The fragmentation pathways of two taxanes drugs have been studied in positive ion mode by Q-TOF with the advantages of high mass accuracy and high resolution analysis. The [M+H] + ions were observed by ESI-MS, from which the molecular weights were obtained. Using the protonated pseudo-molecular ions [M+H]+ as internal reference compounds, the accurate mass and element composition of the fragment ions were determined. The collision induced dissociation (CID) data of the [M+H] ions provided fragmentation pathways of related compounds. Results showed that the major cleavage pathways of paclitaxel and docetaxel were the same that the cleavage of C-O bond between the side chain and taxol skeleton easily occurred, then stripping of the functional groups on the parent ring. Some common fragments were formed, such as m/z 105.033 7, 291.137 3, 309.148 5, 327.159 7, 387.181 2 and 509.217 4, which would provide a basis for future qualitative and quantitative analysis of taxanes in vitro and in vivo.
Paclitaxel ; chemistry ; Peptide Fragments ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; methods ; Taxoids ; chemistry

OBJECTIVEDynamic changes of quality and growth of Whitmania pigra were investigated to provide theoretical basis for quality control and determination of optimal harvest time.

METHODThe contents of moisture, ethanol-soluble extractive, total ash, and acid-insoluble ash, as well as antithrombin activity were determined according to Chinese pharmacopoeia (2005 edition).

RESULTQualities of W. pigra collected from different growth stages met the standards of Chinese pharmacopoeia (2005 edition). The highest amount of ethanol-soluble extract was found in 9-month-old, W. pigra, followed by 6-month-aged ones. And there was significant difference between 9-month-old and 4-month-old, 7 month-old and 11 month-old (P < 0.05). The highest antithrombin activity was detected in 6 month-old W. pigra, followed by 10 month-old. Significant differences of antithrombin activity were found between 6-month-old and 4 month-old ones, and 5 month-old (P < 0.05), however, there were no significant difference between 6 month-old ones with other samples. Eleven-month-old W. pigra got the most dry weight, and there were significant difference between 11 month-old ones with other samples (P < 0.05).

CONCLUSIONNovember, namely for 6 month old artificial cultivated W. pigra was the best harvest time in Jiangsu and Zhejiang provinces.

Animals ; Antithrombin III ; Antithrombins ; analysis ; China ; Leeches ; chemistry ; growth & development ; Medicine, Chinese Traditional ; Peptide Fragments

The chronic effects of carboxyl-terminal polypeptide of Cardiotrophin-1 (CT-1-CP) on ventricular electrical remodeling were investigated. CT-1-CP, which contains 16 amino acids in sequence of the C-terminal of Cardiotrophin-1, was selected and synthesized, and then administered to Kunming mice (aged 5 weeks) by intraperitoneal injection (500 ng·g⁻¹·day⁻¹) (4 groups, n=10 and female: male=1:1 in each group) for 1, 2, 3 and 4 weeks, respectively. The control group (n=10, female: male=1:1) was injected by physiological saline for 4 weeks. The epicardial monophasic action potential (MAP) was recorded by using a contact-type MAP electrode placed vertically on the left ventricular (LV) epicardium surface, and the electrocardiogram (ECG) signal in lead II was monitored synchronously. ECG intervals (RR, PR, QRS and QT) and the amplitude of MAP (Am), the maximum upstroke velocity (Vmax), as well as action potential durations (APDs) at different repolarization levels (APD30, APD50, APD70, and APD90) of MAP were determined and analyzed in detail. There were no significant differences in RR and P intervals between CT-1-CP-treated groups and control group, but the PR segment and the QRS complex were greater in the former than in the latter (F=2.681 and 5.462 respectively, P<0.05). Though QT interval and the corrected QT interval (QTc) were shorter in CT-1-CP-treated groups than in control group, the QT dispersion (QTd) of them was greater in the latter than in the former (F=3.090, P<0.05) and increased with the time. The ECG monitoring synchronously with the MAP showed that the compression of MAP electrode on the left ventricular epicardium induced performance similar to myocardium ischemia. As compared with those before chest-opening, the PR segment and QT intervals remained basically unchanged in control group, but prolonged significantly in all CT-1-CP-treated groups and the prolongation of QT intervals increased gradually along with the time of exposure to CT-1-CP. The QRS complex had no significant change in control group, one-week and three-week CT-1-CP-treated groups, but prolonged significantly in two-week and four-week CT-1-CP-treated groups. Interestingly, the QTd after chest-opening was significantly greater than that before chest-opening in control group (t=5.242, P<0.01), but decreased along with the time in CT-1-CP-treated groups. The mean MAP amplitude, Vmax and APD were greater in CT-1-CP-treated groups than those in control group, and became more obvious along with the time. The APD in four CT-1-CP-treat groups was prolonged mainly in middle to final repolarization phase. The difference among these groups became significant in middle phase (APD50) (F=6.076, P<0.01) and increased furthermore in late and final phases (APD70: F=10.054; APD90: F=18.691, P<0.01) along with the time of injection of CT-1-CP. The chronic action of CT-1-CP might induce the adapting alteration in cardiac conductivity and ventricular repolarization. The amplitude and the Vmax of the anterior LV epicardial MAP increased obviously, and the APD prolonged mainly in late and final phase of repolarization.
Animals ; Cytokines ; chemistry ; physiology ; Electrocardiography ; Heart Ventricles ; metabolism ; Mice ; Peptide Fragments ; physiology ; Ventricular Function

