Search

Bitter tea oil meal (BTOM) is the main waste from the production of bitter tea oil which is squeezed from bitter tea seeds. The purpose of this study is to reuse the BTOM as an additive of the polylactic acid (PLA) to prepare eco-friendly composites. The effects of the addition of BTOM and maleic anhydride grafted polybutylene succinate (MAPBS) on the properties of PLA were investigated. The addition of MAPBS is mainly to increase the toughness of the PLA, and to increase the compatibility between BTOM and PLA. The experimental results show that the compatibility of PLA and BTOM and impact resistance of the composites can be improved by addition of MAPBS. The composite with 5% BTOM and 8% MAPBS exhibited the best tensile strength. In addition, the composite with 5% BTOM and 5% MAPBS has the best impact strength. It was found that the addition of BTOM and MAPBS can promote the crystallization of PLA. Moreover, the addition of BTOM not only can reduce the usage of PLA and the cost of the materials, but also reuse and reduce the waste from food industry.

Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.

Thrombin activates protease-activated receptor-1 (PAR-1) through binding to exosite I and the active site to promote tumor growth. We have developed a new class of anti-cancer glyco-peptides to target exosite I selectively without affecting the active-site-mediated coagulation activity and showed the importance of glycans for the stability and anti-cancer activity of the glyco-peptides.

It is more challenging to design peptide drugs than small molecules through molecular docking and in silico analysis. Here, we developed a structure-based approach with various computational and analytical techniques to optimize cancer-targeting peptides for molecular imaging and therapy. We first utilized a peptide-binding protein database to identify GRP78, a specific cancer cell-surface marker, as a target protein for the lead, L-peptide. Subsequently, we used homologous modeling and molecular docking to identify a peptide-binding domain within GRP78 and optimized a series of peptides with a new protein-ligand scoring program, HotLig. Binding of these peptides to GRP78 was confirmed using an oriented immobilization technique for the Biacore system. We further examined the ability of the peptides to target cancer cells through in vitro binding studies with cell lines and clinical cancer specimens, and in vivo tumor imaging and targeted chemotherapeutic studies. MicroSPECT/CT imaging revealed significantly greater uptake of (188)Re-liposomes linked to these peptides as compared with non-targeting (188)Re-liposomes. Conjugation with these peptides also significantly increased the therapeutic efficacy of Lipo-Dox. Notably, peptide-conjugated Lipo-Dox significantly reduced stem-cell subpopulation in xenografts of breast cancer. The structure-based optimization strategy for peptides described here may be useful for developing peptide drugs for cancer imaging and therapy.

Upconversion nanoparticles (UCNPs) have extensive biological-applications because of their bio-compatibility, tunable optical properties and their ability to be excited by infrared radiation. Matrix metalloproteinases (MMPs) play important roles in extracellular matrix remodelling; they are usually found to significantly increase during cancer progression, and these increases may lead to poor patient survival. In this study, we produced a biosensor that can be recognized by MMP2 and then be unravelled by the attached quencher to emit visible light. We used 3.5-nm gold nanoparticles as a quencher that absorbed emission from UCNPs at a wavelength of 540 nm. The biosensor consists of an upconversion nanoparticle, MMP2-recognized polypeptides and quenchers. Here, UCNPs consisting of NaYF4:Yb(3+)/Er(3+) were prepared via a high temperature co-precipitation method while protecting the oleic acid ligand. To improve the biocompatibility and modify the UCNPs with a polypeptide, they were coated with a silica shell and further conjugated with MMP-recognizing polypeptides. The polypeptide has two ends of featuring carboxylic and thiol groups that react with UCNPs and AuNPs, and the resulting nanoparticles were referred to as UCNP@p-Au. According to the in vitro cell viability analysis, UCNP@p-Au exhibited little toxicity and biocompatibility in head and neck cancer cells. Cellular uptake studies showed that the MMP-based biosensor was activated by 980-nm irradiation to emit green light. This MMP-based biosensor may serve as sensitive and specific molecular fluorescent probe in biological-applications.

Plant shoot stem cell pool is constantly maintained by a negative feedback loop through peptide-receptor mediated signaling pathway. CLAVATA3 (CLV3) encode a 96 amino-acid protein which is processed to 12-amino-acid or arabinosylated 13-amino-acid peptides, acting as a ligand signal to regulate stem cell homeostasis in the shoot apical meristem (SAM). Although arabinosylated 13-amino-acid CLV3 peptide (CLV3p) shows more significant binding affinity to its receptors and biological activities in the SAM, the physiological function of two mature forms of CLV3p remained an unresolved puzzle in the past decade due to the technical difficulties of arabinosylation modification in the peptide synthesis. Here, we analyzed the role of two mature CLV3 peptides with newly synthesized arabinosylated peptide. Beside shoot meristem phenotypes, arabinosylated CLV3p showed the conventional trait of CLV2-dependent root growth inhibition. Moreover, both 12-amino-acid and arabinosylated 13-amino-acid CLV3 peptides have analogous activities in shoot stem cell signaling. Notably, we demonstrated that non-arabinosylated 12-amino acid CLV3p can affect shoot stem cell signaling at the physiological level unlike previously suggested (Ohyama et al., 2009; Shinohara and Matsubayashi, 2013; Shinohara and Matsubayashi, 2015). Therefore, these results support the physiological role of the 12-amino-acid CLV3p in shoot stem cell signaling in the deficient condition of arabinosylated 13-amino-acid CLV3p in Arabidopsis thaliana.

