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Gene therapy is a potent tool for the treatment of cancer and other gene defect diseases, which involves using DNA that encodes a functional, therapeutic gene to replace a mutated gene. However, the DNA transfection efficiency is restricted by its negative charges and low susceptibility to endonucleases which prevent them penetrating tissue and cellular membranes. Both viral and non-viral vectors have been used for gene delivery, but the former are limited by their immunogenicity, while the latter are less efficient than their viral counterpart. Cationic amphiphilic lipopeptides whose structures can be easily modified and transformed have been used as non-viral vectors in gene delivery system due to their low cytotoxicity and high transfection efficiency. In this study, a series of cationic amphiphilic N-phosphoryl oligopeptides with varied lengths of hydrophobic tails and oligopeptide headgroups (C12-K6, C14-K6, C16-K6, Chol-K6 and C12-H6) were synthesized and used as gene delivery vectors. The affinities, abilities to condense pDNA and transfection efficiencies of the K6-lipopeptides were better than those of the H6-lipopeptides. In addition, the hydrophobic chains of the lipopeptides also affected their transfection efficiencies. The K6-lipopeptide with a hydrophobic chain of twelve carbons (C12-K6) showed the highest transfection efficiency in all these synthetic lipopeptides. At an optimal P/N ratio of 20, C12-K6 showed comparable pDNA transfection efficiency to PEI-25k, a well-defined gene delivery vector, but the cytotoxicity of C12-K6 was much lower. With acceptable gene transfection efficiency and low cytotoxicity, this cationic amphiphilic lipopeptide will have promising applications in gene therapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: We aimed to determine the efficacy and safety of endoscopic nasobiliary drainage (ENBD) with or without endoscopic sphincterotomy (EST) for temporary biliary decompression in patients with acute obstructive cholangitis., Methods: In total, 72 patients with acute obstructive cholangitis were prospectively randomized to undergo emergency ENBD with EST (EST group, n = 36) or without EST (non-EST group, n = 36). The clinical outcomes and complications between the two groups were compared., Results: Endoscopic nasobiliary decompression was successful in all 72 patients. Four patients underwent a second endoscopic retrograde cholangiopancreatography (ERCP) to replace the nasobiliary catheter due to blockage (one in the EST group and two in the non-EST group) or migration (one in the EST group). The mean serum γ-glutamyltransferase and total bilirubin levels after treatment were significantly higher in the non-EST group than in the EST group (P < 0.05). However, no significant differences were observed for other parameters evaluated. The total complication rate was similar between the two groups (EST 25.0% vs non-EST 19.4%). Although hemorrhage occurred more frequently in the EST group and acute pancreatitis in the non-EST group, these differences were not significant., Conclusions: EST is helpful and safe for biliary drainage while ENBD without EST is the first choice for acute cholangitis. EST may increase the efficacy of ENBD in patients with papillary inflammatory stricture and thick bile., (© 2013 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.)

Peptide-based hydrogels have gained much interest for biomedical applications as a result of their biocompatibility. Herein, we reported a synthetic pH-sensitive and calcium-responsive peptide-amphiphilic hydrogel. The sequences of the peptide amphiphiles were derived from the repeat-in-toxin (RTX) motif. At a certain peptide-amphiphile concentration, self-assembly was accompanied by the formation of a rigid, viscoelastic hydrogel at low pH or the presence of calcium ions. Circular dichroism spectra showed that the peptide amphiphiles adopted beta-sheet structure. Meanwhile, as revealed by transmission electron microscopy, the peptide-amphiphile self-assembly was accompanied by the formation of long interconnected nanofibrillar superstructure. Material properties of the resulting peptide-amphiphile hydrogel were characterized using oscillatory sheer rheology, and the storage modulus (G') was found to be one order of magnitude higher than the loss modulus (G"), indicating a moderately rigid viscoelastic material. Furthermore, with systematical residue substitution, it was found that the aspartic acid within the repeat-in-toxin sequence of peptide amphiphiles was responsible for the pH and calcium selectivity. The environmental responsiveness, secondary structure, morphology, and mechanical nature of the peptide-amphiphile hydrogel make it a possible material candidate for biomedical and engineering application., (Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.)

