxicities, which could possibly result in brain disorders, such as depression.

Authentication of Cordyceps sinensis by DNA Analyses: Comparison of ITS Sequence Analysis and RAPD-Derived Molecular Markers.

Cordyceps sinensis is an endoparasitic fungus widely used as a tonic and medicinal food in the practice of traditional Chinese medicine (TCM). In historical usage, Cordyceps specifically is referring to the species of C. sinensis. However, a number of closely related species are named themselves as Cordyceps, and they are sold commonly as C. sinensis. The substitutes and adulterants of C. sinensis are often introduced either intentionally or accidentally in the herbal market, which seriously affects the therapeutic effects or even leads to life-threatening poisoning. Here, we aim to identify Cordyceps by DNA sequencing technology. Two different DNA-based approaches were compared. The internal transcribed spacer (ITS) sequences and the random amplified polymorphic DNA (RAPD)-sequence characterized amplified region (SCAR) were developed here to authenticate different species of Cordyceps. Both approaches generally enabled discrimination of C. sinensis from others. The application of the two methods, supporting each other, increases the security of identification. For better reproducibility and faster analysis, the SCAR markers derived from the RAPD results provide a new method for quick authentication of Cordyceps.

The extract of Ziziphus jujuba fruit (jujube) induces expression of erythropoietin via hypoxia-inducible factor-1α in cultured Hep3B cells.

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as health supplement or herbal medicine worldwide. To study the beneficial role of jujube in enhancing hematopoietic function, we investigated its roles on the expression of erythropoietin in cultured Hep3B human hepatocellular carcinoma cells. Application of chemically standardized jujube water extract stimulated erythropoietin expression in a dose-dependent manner, with the highest response by ~ 100 % of increase. A plasmid containing hypoxia response element, a critical regulator for erythropoietin transcription, was transfected into Hep3B cells. Application of jujube water extract onto the transfected cells induced the transcriptional activity of the hypoxia response element. To account for its transcriptional activation, the expression of hypoxia-inducible factor-1α was increased after treatment with jujube water extract: the increase was in both mRNA and protein levels. These results confirmed the hematopoietic function of jujube in the regulation of erythropoietin expression in liver cells.

Authentication of Bulbus Fritillariae Cirrhosae by RAPD-derived DNA markers.

Bulbus Fritillariae is the most commonly used antitussive herb in China. Eleven species of Fritillaria are recorded as Bulbus Fritillariae in the Chinese Pharmacopoeia. Bulbus Fritillariae Cirrhosae is a group of six Fritillaria species with higher efficiency and lower toxicity derived mainly from wild sources. Because of their higher market price, five other Fritillaria species are often sold deceptively as Bulbus Fritillariae Cirrhosae in the herbal market. To ensure the efficacy and safety of medicinal herbs, the authentication of botanical resources is the first step in quality control. Here, a DNA based identification method was developed to authenticate the commercial sources of Bulbus Fritillariae Cirrhosae. A putative DNA marker (0.65 kb) specific for Bulbus Fritillariae Cirrhosae was identified using the Random Amplified Polymorphic DNA (RAPD) technique. A DNA marker representing a Sequence Characterized Amplified Region (SCAR) was developed from a RAPD amplicon. The SCAR marker was successfully applied to differentiate Bulbus Fritillariae Cirrhosae from different species of Fritillaria. Additionally, the SCAR marker was also useful in identifying the commercial samples of Bulbus Fritillariae Cirrhosae. Our results indicated that the RAPD-SCAR method was rapid, accurate and applicable in identifying Bulbus Fritillariae Cirrhosae at the DNA level.

Molecular Assembly and Biosynthesis of Acetylcholinesterase in Brain and Muscle: the Roles of t-peptide, FHB Domain, and N-linked Glycosylation.