Wheatgrass is one of the most widely used health foods, but its functional components and mechanisms remain unexplored. Herein, wheatgrass-derived oligosaccharides (WG-PS3) were isolated and found to induce CD69 and Th1 cytokine expression in human peripheral blood mononuclear cells. In particular, WG-PS3 directly activated the purified monocytes by inducing the expression of CD69, CD80, CD86, IL-12, and TNF-α but affected NK and T cells only in the presence of monocytes. After further purification and structural analysis, maltoheptaose was identified from WG-PS3 as an immunomodulator. Maltoheptaose activated monocytes via Toll-like receptor 2 (TLR-2) signaling, as discovered by pretreatment of blocking antibodies against Toll-like receptors (TLRs) and also determined by click chemistry. This study is the first to reveal the immunostimulatory component of wheatgrass with well defined molecular structures and mechanisms.

Surface IgD and IgM doubly positive cells comprise the major population of B cells in the human immune system. The heavy chain of membrane-bound IgD (mδ) differs from that of IgD (δ) in that mδ contains a C-terminal membrane-anchor peptide. Our group previously proposed that the N-terminal extracellular segment of 27 aa residues of the membrane-anchor peptide of mδ, referred to as the mIg isotype-specific-δ (migis-δ) segment, may provide a unique antigenic site for isotype-specific targeting of mIgD + B cells. Here we report the preparation of mouse mAbs specific for human migis-δ. The mAbs bound to human migis-δ-containing recombinant proteins in an ELISA and to mIgD-expressing transfectants of a CHO cell line as analyzed by flow cytometry. MAb 20E6, which binds to an epitope toward the N-terminal of human migis-δ, could stain human B cell line MC116, which expressed mIgD and mIgM. MC116 cells could be induced to undergo apoptosis by treatment with 20E6 in the presence of a second crosslinking antibody. Chimeric 20E6 caused antibody-dependent cellular cytotoxicity of MC116 cells in the presence of human PBMCs as the source of effector cells. In cultures of PBMCs, 20E6 down-regulated the population of mIgD + B cells. The production of human IgM by transplanted MC116 cells in NOD-SCID (NOD.CB17- Prkdc scid /IcrCrlBltw) mice could be suppressed by 20E6. These results encourage further investigation of the potential of anti-migis-δ mAbs to control mIgD + B cells, when such a manipulation may alleviate a disease state.

Amyloid-β (Aβ), the main constituent in senile plaques found in the brain of patients with Alzheimer's disease (AD), is considered as a causative factor in AD pathogenesis. The clinical examination of the brains of patients with AD has demonstrated that caspase-3 colocalizes with senile plaques. Cellular studies have shown that Aβ can induce neuronal apoptosis via caspase-3 activation. Here, we performed biochemical and in silico studies to investigate possible direct effect of Aβ on caspase-3 to understand the molecular mechanism of the interaction between Aβ and caspase-3. We found that Aβ conformers can specifically and directly sequester caspase-3 activity in which freshly prepared Aβ42 is the most potent. The inhibition is noncompetitive, and the C-terminal region of Aβ plays an important role in sequestration. The binding of Aβ to caspase-3 was examined by cross-linking and proteolysis and by docking and all-atom molecular dynamic simulations. Experimental and in silico results revealed that Aβ42 exhibits a higher binding affinity than Aβ40 and the hydrophobic C-terminal region plays a key role in the caspase-Aβ interaction. Overall, our study describes a novel mechanism demonstrating that Aβ sequesters caspase-3 activity via direct interaction and facilitates future therapeutic development in AD.

Spatial data fusion is an important method of spatial data acquisition. The aim of multisource spatial data integration and fusion is to improve the information precision and information's utilization efficiency. Vector and raster are the two main spatial data structures. This article discusses vector data fusion from of data model fusion, semantic information fusion and coordinates unification, reviews the main methods of raster data fusion and discusses the key technologies of vector and raster data fusion, and proposes the future developments of spatial data fusion technique.

MALDI-TOF MS provides rapid and sensitive analyses of larger biomolecules. However, MS analyses of polysaccharide have been reported to have lower sensitivity compared to peptides and proteins. Here, we investigated some polysaccharides chemically derivatized by permethylation and ortho-phenylene diamine (OPD) tagging. Methylated glycan is obviously able to improve the sensitivity for mass spectrometry detection. Oxidative condensation by UV-activation tagging to saccharides by OPD and peptide-OPD also improve the sensitivity of MALDI-TOF MS analyses. Polysaccharides including dextran, glucomannan, arabinoxylan, arabinogalactan and beta-1,3-glucan, isolated from nutritional supplements of Ganoderma lucidum and Saccharomyces pastorianus were measured using MALDI-TOF MS with 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix. These glycans were also derivatized to methylated and benzimidazole-tagged glycans by chemical transformation for molecular weight analysis. The derivatized polysaccharides showed excellent MALDI-TOF MS signal enhancement in the molecular weight range from 1 to 5 kDa. Here, we demonstrate an efficient method to give glycan-benzimidazole (glycan-BIM) derivatives for polysaccharide determination in MALDI-TOF MS. Therefore, permethylated or benzimidazole-derivatized polysaccharides provide a new option for polysaccharide analysis using MALDI-TOF MS.