BACKGROUNDWe previously reported that iodine-131((131)I)-labeled anti-pro-gastrin-releasing peptide (ProGRP(31-98)) monoclonal antibody D-D3 could selectively accumulate in the tumor sites of nude mice bearing small cell lung cancer (SCLC) xenografts. However, (131)I-D-D3 was cleared slowly from the body, and the best radioimmunoimaging time for SCLC was 72 - 96 hours after injection. The aims of this study were to radiolabel anti-ProGRP(31-98) D-D3 monoclonal antibody with technetium-99m ((99m)Tc) and to investigate the biodistribution of this antibody in healthy ICR mice.

METHODSD-D3 was labeled with (99m)Tc via the 2-mercaptoethanol reduction method. (99m)Tc-D-D3 was purified by the gel column separation method. The labeling efficiency and radiochemical purity were measured by thin-layer chromatography. The immunological activity of (99m)Tc-D-D3 was determined with cell conjugation assays. (99m)Tc-D-D3 was injected into healthy ICR mice via a tail vein, and all the healthy ICR mice were sacrificed by cervical dislocation at a designated time. Then, the blood and major organs were removed and weighed, and counted in a gamma scintillation counter to determine the percentage of the injected dose per gram (%ID/g).

RESULTSThe labeling rate and the radiochemical purity of (99m)Tc-D-D3 were (73.87 ± 2.89)% and (94.13 ± 4.49)%, respectively. The immunobinding rates of (99m)Tc-D-D3 to the human small cell lung cancer NCI-H446 cell line and lung adenocarcinoma A549 cell line were (81.2 ± 2.37)% and (24.3 ± 1.46)%, respectively. The distribution data of normal ICR mice demonstrated that (99m)Tc-D-D3 was mainly distributed in the liver, kidney and lung, and less in the brain tissue and muscle.

CONCLUSIONS(99m)Tc-D-D3 antibody not only had high radiochemical purity, but also had good stability both in vitro and in vivo, and maintained good immunological activity. (99m)Tc-D-D3 was metabolized mainly in the kidney and liver, and the blood radioactivity decreased rapidly. Thus, (99m)Tc-D-D3 is conducive to the radioimmunoimaging of SCLC.