Acetylcholinesterase (AChE) is responsible for the hydrolysis of the neurotransmitter, acetylcholine, in the nervous system. The functional localization and oligomerization of AChE T variant are depending primarily on the association of their anchoring partners, either collagen tail (ColQ) or proline-rich membrane anchor (PRiMA). Complexes with ColQ represent the asymmetric forms (A(12)) in muscle, while complexes with PRiMA represent tetrameric globular forms (G(4)) mainly found in brain and muscle. Apart from these traditional molecular forms, a ColQ-linked asymmetric form and a PRiMA-linked globular form of hybrid cholinesterases (ChEs), having both AChE and BChE catalytic subunits, were revealed in chicken brain and muscle. The similarity of various molecular forms of AChE and BChE raises interesting question regarding to their possible relationship in enzyme assembly and localization. The focus of this review is to provide current findings about the biosynthesis of different forms of ChEs together with their anchoring proteins.

The assembly of proline-rich membrane anchor (PRiMA)-linked acetylcholinesterase enzyme: glycosylation is required for enzymatic activity but not for oligomerization.

Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal "t-peptide" in AChE catalytic subunit (AChE(T)). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChE(T) plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChE(T) mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher K(m) value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.

ATP induces synaptic gene expressions in cortical neurons: transduction and transcription control via P2Y1 receptors.

Studies in vertebrate neuromuscular synapses have revealed previously that ATP, via P2Y receptors, plays a critical role in regulating postsynaptic gene expressions. An equivalent regulatory role of ATP and its P2Y receptors would not necessarily be expected for the very different situation of the brain synapses, but we provide evidence here for a brain version of that role. In cultured cortical neurons, the expression of P2Y(1) receptors increased sharply during neuronal differentiation. Those receptors were found mainly colocalized with the postsynaptic scaffold postsynaptic density protein 95 (PSD-95). This arises through a direct interaction of a PDZ domain of PSD-95 with the C-terminal PDZ-binding motif, D-T-S-L of the P2Y(1) receptor, confirmed by the full suppression of the colocalization upon mutation of two amino acids therein. This interaction is effective in recruiting PSD-95 to the membrane. Specific activation of P2Y(1) (G-protein-coupled) receptors induced the elevation of intracellular Ca(2+) and activation of a mitogen-activated protein kinase/Raf-1 signaling cascade. This led to distinct up-regulation of the genes encoding acetylcholinesterase (AChE(T) variant), choline acetyltransferase, and the N-methyl-d-aspartate receptor subunit NR2A. This was confirmed, in the example of AChE, to arise from P2Y(1)-dependent stimulation of a human ACHE gene promoter. That involved activation of the transcription factor Elk-1; mutagenesis of the ACHE promoter revealed that Elk-1 binding at its specific responsive elements in that promoter was induced by P2Y(1) receptor activation. The combined findings reveal that ATP, via its P2Y(1) receptor, can act trophically in brain neurons to regulate the gene expression of direct effectors of synaptic transmission.

The expression of erythropoietin triggered by danggui buxue tang, a Chinese herbal decoction prepared from radix Astragali and radix Angelicae Sinensis, is mediated by the hypoxia-inducible factor in cultured HEK293T cells.

ETHNOPHARMACOLOGICAL EVIDENCE: Danggui buxue tang (DBT), a Chinese medicinal decoction that is being commonly used as hematopoietic medicine to treating woman menopausal irregularity, contains two herbs: radix Astragali and radix Angelicae Sinensis. Pharmacological results indicate that DBT can stimulate the production of erythropoietin (EPO), a specific hematopoietic growth factor, in cultured cells. AIM OF THE STUDY: In order to reveal the mechanism of DBT's hematopoietic function, this study investigated the activity of the DBT-induced EPO expression and the upstream regulatory cascade of EPO via hypoxia-induced signaling in cultured kidney fibroblasts (HEK293T). MATERIALS AND METHODS: DBT-induced mRNA expressions were revealed by real-time PCR, while the change of protein expressions were analyzed by Western blotting. For the analysis of hypoxia-dependent signaling, a luciferase reporter was used to report the transcriptional activity of hypoxia response element (HRE). RESULTS: The plasmid containing HRE, being transfected into HEK293T, was highly responsive to the challenge of DBT application. To account for the transcriptional activation of HRE, DBT treatment was shown to increase the mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α). In addition, the activation of Raf/MEK/ERK signaling pathway by DBT could also enhance the translation of HIF-1α, suggesting the dual actions of DBT in stimulating the EPO expression in kidney cells. CONCLUSION: Our study indicates that HIF pathway plays an essential role in directing DBT-induced EPO expression in kidney. These results provide one of the molecular mechanisms of this ancient herbal decoction for its hematopoietic function.