Membrane-bound IgA (mIgA) is associated with Igα/Igβ as the B cell receptor (BCR) complex on mIgA-expressing B cells. The α chain of mIgA (mα) contains a C-terminal membrane-anchor peptide, which encompasses extracellular, transmembrane and intracellular segments. The extracellular segment, referred to as the mIg isotype-specific (migis-α) segment or the extracellular membrane proximal domain of mα, has been proposed to be a specific antigenic site suitable for isotype-specific targeting of mIgA-expressing B cells by antibodies. In this study, we developed several anti-migis-α monoclonal antibodies (mAbs), such as mAb 29C11, specific to a segment towards the N-terminus of the 26 amino acid long migis-α. The mAbs bound strongly to synthetic peptides of migis-α and to various recombinant proteins containing migis-α as revealed by ELISA. On B cells, however, flow cytometric analysis suggested that these mAbs did not bind strongly to mIgA. After lipid rafts of B cells were disrupted by cholesterol extraction, the mAbs were able to bind strongly to the treated B cells. Moreover, immunoprecipitation analysis of these mAbs indicated that mIgA could only be pulled down by the mAbs when mIgA-expressing B cells were solubilized by strong detergents, such as sodium dodecyl sulfate (SDS), or when lipid rafts were disrupted. Together, these results suggest that the migis-α region of mIgA in the BCR is associated with lipid rafts, which hinder binding of migis-α-specific antibodies to mIgA on the cell surface. Further studies are in progress to evaluate the suitability of 29C11 or its affinity-improved variants for targeting mIgA-expressing B cells.

MALDI-MS provides a rapid and sensitive analysis of large biomolecules such as proteins and nucleic acids. However, oligo- and polysaccharides are less sensitive in MS analysis partly due to their neutral and hydrophilic nature to cause low ionization efficiency. In this study, four types of oligosaccharides including aldoses, aminoaldoses, alduronic acids and α-keto acids were modified by appropriate tags at the reducing termini to improve their ionization efficiency. Bradykinin (BK), a vasoactive nonapeptide (RPPGFSPFR), containing two arginine and two phenylalanine residues turned out to be an excellent MS signal enhancer for maltoheptaose, GlcNAc oligomers and oligogalacturonic acids. In the MALDI-TOF-MS analysis using 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix, the GalA4-BK and GalA5-BK conjugates prepared by reductive amination showed the detection limit at 0.1 fmol, i.e. ∼800-fold enhancement over the unmodified pentagalacturonic acids. The remarkable MS enhancement was attributable to the synergistic effect of the basic arginine residues for high proton affinity and the hydrophobic property phenylalanine residues for facile ionization. A tetrapeptide GFGR(OMe) and an arginine linked phenylenediamine (H(2) N)(2) Ph-R(OMe) were thus designed to act as potent tags of oligosaccharides in MS analysis. Interestingly, concurrent condensation and lactonization of α2,8-linked tetrasialic acid (SA4) was carried out with (H(2) N)(2) Ph-R(OMe) to obtain a quinoxalinone derivative, which showed > 200-fold enhancement over unmodified SA4 in the MALDI-TOF-MS analysis.

Abnormally high concentrations of Zn2+, Cu2+, and Fe3+ are present along with amyloid-β (Aβ) in the senile plaques in Alzheimer disease, where Al3+ is also detected. Aβ aggregation is the key pathogenic event in Alzheimer disease, where Aβ oligomers are the major culprits. The fundamental mechanism of these metal ions on Aβ remains elusive. Here, we employ 4,4′-Bis(1-anilinonaphthalene 8-sulfonate) and tyrosine fluorescence, CD, stopped flow fluorescence, guanidine hydrochloride denaturation, and photo-induced cross-linking to elucidate the effect of Zn2+, Cu2+, Fe3+, and Al3+ on Aβ at the early stage of the aggregation. Furthermore, thioflavin T assay, dot blotting, and transmission electron microscopy are utilized to examine Aβ aggregation. Our results show that Al3+ and Zn2+, but not Cu2+ and Fe3+, induce larger hydrophobic exposures of Aβ conformation, resulting in its significant destabilization at the early stage. The metal ion binding induces Aβ conformational changes with micromolar binding affinities and millisecond binding kinetics. Cu2+ and Zn2+ induce similar assembly of transiently appearing Aβ oligomers at the early state. During the aggregation, we found that Zn2+ exclusively promotes the annular protofibril formation without undergoing a nucleation process, whereas Cu2+ and Fe3+ inhibit fibril formation by prolonging the nucleation phases. Al3+ also inhibits fibril formation; however, the annular oligomers co-exist in the aggregation pathway. In conclusion, Zn2+, Cu2+, Fe3+, and Al3+ adopt distinct folding and aggregation mechanisms to affect Aβ, where Aβ destabilization promotes annular protofibril formation. Our study facilitates the understanding of annular Aβ oligomer formation upon metal ion binding.