Objective: To study the correlation between the expression of Fcgamma receptor II b (FcgammaRII b) on B cells and rheumatoid arthritis (RA) patients of Shen deficiency syndrome (SDS)., Methods: There were 43 RA patients, including 26 of SDS and 17 of non-SDS. The expression levels of FcgammaRII b on naive B cells, memory B cells, and plasma blasts in the peripheral blood were detected by flow cytometry. The numbers of tender joints, numbers of swollen joints, erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and disease activity score (DAS28), the correlation between the distribution of B cells and the expression level of FcgammaRII b in RA patients were analyzed. Besides, another 21 healthy volunteers were recruited as the control group., Results: The expression level of FcgammaRII b was 49.65% +/- 15.86% on memory B cells and 43.69% +/- 22.57% on plasma blasts in RA patients of SDS, significantly down-regulated when compared with those of the control group (64.03% +/- 6.01%, 66.59% +/- 10.18%, P < 0.01). The expression level of FcgammaRII b on memory B cells of RA patients of non-SDS was down-regulated more obviously when compared with that of the control group (52.70% +/- 9.52% versus 64.03% +/- 6.01%, P < 0.01). The expression level of FcgammaRII b on plasma blasts was obviously lower in RA patients of SDS than in RA patients of non-SDS (56.10% +/- 17.05%, P < 0.05). The expression level of FcgammaRII b on memory B cells was not correlated with numbers of tender joints, numbers of swollen joints, ESR, RF, or DAS28., Conclusions: The defective immunological tolerance of B cells in RA patients of SDS might be closely correlated with down-regulation of FcgammaRII b on memory B cells and plasma blasts. There might exist genetic abnormality of FcgammaRII b gene in RA patients of SDS, thus inducing loss of autoimmunity tolerance.

Objective: To investigate the correlation between auto-immune antibodies and rheumatoid arthritis (RA) patients of Shen deficiency syndrome (SDS), thus providing clinical evidence for further researches on molecular biological mechanisms of RA patients of SDS., Methods: Totally 451 RA patients were assigned to the SDS group and the non-SDS group. Their general conditions (including gender, age, duration, and age of onset), C reactive protein (CRP), erythrocyte sedimentation rate (ESR), platelet (PLT), disease activities (DAS28), auto-antibodies [rheumatoid factor (RF), anti-CCP antibodies, anti-nuclear antibody (ANA)] were compared between the two groups., Results: (1) The scores for EST, PLT, and DAS28 were obviously higher in the SDS group than in the non-SDS group (P < 0.05, P <0. 01). (2) The level of average RF was (697.32 +/-1 061.38 IU/mL) in the SDS group, higher than that in the non-SDS group (439.91 +/- 672.24 IU/mL, P <0.01). There was no statistical difference in anti-CCP antibody between the two groups (P >0.05).(3) The ANA positive rate of RA patients was 29. 63% (120/405). It was 37.19% (74/199) in RA patients of SDS and 22. 33% (46/206) in RA patients of non-SDS, showing statistical difference between the two groups (P <0.01). (4) The odds ratio for high level RF positive and ANA positive was 1. 574 and 2. 059 folds in RA patients of SDS as high as that in RA patients of non-SDS., Conclusions: RA patients of SDS would have higher risk of having auto-immune antibodies, fastened development, more worsen joint damage, and more poor prognosis. Its mechanisms might be closely associated with autoimmune tolerance.