The PRiMA-linked cholinesterase tetramers are assembled from homodimers: hybrid molecules composed of acetylcholinesterase and butyrylcholinesterase dimers are up-regulated during development of chicken brain.

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE(T) and BChE(T) with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q(N-GPI)). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q(N-GPI) always consist of AChE(T) and BChE(T) homodimers. The dimer formation of AChE(T) and BChE(T) depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal "t-peptides" in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE(T) or BChE(T) homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.

PRiMA directs a restricted localization of tetrameric AChE at synapses.

Acetylcholinesterase (AChE), a highly polymorphic enzyme with various splicing variants and molecular isoforms, plays an essential role in the cholinergic neurotransmission by hydrolyzing acetylcholine into choline and acetate. The AChE(T) variant is expressed in the brain and muscle: this subunit forms non-amphiphilic tetramers with a collagen tail (ColQ) as asymmetric AChE (A(12) AChE) in muscle, and amphiphilic tetramers with a proline-rich membrane anchor (PRiMA) as globular AChE (G(4) AChE) in the brain and muscle. During the brain development, the expression of amphiphilic G(4) AChE is up regulated and becomes the predominant form of AChE there. This up-regulation of G(4) AChE can be attributed to the increased expressions of both AChE(T) and PRiMA. A significant portion of this membrane-bound G(4) AChE is localized at the membrane rafts of the cell membranes derived from the brain. This raft association could be directed by PRiMA via its CRAC (cholesterol recognition/interaction amino acid consensus) motif and C-terminus. In cultured cortical neurons and muscles, the PRiMA-linked AChE was clustered and partially co-localized with synaptic proteins. The restricted localizations suggest that the raft association of PRiMA-linked AChE could account for its synaptic localization and function.

Andrographolide induces cell cycle arrest at G2/M phase and cell death in HepG2 cells via alteration of reactive oxygen species.

The cytotoxicity of andrographolide to HepG2 human hepatoma cells was investigated in the present study. Growth of HepG2 cells was affected in the presence of andrographolide with an IC(50) of 40.2 microM after 48 h treatment. Flow cytometric analysis and DNA fragmentation assay revealed that andrographolide induced cell cycle arrest at G2/M phase and a late apoptosis of the cells. The occurrence of cell cycle arrest was accompanied by the collapse of mitochondrial membrane potential (MMP) and an intracellular increase of hydrogen peroxide (H(2)O(2)) but a decrease of superoxide radicals (O(2)(-)) and reduced glutathione. In the treated cells, expression of Bax as well as the transcriptional controller of this pro-apoptotic gene, p53, was upregulated but not other apoptotic proteins such as Bad, Bcl-2 and Bcl-X(L). Although the activity of caspase-3, which has direct effect on apoptosis, was also enhanced by the presence of andrographolide, cell death of HepG2 could neither be prevented by a specific inhibitor of capsase-3 nor the pan-caspase inhibitor-zVAD (Val-Ala-Asp), indicating that it was a caspase-independent cell death. Since the overall percentage of apoptotic cells was relatively small throughout the experimental studies, we conclude that the cytotoxic effect of andrographolide on HepG2 cells is primary attributed to the induction of cell cycle arrest via the alteration of cellular redox status.