The e chain of membrane-bound IgE (mIgE) is expressed predominantly as a “long” isoform, containing an extra segment of 52 amino acid (a.a.) residues, referred to as CemX, between the CH4 domain and the C-terminal membrane-anchoring transmembrane peptide. CemX results from an alternative splicing of the e RNA transcript at 156-bp upstream of the splicing acceptor site used by the “short” isoform. Here, based on an analysis of the CemX genomic DNA sequences of 320 subjects residing in Taiwan, we report that single-nucleotide polymorphisms have been found at two positions, namely, G/T at #46 and A/G at #93 (along the 156 bp of CemX), with the former creating an amino acid change from Val to Leu at #16 (along the 52 a.a. of CemX) and the latter resulting in no change (Gly). Among the 640 CemX sequences identified, the previously known 46G93A allelic form appeared 293 times, the newly discovered 46T93A allelic form (GeneBank accession no. GU208817) 26 times, and the 46G93G allelic form (GU208818) 321 times. No 46T93G allelic form was found. Serum IgE measurements showed that the polymorphisms did not correlate with the levels of serum IgE. The anti-CemX monoclonal antibody, 4B12, could bind equally well to mIgE.FcL(16V) and mIgE.FcL(16L). While genetic variation of CemX of broader populations should also be investigated, these newly discovered genetic variants of CemX in the Taiwanese population do not seem to affect the feasibility of using an anti-CemX mAb, such as 4B12, to target mIgE-expressing B cells.

Ion channels are involved in diverse biological processes and play essential roles in the physiology of all cells. An increasing number of human and animal diseases have been identified as relating to the defective function of ion channels. Scorpion venoms contain various polypeptides with distinct biological functions that particularly affect the permeability of ion channels in cell membranes (Catterall 1980; Valdivia et al. 1992; Garcia et al. 1997). These polypeptides possess the potency to recognize ion channels and receptors in excitable membranes and are classified into four groups on the basis of ion-channel types: (1) group I modulates Na+-channel activity (Possani et al. 1999) and contains peptides of 60 to 70 amino acids linked by four disulfide bridges; (2) group II blocks K+ channels (Miller 1995; Romi-Lebrun et al. 1997) and are short peptides with 31 to 41 amino acid residues with three or four disulfide bonds; (3) group III supposedly inhibits Cl−channels (DeBin et al. 1993) and contains short-chain insect toxin peptides of ∼36 amino acids with four disulfide bonds; and (4) group IV includes peptides that modulate ryanodine-sensitive Ca2+ channels (Valdivia and Possani 1998). It is believed that the toxin has a unique tertiary structure that may provide valuable information for understanding channels. Thus, understanding the structural basis of the specificity of scorpion toxins for these receptors could lead to the design of new ligands with controlled activity and potency with potential for clinical applications. Scorpion K+-channel blockers of group II, named α-KTx, have been classified into 12 subfamilies (Miller 1995; Tytgat et al. 1999). These K+-channel blockers block two major classes of K+ channels: voltage-gated (Kv-type) and high-conductance Ca2+-activated (BK-type) K+ channels. The three-dimensional structures of several scorpion K+-channel blockers have been determined by NMR spectroscopy; these include charybdotoxin (ChTx; Bontems et al. 1991), iberiotoxin (IbTx; Johnson et al. 1992), noxiustoxin (NTx; Dauplais et al. 1995), PO5-NH2 (Meunier et al. 1993), kaliotoxin (KTx; Fernandez et al. 1994), margatoxin (MgTx; Johnson et al. 1994), and tityustoxin K-α (TsTx-Kα; Ellis et al. 2001). Although the overall fold of these α-KTx toxins is very similar, there are subtle variations among them in amino acid sequence, the size of the β-sheet, the type of β-turn, or the type of α-helix (i.e., α-helix versus 310-helix). These differences in toxin structure affect the placement of side-chain moieties. Thus, the selectivity that various scorpion toxins have for the outer vestibule of different K+ channels is typically quite distinct. Previously, Doyle et al. (1998) applied X-ray crystallographic methods to determine the three-dimensional structure of the KcsA bacterial K+ channel, which may serve as a good model for understanding the binding site of scorpion toxin on Kv-type channels. Recently, a new K+-channel blocker was identified from the scorpion venom of Tityus cambridgei (Tc1; Batista et al. 2000). Tc1 contains 23 amino acids linked with three disulfide bridges and is the smallest K+-channel blocker toxin from scorpion venoms. All previously known K+-channel blockers from scorpion venoms are longer than 30 amino acid residues and are classified into 12 subfamilies as described above. Tc1 is classified as the first member of the new subfamily 13. In K+-channel blocking activity, Tc1 recognizes the Shaker B K+ channels with a dissociation constant (Kd) of 65 nM and competes with NTx for binding to the synaptosomal membranes, with an inhibitory concentration 50% (IC50) value in the order of 200 nM (Batista et al. 2000). Tc1 is a highly basic peptide because it contains seven positively charged residues with a pI value of 9.50. The sequence alignment of Tc1 with eight other K+-channel blockers from scorpion toxins is shown in Figure 1 ▶. We found that six cysteine residues (Cys2, Cys5, Cys9, Cys15, Cys20, and Cys22), Gly13, and Lys14 (Tc1 numbering) are conserved, and the C-terminal regions are highly similar among these toxins. In addition, the sequence of Tc1 shows some unique properties. For example, Tc1 possesses Arg at position 19, whereas the corresponding residue in the other toxins is Lys. At position 16, Tc1 has Ile, whereas the other toxins, with the exception of the PO5 peptide, have Met at the corresponding position. Furthermore, Tc1 contains dense positively charged residues at residues 5–10. Unlike other scorpion toxins, Tc1 does not contain either negatively charged residues or proline. These properties make Tc1 an excellent candidate for three-dimensional structure determination and site-directed mutagenesis and for gaining clearer understanding of K+ channels. Fig. 1. Sequence alignment of Tc1 with eight other K+-channel blockers was generated using the CLUSTAL-W (Thompson et al. 1994) and ESPript (Gouet et al. 1999) programs. Three-dimensional solution structures for six of them have been determined: They are ChTx ... In this study, synthetic Tc1 was made by conventional solid-phase peptide synthesis and folded into its active conformation. We checked the channel-blocking activity of the synthesized Tc1 and found that both synthetic and native Tc1 possess similar blocking activity against the Shaker K+ channel. Next, we applied circular dichrosim (CD) and NMR techniques to solve the solution structure of Tc1. To further understand the various structure-function relationships among the K+-channel blockers from scorpion venoms, we compared the three-dimensional structure of Tc1 and those of other structurally and functionally related scorpion toxins, ChTx and NTx. We concluded that the C-terminal structure is the most important region for the blocking activity of Kv-type channels for scorpion K+-channel blockers. In addition, we also asserted that some of the residues in the larger scorpion K+-channel blockers, which contain 31 to 40 amino acids, are clearly not involved in K+-channel blocking activity.