Dimerization of the transmembrane (TM) adaptor protein DAP12 plays a key role in mediating activation signals through TM-TM association with cell-surface receptors. Herein, we apply the TOXCAT assay and molecular dynamics simulation to analyze dynamics and dimerization of the TM helix of DAP12 in the membrane bilayer. In the TOXCAT assay, we performed site-specific mutagenesis of potential dimerization motifs in the DAP12 TM domain. Instead of the common GxxxG dimerization motif, mutating either of the polar residues Asp-50 and Thr-54 significantly decreased the TOXCAT signal for the dimerization of DAP12 TM domain. Furthermore, through the conformational difference between wild-type and mutant DAP12 TM homodimers, a combined coarse-grained and atomistic molecular dynamics simulation has identified both Asp-50 and Thr-54 at the dimerization interface. The experimental and computational results of the DAP12 TM dimer are in excellent agreement with the previously reported NMR structure obtained in detergent micelles. Such a combination of dynamics simulation and cell-based experiments can be applied to produce insights at the molecular level into the TM-TM association of many other transmembrane proteins., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Endoscopic biliary stent placement during the procedure of endoscopic retrograde cholangiopancreatography (ERCP) is preferred to provide biliary drainage for unresectable malignant biliary obstruction. There is considerable controversy over the use of endoscopic sphincterotomy (ES) prior to stent placement. This study aimed to determine whether ES before intraductal self-expanding metal stent (SEMS) placement affects the clinical outcome and complications in patients with proximal malignant obstructive biliary diseases., Methods: In a prospective randomized controlled trial, 82 patients with inoperable malignant biliary strictures were randomly assigned to biliary stenting groups with or without ES. Resolution of jaundice and the incidence of complications including acute cholangitis, pancreatitis and stent occlusion within 6 months were evaluated., Results: SEMSs were successfully deployed in all patients, resulting in clinical and biochemical improvement of obstructive symptoms in both groups. The incidence of cholangitis was higher in the ES group than in the non-ES group (58.5% vs 31.7%, P=0.015). The interval between stent placement and the first acute cholangitis was much shorter in the ES group than in the non-ES group (P=0.024). The use of ES increased the incidence of cholangitis (P=0.004, risk ratio, 8.196). The rate of post-ERCP pancreatitis after stent placement was greater in the non-ES group than in the ES group (31.7% vs 9.8%, P=0.014). No significant differences were found in the rate of restenosis and the mortality rate between the two groups., Conclusions: ES prior to intraductal SEMS placement was associated with an increased incidence of acute cholangitis. ES should be carefully evaluated prior to its use in patients with proximal malignant obstructive biliary diseases.

Although genetically modified (GM) food is becoming increasingly available, consumers are showing a growing awareness about the need to identify GM and non-GM foodstuff: the reliable identification of GM/non-GM food is therefore an important tool in the social, health and safety debates. The present research responds to this need (i) through developing a novel "single-pot" preparation of PAMAM magnetite nanoparticles (PMNPs) and by fully defining their specific characteristics; (ii) by demonstrating the capability of the PMNPs to isolate genomic DNA from different sample foods; and (iii) by experimentally demonstrating the identification of the isolated DNA by gel-electrophoresis, thus being capable of screening GM and non-GM food., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We report herein a new class of metal ion chemosensors and give the first example of a metal-dependent peptidase chemosensor for metal ions. The chemosensor contains the basic specific Ni(II)-dependent peptide bond hydrolysis sequence (Gly-Ala-Ser-Arg-His-Trp-Lys-Phe-Lys). The substrate was labeled with a fluorophore at the N-terminal and a quencher at the C-terminal Lys side chain. Initially, the MOCAc ((7-methoxycoumarin-4-yl)acetyl-) emission was quenched by the nearby quencher. In the presence of Ni(II), the substrate was irreversibly cleaved at the cleavage site, leading to a 20-fold increase in fluorescence intensity. The chemosensor combines the high selectivity of a peptidase (at least greater than tenfold for Ni(II) over other metal ions) with the high sensitivity of fluorescence detection limit of 50 nM and can be applied for the quantitative detection of Ni(II) over a concentration range of three orders of magnitude. Given this degree of selectivity and sensitivity, our molecular engineering design may prove useful in the future development of other peptidase-based probes for different metal ions in toxicological and environmental monitoring.

A new fluorescent chemosensor for Cu(II) ions was designed and synthesized on the basis of the sequence-specific cleavage of the peptide bond by the peptidase (metal or metal complexes). In the chemosensor system, the substrate was labeled with a FAM fluorophore (6-carboxyfluorescein) at the N-terminal and with a Dabcyl quencher 4-(4'-dimethylaminophenylazo)benzoic acid at the ε-N of C-terminal Lys. In the presence of Cu(II), the substrate strand is cleaved, and the release of the cleaved fragment results in a significant fluorescence increase. The design was aided by the FRET study that showed a "turn-on" response for Cu(II) in an aqueous medium. Under optimum conditions, the novel chemosensor described here had a linear response range for Cu(II) from 1.0 × 10(-8) to 1.0 × 10(-6) mol dm(-3) with a detection limit of 1.0 × 10(-8) mol dm(-3).