Animals ; Antibodies, Monoclonal ; chemistry ; immunology ; metabolism ; Female ; Male ; Mice ; Mice, Inbred ICR ; Peptide Fragments ; immunology ; Recombinant Proteins ; immunology ; Technetium ; chemistry

The amyloid β-protein (Aβ)-induced disturbance of intracellular calcium homeostasis has been regarded as the final route whereby Aβ insults neurons. However, the mechanism of Aβ-induced Ca(2+) overloading is still unclear so far. Especially, it remains to be clarified whether nicotinic acetylcholine receptors (nAChRs) are involved in the Aβ-induced elevation of intracellular calcium concentration ([Ca(2+)](i)). In the present study, we observed the effects of Aβ fragments 25-35 (Aβ(25-35)) and 31-35 (Aβ(31-35)) on [Ca(2+)](i) in primary cultured rat cortical neurons using laser-scanning confocal calcium imaging technique, and investigated its probable cholinergic mechanism. The results showed that: (1) Both Aβ(25-35) and Aβ(31-35) induced similar and significant [Ca(2+)](i) elevation in a concentration-dependent manner, and no statistical difference was found between the effects of both peptides; (2) The reverse peptide of Aβ(31-35), i.e. Aβ(35-31), had no effect on [Ca(2+)](i) elevation; (3) Mecamylamine (MCA), a non-specific nAChRs antagonist, significantly and dose-dependently blocked the [Ca(2+)](i) elevation induced by Aβ(25-35) or Aβ(31-35) (4) Dihydro-β-erythroidine (D-β-E), a specific α4β2 subtype nAChRs antagonist, also significantly inhibited the [Ca(2+)](i) elevation induced by Aβ(25-35) and Aβ(31-35), but the effect was weaker than the effect of MCA at the same concentration. These results indicate that Aβ(31-35) may be a shorter active sequence in full length of Aβ molecule, and the overactivation of nAChRs, including α4β2 subtype, may be, at least partly, responsible for the Aβ-induced elevation of [Ca(2+)](i) in cultured rat cortical neurons. Thus, the present study suggests a new potential target of Aβ in the brain, and provides a new insight into the mechanisms by which Aβ impairs the cognitive function in Alzheimer's disease.
Amyloid beta-Peptides ; chemistry ; Animals ; Calcium ; metabolism ; Cells, Cultured ; Neurons ; metabolism ; Peptide Fragments ; chemistry ; Rats ; Receptors, Nicotinic ; metabolism

The huGM-CSF(9-127)-IL-6(29-184) fusion protein was precipitated on column when being purified by Q Sepharose H.P. ion exchange chromatography after renaturation by dilution. To solve this problem, a novel purification and refolding strategy was adopted. Inclusion bodies was first purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding on column by Sephacryl S-200. Renatured fusion protein was obtained in a purity of more than 95%. It was showed that the method of refolding on gel filtration column is efficient, with relative refolding rate at 80%. By the whole procedure, refolding and purification of recombinant protein can be performed within one day. This strategy is also promising to be applied in large scale purification and refolding of recombinant protein from inclusion bodies in E. coli.
Granulocyte-Macrophage Colony-Stimulating Factor ; chemistry ; isolation & purification ; Interleukin-6 ; chemistry ; isolation & purification ; Peptide Fragments ; chemistry ; isolation & purification ; Protein Folding ; Recombinant Fusion Proteins ; chemistry ; isolation & purification

OBJECTIVETo develop a novel non-viral gene delivery vector CY11-PEI-beta-CyD and to test its gene transfection efficiency.

METHODSCY11 (CGMQLPLATWY) was conjugated to polyethylenimine-beta-cyclodextrin to form CY11-PEI-beta-CyD with a cross-linker [N-succinimidy-3-(2-pyridyldithio) propionate, SPDP]. (1)H-NMR and TGA were used to confirm the structure of vector. The DNA condensing ability of CY11-PEI-beta-CyD was investigated by gel retardation assay. Cytotoxicity of CY11-PEI-beta-CyD was determined by MTT assay and transfection efficiency was investigated in COS-7, Hela and B16 cells.

RESULTCY11 was conjugated onto PEI-beta-CyD successfully, confirmed by(1)H NMR and TGA. The novel vector effectively condensed DNA at N/P ratio of 4îIt showed low cytotoxicity up to the concentration was 160 Mgr;g/ml. The transfection efficiency was 17-fold higher than that of PEI 25 kDa at N/P ratio of 20.

CONCLUSIONThe novel vector CY11 -PEI-beta-CyD with low cytotoxic and high transfection efficiency may be used as a potential carrier for gene delivery.