The solution conformation of a synthetic cyclic decapeptide [with sequence mimicking the third disulfide loop of rat transforming growth factor-alpha (rTGF-alpha)] in deuterated dimethyl sulfoxide was studied by 2D NMR. The determination of solution structures was based on NOE interproton distances, using a combination of distance geometry and simulated annealing protocols. The convergence of the selected structures was evident from the small atomic pairwise root-mean-square deviation values among them. Good agreement was noted between the experimental and simulated NOESY spectra, thereby reflecting the accuracy of the calculated solution structures. Analysis of the structures indicates that the residues Tyr5 and Arg9 exhibit similar side chain orientation as that in the corresponding disulfide loop of human transforming growth factor-alpha.

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.

The functional components in soymilk may vary depending upon the fermentation process. A fermented soymilk product (FSP) obtained by incubation with the microorganisms of intestinal microflora was found to reduce the risk of breast cancer. Guided by the inhibitory activities against breast cancer cells, two cytotoxic compounds, daidzein and (S)-latifolicinin A, were isolated from the FSP by repetitive extraction and chromatography. Latifolicinin A is the n-butyl ester of β-(4-hydroxyphenyl)lactic acid (HPLA). A series of the ester and amide derivatives of (S)-HPLA and L-tyrosine were synthesized for evaluation of their cytotoxic activities. In comparison, (S)-HPLA derivatives exhibited equal or superior inhibitory activities to their L-tyrosine counterparts, and (S)-HPLA amides showed better cytotoxic activities than their corresponding esters. In particular, (S)-HPLA farnesyl amide was active to triple-negative MDA-MB-231 breast cancer cells (IC50 = 27 μM) and 10-fold less toxic to Detroit-551 normal cells.

Garnets in UHP eclogites from Bixiling in Dabieshan were investigated by Fourier transform infrared spectroscopy (FTIR). The results indicate that all garnets contain structural water that occurs as hydroxyl (OH − ) and non-structural molecular water (H 2 O) possibly in the form of sub-microscopic fluid inclusions. The structural hydroxyl contents range from 92 to 1735 ppm (H 2 O wt.) and most are between 200 and 1000 ppm. Therefore, garnet in eclogite can recycle surface water into the mantle. Various water contents were observed among different samples of the same outcrop (∼150 m) and in different domains of the same sample (∼1 cm). This variability in structural H 2 O contents suggests that the mobility of fluids during UHP metamorphism was very limited, and that both subduction and exhumation processes of UHP rocks occurred in a short time interval.

Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full‐length nucleocapsid proteins (N protein) from SARS and 229E human coronaviruses. N proteins form oligomers, mostly as dimers at low concentration. These two N proteins degrade rapidly upon storage and the major degraded N protein is the C‐terminal fragment of amino acid (aa) 169–422. Taken together with other data, we suggest that N protein is a two‐domain protein, with the N‐terminal aa 50–150 as the RNA‐binding domain and the C‐terminal aa 169–422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti‐nucleocapsid protein (NP) antibodies produced were mapped to aa 1–20, aa 150–170 and aa 390–410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti‐NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross‐react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15–20 amino acids) of S, M and N proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large‐scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.

The SARS-CoV spike protein, a glycoprotein essential for viral entry, is a primary target for vaccine and drug development. Two peptides denoted HR-N(SN50) and HR-C(SC40), corresponding to the Leu/Ile/Val-rich heptad-repeat regions from the N-terminal and C-terminal segments of the SARS-CoV spike S2 sequence, respectively, were synthesized and predicted to form trimeric assembly of hairpin-like structures. The polyclonal antibodies produced by recombinant S2 protein were tested for antigenicity of the two heptad repeats. We report here the first crystallographic study of the SARS spike HR-N/HR-C complex. The crystal belongs to the triclinic space group P1 and the data-set collected to 2.98 A resolution showed noncrystallographic pseudo-222 and 3-fold symmetries. Based on these data, comparative modeling of the SARS-CoV fusion core was performed. The immunological and structural information presented herein may provide a more detailed understanding of the viral fusion mechanism as well as the development of effective therapy against SARS-CoV infection.