Experiments with the transmembrane (TM) domains of the glycoprotein (GP) Ib-IX complex have indicated that the associations between the TM domains of these subunits play an important role in the proper assembly of the complex. As a first step toward understanding these associations, we previously found that the Ibβ TM domain dimerized strongly in Escherichia coli cell membranes and led to Ibβ TM-CYTO (cytoplasmic domain) dimerization in the SDS-PAGE assay, while neither Ibα nor IX TM-CYTO was able to dimerize. In this study, we used the TOXCAT assay to probe the Ibβ TM domain dimerization interface by Ala- and Leu-scanning mutagenesis. Our results show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids. Mutating either one of polar residues Gln129 or His139 to Leu or Ala disrupted Ibβ TM dimerization dramatically, indicating that polar residues might form part of the leucine zipper-based dimerization interface. Furthermore, these specific mutational effects in the TOXCAT assay were confirmed in the thiol-disulfide exchange and SDS-PAGE assays. The computational modeling studies further revealed that the most likely leucine zipper interface involves hydrogen bonding of Gln129 and electrostatic interaction of the His139 side chain. Correlation of computer modeling results with experimental mutagenesis studies on the Ibβ TM domain may provide insights for understanding the role of the association of TM domains on the assembly of GP Ib-IX complex., (Copyright © 2011 The Protein Society.)

Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIbα, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIbα sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIbα, GPIbβ, and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIbβ is critical for ADAM17-dependent shedding of GPIbα induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIbα and GPIbβ subunits further lessened the restraint of GPIbβ on GPIbα shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIbβ cytoplasmic domain disrupted calmodulin binding to GPIbβ but had little effect on GPIbα shedding. Analysis of induction of GPIbα shedding by membrane-permeable GPIbβ-derived peptides implicated the association of GPIbβ with an unidentified intracellular protein in mediating regulation of GPIbα shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.

Fe(3)O(4)@Fe(OH)(3) composite microspheres are highly active, environmentally friendly and easy to recycle catalysts for aldol reactions, which are catalyzed by a solid-liquid interfacial hydrogen bond catalyst at room temperature.

Platelet glycoprotein (GP) Ib-IX complex requires all its three subunits for efficient expression on the cell surface, but the underlying molecular basis is not fully clear. Using transfected Chinese hamster ovary cells as the model system, we demonstrate that juxtamembrane residues 149-154 in the cytoplasmic domain of the GPIbbeta subunit is required for assembly and surface expression of the GPIb-IX complex. The complex, or GPIbbeta by itself, lacking these residues is retained in the endoplasmic reticulum. Our results thus have illustrated an important role of the GPIbbeta cytoplasmic domain in biosynthesis of the GPIb-IX complex.

As the receptor on the platelet surface for von Willebrand factor, glycoprotein (GP) Ib-IX complex is critically involved in hemostasis and thrombosis. How the complex is assembled from GP Ibalpha, GP Ib beta and GP IX subunits, all of which are type I transmembrane proteins, is not entirely clear. Genetic and mutational analyses have identified the transmembrane (TM) domains of these subunits as active participants in assembly of the complex. In this study, peptides containing the transmembrane domain of each subunit have been produced and their interaction with one another characterized. Only the Ib beta TM sequence, but not the Ibalpha and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ib beta-Ibalpha-Ib beta peptide complex (alphabeta(2)) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibalpha and Ib beta TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native alphabeta(2) complex, reproducing the same effect by the IX subunit in cells expressing the GP Ib-IX complex. Specific fluorescence resonance energy transfer was observed between donor-labeled alphabeta(2) peptide complex and acceptor-conjugated IX TM peptide in micelles. Finally, the mutation D135K in the IX TM peptide could hamper both the formation of the alphabeta(2) complex and the energy transfer, consistent with its reported effect in the full-length complex. Overall, our results have demonstrated directly the native-like heteromeric interaction among the isolated Ibalpha, Ib beta and IX TM peptides, which provides support for the four-helix bundle model of the TM domains in the GP Ib-IX complex and paves the way for further structural analysis. The methods developed in this study may be applicable to other studies of heteromeric interaction among multiple TM helices.