Cell Line ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Peptide Fragments ; chemistry ; Polyethyleneimine ; chemistry ; Receptors, Fibroblast Growth Factor ; chemistry ; beta-Cyclodextrins ; chemistry

OBJECTIVETo develop a novel gene delivery vector TAT-PEI-beta-CyD.

METHODSbeta-cyclodextrin (beta-CyD) was linked by low molecular weight (PEI 600) via 1, 1-carbonyldiimidazole (CDI), and TAT peptide (RRRQRRKKRC) was coupled to PEI 600 by [N-succinimidy-3-(2-pyridyldithio) propionate, SPDP]. The copolymer was characterized by (1)H-NMR and FT-IR. Physiochemical characteristics of TAT-PEI-beta-CyD/DNA complexes were tested by agarose gel electrophoresis and particle size measurements. Cell viability and transfection efficiency were evaluated in A293 and B16 cells using PEI 25 kDa as a control.

RESULTTAT peptide was successfully coupled to PEI-beta-CyD. The result of gel electrophoresis showed that the TAT-PEI-beta-CyD was able to condense DNA efficiently at N/P ratio of 4. The particle size of TAT-PEI-beta-CyD/DNA complexes was around 100 nm. The cytotoxicity of TAT-PEI-beta-CyD was lower than that of PEI 25 kDa. The transfection efficiency of TAT-PEI-beta-CyD was higher than that of PEI 25 kDa in A293 and B16 cells at N/P ratio of 30.

CONCLUSIONThe novel vector TAT-PEI-beta-CyD has been developed successfully with low cytotoxicity and high transfection efficiency.

Cell Line ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Peptide Fragments ; chemistry ; Polyethyleneimine ; chemistry ; beta-Cyclodextrins ; chemistry ; tat Gene Products, Human Immunodeficiency Virus ; chemistry

OBJECTIVETo investigate both PrP and PrP106-126 peptide effect on 14-3-3beta dimeration.

METHODS14-3-3beta were incubated with different does recombinant PrP or PrP106-126 peptide, both 14-3-3beta dimer and polymer were separated 15% non-denaturing polyacrylamide gel electrophoresis (PAGE) and the 14-3-3 dimers were evaluated using 14-3-3beta-specific Western blotting. And then,14-3-3beta dimeration buffer were incubated with different does recombinant PrP and 250 micromol/L PrP106-126 peptide, 14-3-3beta dimer and polymer were detected by above methods. Cellular 14-3-3 dimer were also detected after PrP106-126 peptide were added to HeLa cell for 8 hours.

RESULTSRecombinant full-length PrP facilitated the dimerization of 14-3-3beta and PrP106-126 disturbed 14-3-3beta dimeration as both have dose dependence effect. PrP antagonized PrP106-126-induced 14-3-3beta dimer with PrP protein increase in vitro. Cellular 14-3-3 dimerization also decreased after treatment of peptide PrP106-126 on HeLa cells for 8 hours.

CONCLUSION[corrected] Dimerization process of 14-3-3beta was promoted by full-length PrP (PrP23-231) but inhibited by peptide PrP106-126 in vitro. PrP agonized PrP106-126-induced inhibition of 14-3-3 dimeration. PrP106-126 inhibited cellular 14-3-3 dimerization.

14-3-3 Proteins ; chemistry ; HeLa Cells ; Humans ; Peptide Fragments ; pharmacology ; Prions ; pharmacology ; Protein Multimerization ; drug effects ; Recombinant Proteins ; pharmacology

As the dominant antigens, early secreted antigenic target 6 (ESAT6, E6) and culture filtrate protein 10 (CFP10, C10) had once been the focus of tuberculosis (TB) vaccine due to their capability of inducing strong cell immune response in the host. They are also endowed with promising future of prevention against and diagnosis of TB. In this review, we systematically introduce recent research progress of E6 and C10, especially in structure-function, biological characteristics, protein expression and secretion, host immunity and vaccine development, and the prospects of their application are also discussed.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; Bacterial Proteins ; chemistry ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; immunology ; Molecular Biology ; Peptide Fragments ; chemistry ; genetics ; immunology ; Tuberculosis Vaccines ; genetics ; immunology ; Vaccines, DNA ; immunology