The SmI 2 -promoted three-component coupling reaction of thiophene-2-carboxylate, indole-2-carbaldehyde and acetophenone provides an expedient route to a series of tetracyclic carbazolothiophene compounds bearing the indole and thiophene rings. Among these samples, 9-benzyl-4-methyl-4-(4-hydroxyphenyl)-10-oxo-4,10-dihydrocarbazolo[2,3- b ]thiophene-2-carboxylic acid ( 18 ) shows the most potent inhibition against the endothelin-1 induced increase of intracellular calcium ion concentration.

The first total synthesis of ganglioside DSG-A (1) is achieved via chemoselective glycosylation and a [1 + 1 + 2] synthetic strategy. We have also developed an efficient method that can be handled on large scale (50 g) for the synthesis of the phytosphingosine.

CD8(+) T cells play important roles in anti-tumor immunity but distribution profile or functional characteristics of effector memory subsets during tumor progression are unclear. We found that, in oral squamous carcinoma patients, circulating CD8(+) T cell pools skewed toward effector memory subsets with the distribution frequency of CCR7(-)CD45RA(-)CD8(+) T cells and CCR7(-) CD45RA(+)CD8(+) T cells negatively correlated with each other. A significantly higher frequency of CD127(lo) CCR7(-)CD45RA(-)CD8(+) T cells or CCR7(-)CD45RA(+)CD8(+) T cells among total CD8(+) T cells was found in peripheral blood or tumor infiltrating lymphocytes, but not in regional lymph nodes. The CD127(hi) CCR7(-)CD45RA(-)CD8(+) T cells or CCR7(-)CD45RA(+)CD8(+) T cells maintained significantly higher IFN-γ, IL-2 productivity and ex vivo proliferative capacity, while the CD127(lo) CCR7(-)CD45RA(-)CD8(+) T cells or CCR7(-)CD45RA(+)CD8(+) T cells exhibited higher granzyme B productivity and susceptibility to activation induced cell death. A higher ratio of CCR7(-)CD45RA(+)CD8(+) T cells to CCR7(-)CD45RA(-)CD8(+) T cells was associated with advanced cancer staging and poor differentiation of tumor cells. Therefore, the CD127(lo) CCR7(-)CD45RA(-)CD8(+) T cells and CCR7(-)CD45RA(+)CD8(+) T cells are functionally similar CD8(+) T cell subsets which exhibit late differentiated effector phenotypes and the shift of peripheral CD8(+) effector memory balance toward CCR7(-)CD45RA(+)CD8(+) T cells is associated with OSCC progression.

The 24p3 protein is a 25 KDa glycoprotein, having been purified from mouse uterine fluid. Thr54, Ser88, and Thr128/Ser129 on the protein molecule were predicted to be the phosphorylation site of casein kinase II, protein kinase C, and cAMP-dependent protein kinase, respectively. Incorporation of phosphate to this protein from [gamma-32P]-ATP was tested in the solution suitable for the three kinases. Neither casein kinase II nor cAMP-dependent protein kinase reacted to the 24p3 protein; however, protein kinase C demonstrated phosphorylation to this protein. This phosphorylation may be competing with a polypeptide segment: Arg79-Tyr-Trp-Ilu-Arg-Thr-Phe-Val-Pro-Ser88-Ser-Arg-Ala-Gly-Gln-Phe-Thr-Leu-Gly97 in the 24p3 protein molecule. To support this theory, Ser88 is a phosphorylation site of protein kinase C on 24p3 protein. The enzyme kinetic parameter, based on the Michaelis-Menten equation, determined Km to be 2.96 microM in the phosphorylation of 24p3 protein by the kinase. Both of the phosphorylated and dephosphorylated form of 24p3 protein can enhance the cAMP-dependent protein kinase activity in vitro. In addition, this experiment will show for the first time that serine-phosphorylated 24p3 protein exists in mouse uterine tissue.

New enzymatic reactions in supercritical fluid carbon dioxide catalyzed by lipases (PPL, Lipase MY, Candida cylindracea Lipase), and Proteases (subtilisin Carlsberg, subtilisin 8397, immobilized papain) with high efficiency and yields in a simple high pressure reactor using readily available dry-ice have been developed.

Microwave heating involves direct absorption of energy by functional groups that bear ionic conductivity or a dipole rotational-effect, and this energy is then released into the surrounding solution. This absorption of energy causes the functional groups involved to have higher reactivity to other surrounding reactants than when they are simply incubated with the reactants at the same temperature. In other word the enhanced rate of the reaction can be due to the reactant stirred by the molecular dipole rotation and molecules themselves acting as a stirring bar. In contrast to conventional heating, the salient feature of “dipole rotation” constitutes one efficient form of “molecular agitation” or “molecular stirring” many aspects of which can be explore in chemical reactions. We will discuss some of the useful applications of this “molecular agitation” by means of microwave irradiation. Using this unique technology, we have developed: 1) a method to control the cleavage sites of peptide bonds, especially those bonds connected to aspartic acid residues inside the native peptides and proteins, 2) a method to increase coupling efficiency in solid-phase peptide synthesis using a common microwave oven, 3) a novel procedure that increases the rate of alcalase-catalyzed reactions using microwave irradiation in peptide-bond formation with proline as a nucleophile and selective benzoylation of a pyranoside derivative, 4) a procedure to solubilize and hydrolyze retrograded starch, 5) a novel procedure to enhance the rate of saponification in a serum sample for very long chain fatty acid analysis.