Background: In the platelet glycoprotein (GP)Ib-IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted to the N-terminal domain of the GPIbalpha subunit. How the other subunits in the complex, GPIbbeta and GPIX, regulate the GPIbalpha-VWF interaction is not clear., Objectives and Methods: As GPIbalpha connects with two GPIbbeta subunits via disulfide bonds, we tested whether these intersubunit covalent links were important to the proper VWF-binding activity of the GPIb-IX complex by characterizing the structure and VWF-binding activity of a mutant GPIb-IX complex that lacked the GPIbalpha-GPIbbeta disulfide bonds., Results: Mutating both Cys484 and Cys485 of GPIbalpha to serine prevents GPIbalpha from forming covalent disulfide bonds with GPIbbeta, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIbbeta subunits to form a disulfide bond between themselves. As compared to Chinese hamster ovary (CHO) cells stably expressing the wild-type GPIb-IX complex at a comparable level, CHO cells stably expressing the mutant GPIb-IX complex bind to significantly less soluble VWF in the presence of ristocetin and roll on the immobilized VWF under flow at a higher velocity., Conclusions: The disulfide bonds between GPIbalpha and GPIbbeta are necessary for optimal GPIbalpha binding to VWF. The structural plasticity around the disulfide bonds may also help to shed light on the inside-out mechanism underlying GPIbbeta modulation of VWF binding.

Purpose: This study focusses on a promising carrier system for therapeutic and imaging purposes using meso-tetrakis[3,4-bis(carboxymethyleneoxy)phenyl] porphyrin (T(3,4)CPP). To assess its potential for clinical use, we labelled T(3,4)CPP with (188)Re and analysed some kinetic biodistribution parameters after intravenous injection in mice., Materials and Methods: T(3,4)CPP was synthesized and labelled with (188)Re. Normal Kunming (KM) mice and melanoma- or hepatoma-bearing BALB/c nude mice were injected intravenously with 5.55 MBq (188)Re-labelled T(3,4)CPP and sacrificed at 0.5, 2, 4, and 24 h and 8, and 24 h, respectively., Results: The (188)Re-T(3,4)CPP yield was more than 95% with specific activity 16.9 GBq (mol)(-1), and Vitamin C (VC) could increase its stability in vitro. In normal KM mice, (188)Re-T(3,4)CPP had fast blood clearance (approximately 99%, 24 h postinjection), low retention in the vital organs and hepatotropic characteristics. In nude mice, more than 4.4 and 6.1% uptake per gram of tumour (%ID g(-1)) at 8 h postinjection was in melanoma and hepatoma, respectively; this remained as high levels after 24 h as 4.6 and 6.5%, respectively. At 8 h, the tumour/blood and tumour/muscle (T/M) ratios in melanomas and hepatoma bearing mice were 7.3, 13,and 7.0, 20, respectively. Twenty-four hours later, these high ratios still continued in existence which were 9.6, 19 and 10, 25, respectively., Conclusion: The results obtained in this study indicate that (188)Re-T(3,4)CPP has better tumour affinity and retainable accumulation characteristics in carcinoma which can potentially be used for tumour-targeted radiotherapy.

Background: The glycoprotein (GP) Ib-IX complex is critically involved in platelet adhesion to von Willebrand factor and in the initial step of platelet activation. How this complex is assembled is not clear. We previously showed that the transmembrane (TM) domains of the GPIbalpha and GPIbbeta subunits interact and participate in complex assembly., Objectives and Methods: Here, we have investigated the role of the TM and cytoplasmic domains of GPIX in assembly of the GPIb-IX complex, by analyzing the mutational effects on complex expression and assembly in transiently transfected Chinese hamster ovary cells., Results: Replacing the cytoplasmic domain of GPIX with a poly-alanine sequence had little effect on surface expression and structural integrity of the GPIb-IX complex. In contrast, replacing the GPIX TM domain (residues 132-153) with a poly-leucine-alanine sequence markedly disrupted complex formation of GPIX with GPIbalpha, interfered with GPIb formation, and decreased surface expression of the host complex. We further analyzed the contributions of a number of GPIX TM residues to complex formation by mutagenesis and found significant roles for Asp135 and several Leu residues., Conclusions: The TM domain, rather than the cytoplasmic domain, of GPIX plays an important role in expression and assembly of the GPIb-IX complex by interacting with its counterparts of GPIb. These TM domains may form a parallel four-helical bundle structure in the complex.