This study demonstrates that compounds 1-4 from an extract of Plectranthus amboinicus inhibit the binding of AP-1 to its consensus DNA sequence. Thymoquinone (5) was further identified as a nonpolar ingredient from the hexane extract of P. amboinicus to suppress the expression of lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-α). We then synthesized 2-alkylidenyl-4-cyclopentene-1,3-diones as the designed biomimetics of thymoquinone, and found that compounds 8a, 8b and 8d were more potent TNF-α inhibitors.

To develop a new technique of bilateral angiography in a single radial access (BASiRalA) which can reduce a puncture site.From March 2011 to February 2012, 13 cases of coronary heart disease patients with chronic total occlusion (CTO) were treated (6 CTOs in right coronary artery and 7 in left anterior descending artery). All patients underwent percutaneous coronary intervention (PCI) via the right radial artery access and 6 F guiding catheters were delivered to the diseased artery. Once the wires crossed the CTO lesions and were uncertain if the wires were in true lumen or not, BASiRalA was performed. The Finecross microcatheters were advanced out of the 6 F guiding catheter, then withdraw 6F guiding catheter to the opening of diseased artery, the soft wires were manipulated into the middle portion of opposite coronary artery. After that, the microcatheters were advanced to this segment or the branches relative to the collateral vessels connected with CTOs. After pulling out the wires, microcatheter injections can be performed for contralateral angiography. BASiRalA related complications were observed after the procedure.BASiRalA technique was applied to 13 CTOs and 10 procedures succeeded (76.92%). BASiRalA failed in 3 cases and the wires and microcatheters could not be advanced to the opposite coronary arteries within 20 minutes. Alternatively, contralateral angiography via femoral arteries was performed in these 3 patients. The average time of BASiRalA technique was 7 (5 - 13) minutes and the shortest time of wires crossing to the opposite coronary artery was 5 seconds. There was no procedure induced complication during procedure or post procedure.BASiRalA technique is feasible in treating CTO patients by PCI.

Mastoparan B (MP-B) is a cationic tetradecapeptide isolated from the black-bellied hornet (Vespa basalis) venom. It has a primary structure (LKLKSIVSWAKKVL-CONH2) distinct from other vespine mastoparans. The peptide caused a dose-dependent swelling in rat hind paw and showed a potent hemolytic activity in guinea pig red blood cells. Studies on the structure activity relationship of the peptide showed that replacing lysine at position 2 (Lys2) by asparagine (Asn) in the MP-B sequence caused about 40% decrease in its edema-inducing activity at 50 micrograms/paw and 90% decrease in hemolytic activity at 30 microM of the peptide, while the same substitution at Lys4 did not cause a significant change in either activity. Replacing either Lys11 or Lys12 by leucine (Leu) caused little or no decrease in the edema-inducing and hemolytic activities. Decreases in both activities were observed when both Lys11 and Lys12 were replaced by Leu. On the other hand, replacing tryptophan at position 9 (Trp9) by tyrosine or phenylalanine in MP-B sequence almost abolished its hemolytic activity, while the edema-inducing activity was only partially inhibited. Circular dichroism spectra of the peptides measured in 20% trifluoro-ethanol revealed that substitution of Lys and Trp did not cause a significant change in the conformation of MP-B. it appears that Lys2 is crucial for both hemolytic and edema-inducing activities of MP-B, while Trp9 is of special importance to the hemolytic activity of MP-B. Lys11 and Lys12 in MP-B probably play a lesser role in both activities.

The solution conformation of a synthetic snake venom toxin waglerin I, has been determined by using proton nuclear magnetic resonance spectroscopy. By a combination of various two-dimensional NMR techniques, the 1H-NMR spectrum of waglerin I was completely assigned. A set of 247 interproton distance restraints was derived from nuclear Overhauser enhancement (NOE) measurements. These NOE constraints, in addition to the 2 dihedral angle restraints (from coupling constant measurements) and 7 omega torsion angel restraints for prolines, formed the basis of three-dimensional structure determined by molecular dynamics techniques. The 19 structures that were obtained satisfy the experimental restraints, and display small deviation from idealized covalent geometry. Analysis of converged structures indicates that the toxin has no special secondary structure. In the solution structure of waglerin I, the central ring region is well defined but the N- and C-termini possesses more disorder.