Objective: To investigate the biological character of new agent 153Sm-citrate-nano-Hydroxyapatite (nano-HA) in vitro and in vivo, and to explore a new radiopharmaceutical for the target therapy of bone metastasis cancer., Methods: The nano-HA was synthesized by collosol-gelatum method, and evaluated by transmission electron microscopy (TEM) and X-ray diffraction (XRD). 153Sm was produced at a high specific activity and excellent radionuclidic purity, with which nano-HA was labeled by citrate transfer ligands in the best environment. By adopting the independent variable method, we also studied the optimally labeled condition and stability of 153Sm-citrate-nano-HA in vitro. And 153Sm-citrate-nano-HA was injected into normal rabbits for radio scanning performed. The cancer cell SMMC-7721 and MCF-7 cell lines were divided into two groups, and treated with nano-HA, 153Sm-citrate and 153Sm-citrate-nano-HA in vitro respectively, of which the cell survival rate was measured by MTT methods., Results: Through the detections of TEM, XRD showed that nano-HA consisted of needle-like microcrystals. The label rate of 153Sm-citrate-nano-HA was more than 95%, and extremely stable in vitro. The radio scanning of normal rabbits showed the skeletal system to be clear, and other systems, for example liver, spleen and kidney, could also be seen. The cell culture experiments in vitro indicated that 153Sm-citrate-nano-HA strongly inhibited the proliferation of SMMC-7721 and MCF-7 cells. 153Sm-citrate-nano-HA's half effective inhibition concentrations were 1.89 mg/L and 0.094 mg/L respectively; nano-HA's half effective inhibition concentrations were 3. 31 mg/L and 0.52 mg/L respectively; 153Sm-citrate's half effective inhibition concentrations were 4. 32 mg/L and 0. 67 mg/L respectively., Conclusions: Sm-citrate-nano-HA shows fine biological properties, and is well worth for further researched and prepared as a promising potential radiopharmaceutical in nuclidic treatment for cancer bone metastases.

It is widely accepted that glycoprotein (GP) Ib contains one Ibalpha and one Ibbeta subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ibalpha, C484 or C485, forms the disulfide bond with Ibbeta. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibbeta. In the context of isolated peptides containing the Ibalpha or Ibbeta transmembrane domain and nearby Cys residue, C484 and C485 in the Ibalpha peptide were both capable of forming a disulfide bond with the Ibbeta peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibbeta subunits but only 1 Ibalpha and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ibalpha, not the number of Ibbeta subunits therein. Overall, these results demonstrate that Ibalpha is covalently connected to 2 Ibbeta subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The alphabeta2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.

Ethanol is the main by-product in the fermentation broth of Rhizopus oryzae As3.3461 for the production of high-optical purity L-lactic acid. Alcohol Dehydrogenase (ADH) is the branch pathway enzyme that catalyzes the transformation of ethanol from pyruvate in Rhizopus oryzae, which decreases the conversion rate of glucose to L-lactic acid. Thus, screening the mutants with lower ADH activity may increase lactate production dramatically. In present study, Rhizopus oryzae As3.3461 was mutated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and 21 mutants which showed lower ADH activity were isolated with selective medium of Yeast-Peptone-Dextrose (YPD) containing 0.6% allyl alcohol (V/V). Compared with other mutants, the 12th mutant strain (named as HBF-12) shows the highest conversion rate of L-lactic acid. By contrast with Rhizopus oryzae As3.3461, the parent strain, the ethanol production and the ADH activity of HBF-12 decrease 73.6% and 76%, respectively. Whereas, the L-lactic acid production and the LDH activity of HBF-12 increase 41.2% and 19.6% than those of the parent strain, respectively. The activities of ADH and LDH of HBF-12 were regulated by Zn2+ and Mg2+, but showed opposite effects. Added with Zn2+ to the concentration of 0.01% improves the ADH activity dramatically, but inhibits the activity of LDH. By contraries, added with Mg2+ improves the LDH activity markedly, but inhibits the ADH activity slightly. In fermentation experiment, the addition of Zn2+ and Mg2+ show different effects on the accumulation of ethanol, L-lactic acid and the biomass in mutant HBF-12. When improve the concentration of Zn2+, the accumulation of L-lactic acid and the biomass show the decreased trend, but the production of ethanol show positive effect. With the improvement of the concentration of Mg2+, the production of lactic acid and biomass increase markedly, but no effect on the production of ethanol. When ferment under the concentrations of Zn2+ 0.01% and Mg2+ 0.04% in fermentation medium, the lactate production of HBF-12 reached the highest level, 96.21 g/L.

Twenty-three temperate China species of Lachnum, Lachnum abnorme, L. angustum, L. brevipilosum, L. calosporum, L. calyculiforme, L. carneolum, L. ciliare, L. controversum, L. flavidulum, L. cf. fushanese, L. indicum, L. kumaonicum, L. lushanese, L. minutum, L. montanum, L. cf. pteridophyllum, L. pygmaeum, L. sclerotii var. sclerotii, L. sclerotii var. sichuanense, L. subpygmeaum, L. tenuissimum, L. virgineum and L. willisii are reported, whose main characteristics are given in a formula of the described species, some of which are discussed below.

A systematic study of the fragmentation pattern of N-diisopropyloxyphosphoryl (DIPP) dipeptide methyl esters in an electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was presented. A combination of accurate mass measurement and tandem mass spectrometry had been used to characterize the major fragment ions observed in the ESI mass spectrum. It was found that the alkali metal ions acted as a fixed charge site and expelled the DIPP group after transferring a proton to the amide nitrogen. For all the N-phosphoryl dipeptide methyl esters, under the activation of a metal ion, the rearrangement product ion at m/z 163 was observed and confirmed to be the sodium adduct of phosphoric acid mono-isopropyl esters (PAIE), via a specific five-membered penta-co-ordinated phosphorus intermediate. However, no rearrangement ion was observed when a beta-amino acid was at the N-terminal. This could be used to develop a novel method for differentiating isomeric compounds when either alpha- or beta-amino acid are at the N-terminus of peptides. From the [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters (DIPP Xaa1 Xaa2 OMe), the peaks corresponding to the [M+Na Xaa1 C3H6]+ were observed and explained. The [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters with Phe located in the C-terminal, such as DIPPValPheOMe, DIPPLeuPheOMe, DIPPIlePheOMe, DIPPAlaPheOMe and DIPPPhePheOMe, had characteristic fragmentation. Two unusual gas-phase intramolecular rearrangement mechanisms were first proposed for this fragmentation. These rearrangements were not observed in dipeptide methyl ester analogs which did not contain the DIPP at the N-terminal, suggesting that this moiety was critical for the rearrangement.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to obtain spectra of peptide-DNA complexes formed by basic domain (BD15) of c-Fos protein and DNA AP-1 site (5'-TGAGTCA-3'). The noncovalent interaction between single stranded DNA and BD15 was observed and confirmed to be an ionic one between the negatively charged sugar-phosphate backbone of DNA and positively charged side chains of Arg- and lys-rich peptides as demonstrated by Vertes and coworkers and Woods and coworkers. But the specific noncovalent interaction between DNA AP-1 site and the dimer of BD15 was firstly detected in this paper. Various different sequence DNAs were studied and it was found that this interaction is a sequence-specific one, and AP-1 site was essential for this interaction. This specific interaction depends on the matrix. It was only observed in the ATT matrix and not in the other two matrixes (CHCA and DHBA).

A reversed-phase high performance liquid chromatographic (RP-HPLC) method for the determination of the tetramethylpyrazine(TMP) in Chuanxiong extract, the animal(mouse) serum and cerebrospinal fluid has been developed. The TMP was separated on an ODS column Zorbax SB-C18(4.6 mm i.d. x 250 mm, 5 microns) at room temperature and detected by using UV detector at 270 nm. The mobile phase was methanol-water (50:50, V/V) containing 0.2 mmol/L of NH4H2PO4 flowing at a rate of 0.8 mL/min and 20 microL samples were injected. The detection limit of TMP was 1 mg/L and the calibration curve is linear between 5 and 500 mg/L with a correlation coefficient (r) of 0.999. The recovery of TMP ranged 98%-103%. The extract of Chuanxiong and pretreated serum and cerebrospinal fluid sample are stable for a week at room temperature.