Mastoparan-B, a tetradecapeptide isolated from the venom of the hornet Vespa basalis , belongs to the mastoparan analogs of vespid venom with the lysine residues common for all mastoparan family toxins at positions 4, 11 and 12. Here we use 1 H-NMR spectroscopy and hybrid distance geometry-simulated annealing calculation to investigate its three-dimensional structure in trifluoroethanol-containing aqueous solution. The calculated structure shows that residues 3–14 adopt an amphiphilic α-helical structure in which the residues with hydrophilic side chains (i.e. Lys-4, Ser-5, Ser-8, Lys-11, Lys-12) are located on one side and the residues with hydrophobic side chains (i.e. Leu-3, Ile-6, Trp-9, A a-10, Val-13, Leu-14) located on the other side of the molecule. The overall structural features are very similar to the conformation of mastoparan-X reconstituted in vesicles [Wakamatsu et al. (1992) Biochemistry 31, 5654–5660] in spite of the substitutions made for eight residues with distinctly different hydrophobicity. These substitutions lead to a larger hydrophobic moment for the α-helical segment and further mobilized N-terminal. This study will help reveal the conformational significance of mastoparan toxins with respect to their potency and activity in G protein regulation.

Abnormally high concentrations of Zn(2+), Cu(2+), and Fe(3+) are present along with amyloid-β (Aβ) in the senile plaques in Alzheimer disease, where Al(3+) is also detected. Aβ aggregation is the key pathogenic event in Alzheimer disease, where Aβ oligomers are the major culprits. The fundamental mechanism of these metal ions on Aβ remains elusive. Here, we employ 4,4'-Bis(1-anilinonaphthalene 8-sulfonate) and tyrosine fluorescence, CD, stopped flow fluorescence, guanidine hydrochloride denaturation, and photo-induced cross-linking to elucidate the effect of Zn(2+), Cu(2+), Fe(3+), and Al(3+) on Aβ at the early stage of the aggregation. Furthermore, thioflavin T assay, dot blotting, and transmission electron microscopy are utilized to examine Aβ aggregation. Our results show that Al(3+) and Zn(2+), but not Cu(2+) and Fe(3+), induce larger hydrophobic exposures of Aβ conformation, resulting in its significant destabilization at the early stage. The metal ion binding induces Aβ conformational changes with micromolar binding affinities and millisecond binding kinetics. Cu(2+) and Zn(2+) induce similar assembly of transiently appearing Aβ oligomers at the early state. During the aggregation, we found that Zn(2+) exclusively promotes the annular protofibril formation without undergoing a nucleation process, whereas Cu(2+) and Fe(3+) inhibit fibril formation by prolonging the nucleation phases. Al(3+) also inhibits fibril formation; however, the annular oligomers co-exist in the aggregation pathway. In conclusion, Zn(2+), Cu(2+), Fe(3+), and Al(3+) adopt distinct folding and aggregation mechanisms to affect Aβ, where Aβ destabilization promotes annular protofibril formation. Our study facilitates the understanding of annular Aβ oligomer formation upon metal ion binding.

Amyloid formation is initiated by protein misfolding, followed by self-association to ultimately form amyloid fibrils. The discovery of toxic prefibrillar oligomers in many amyloidosis underscores the importance of understanding the folding mechanism prior to such aggregation. Here, we investigated the folding properties of the natively unfolded amyloid-β (Aβ) peptide and the familial variants (A21G, E22Q, E22G, E22K, and D23N) in Alzheimer's disease (AD). In combinations of native electrophoresis, analytical ultracentrifugation, fluorescence emission, and far-UV circular dichroism, we showed that all Aβ40 variants are predominantly monomeric with similar residual secondary structures, but distinct hydrophobic-exposed protein surfaces. Guanidine hydrochloride (GdnHCl) denaturation in the absence and presence of trifluoroethanol (TFE) showed that Aβ variants adopt an apparent 2-state equilibrium model with different stabilities, in which wild type is less stable than A21G but more stable than D23N and E22 mutants. By correlating the folding stability with the nucleation phase in fibrillization, we found the more stable the variant, the slower the nucleation, except for D23N. Besides, the unfolding of Aβ conformation leads to reduced formation of mature fibrils, but an increase in nonfibrillar, amorphous type of aggregates. Overall, we demonstrated that folding stability of Aβ is an important determinant of the nucleation kinetics.

Dermal papilla cells (DPCs) control the development of hair follicles via cell–cell interactions and extracellular molecules. Colchicine affected active anagen DPCs to result in hair loss in the clinical setting. The purpose of this study was to identify the retro-modulator released by DPCs exposed to sub-toxic dose of colchicine and elucidate its effect on dermal papilla culture. The molecular-weight cutoff ultrafiltration and HPLC were used to purify the components of colchicine-treated DPC secretomes and examined their ability to down-regulate the growth and alkaline phosphatase (ALP) activity of DPCs. The active product was identified by in-gel trypsin digestion, nano-LC-ESI-MS/MS and validated by Western blot to be histone H4 ( P62804 ), which inhibited the proliferation and diminished the ALP activity of cultured DPCs. Treating DPCs with recombinant histone H4 reproduced the growth inhibition effect whereas adding antibody to immunoneutralize histone H4 abolished this growth inhibitory consequence. DPCs with high ALP activity can induce the neogenesis of hair follicles and support the hair fiber growth in vivo . Our results indicated that sub-lethal colchicine can inactivate DPCs through releasing histone H4. Through the investigation of the retro-modulation of histone H4 on dermal papillae may give implications for understanding the mechanism of colchicine-induced hair disorder.

Procedures have been developed for increasing coupling efficiency in solid-phase peptide synthesis by microwave irradiation using a kitchen microwave oven