Identification and quantitative analysis of beta-sitosterol oxides in vegetable oils by capillary gas chromatography-mass spectrometry

As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of beta-sitosterol oxides using capillary gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify beta-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time.     

精制原代地鼠肾细胞狂犬病疫苗制备工艺的研究

通过ａＧ 株病毒在金黄地鼠肾细胞中培养,而制备的一种新型灭活狂犬病精制疫苗已获成功。该纯化方法包括醋酸锌沉淀和Ｓｅｐｈａｒｏｓｅ ４ＦＦ柱层析,所生产的疫苗质量完全达到新型狂犬病疫苗的要求。该工艺方法简单,成本低廉,是一种理想的狂犬病疫苗生产方法。     

Synthesis and characterization of new imidophosphanes and phosphine oxides containing 3,3,4,4-tetramethylsuccinimidyl group

A series of new imidophosphanes and phosphine oxides containing 3,3,4,4-tetramethylsuccinimidyl group were synthesized and characterized by ¹H, ¹³C{¹H} and ³¹P NMR spectroscopy, IR and MS. Ph_mPCl_n (m = 3 − n, n = 3, 2, 1) reacted with 3,3,4,4-tetramethylsuccinimide (TH) and potassium 3,3,4,4-tetramethylsuccinimidate 1 to give corresponding Ph_mPT_n. Molecular structures of products were established by single-crystal X-ray diffraction experiments. Attempts to prepare new imidophosphoranes by reactions of 1 with Ph_mPCl_n (m = 5 − n, n = 4, 3, 2) resulted in phosphine oxides. In these reactions the phosphoryl group was formed and we characterized a by-product of this type of reaction.     

Beneficial effects of beta-sitosterol on glucose and lipid metabolism in L6 myotube cells are mediated by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that has been implicated as a key factor for controlling intracellular lipids and glucose metabolism. β-Sitosterol, a plant sterol known to prevent cardiovascular disease was identified from Schizonepeta tenuifolia to an AMPK activator. In L6 myotube cells, β-sitosterol significantly increased phosphorylation of the AMPKα subunit and acetyl-CoA carboxylase (ACC) with stimulating glucose uptake. In contrast, β-sitosterol treatment reduced intracellular levels of triglycerides and cholesterol in L6 cells. These effects were all reversed by pretreatment with AMPK inhibitor Compound C or LKB1 destabilizer radicicol. Similarly, β-sitosterol-induced phosphorylation of AMPK and ACC was not increased in HeLa cells lacking LKB1. These results together suggest that β-sitosterol-mediated enhancement of glucose uptake and reduction of triglycerides and cholesterol in L6 cells is predominantly accomplished by LKB1-mediated AMPK activation. Our findings further reveal a molecular mechanism underlying the beneficial effects of β-sitosterol on glucose and lipid metabolism.     

层析法纯化破伤风毒素的试验研究

为了获得高纯度的破伤风毒素,用疏水层析和离子交换层析纯化破伤风毒素。破伤风毒素培养滤液经Phenyl Sepharose疏水层析除去大部分杂质,再经DEAE Sephadex离子交换层析进一步纯化。经两步层析纯化后,毒素纯度达到2000Lf/mg PN以上,回收率为52%~73%。用此方法,连续纯化五批毒素,均获得高纯度的破伤风毒素。试验证明破伤风毒素经疏水层析和离子交换层析可得到有效纯化。     

Biosynthesis of beta-sitosterol and stigmasterol in Croton sublyratus proceeds via a mixed origin of isoprene units

A green callus culture of Croton sublyratus Kurz established from the leaf explants appeared to actively synthesize two well-known phytosterols, beta-sitosterol and stigmasterol. The phytosterol biosynthesis was highly active during the linear phase of the culture. Feeding of [1-13C]glucose into the callus culture at this growth phase showed that the label from glucose was highly incorporated into both phytosterols. Isolation of the labeled products followed by 13C NMR analysis revealed that the phytosterols had their 13C-labeling patterns consistent with the acquisition of isoprene units via both the mevalonate pathway and the deoxyxylulose pathway with relatively equal contribution. Since the biosynthesis of phytosterol has so far been reported to be mainly from the classical mevalonate pathway, this study provides a new evidence on the biosynthesis of phytosterols via the novel deoxyxylulose pathway.     

Preparation and mass spectrometry of 14 pure and 18O(2)-labeled oxidation products from the phytosterols beta-sitosterol and stigmasterol

To lower cholesterol, phytosterols are currently introduced as food additives, where they may become oxidized. In addition, specific biological effects of oxyphytosterols are suggested by the recent molecular clarification of the phytosterol storage disease as a dysfunctional mutation of an active sterol reexporter potentially regulated by oxidized phytosterols. We therefore studied the hydroxybenzotriazole-mediated PbO(2)-driven oxidation of phytosterols and compared it to the oxidation of cholesterol. We prepared, identified, and purified standards of 14 oxidation products of two major phytosterols. The gas chromatographic mass spectrometric characteristics of the 7alpha- and 7beta-hydroxy-, 5alpha,6alpha-epoxy, 5beta,6beta-epoxy, 7keto-, 3beta,5alpha,6beta-trihydroxy-, 3keto-, and 7-dehydro-derivatives of beta-sitosterol and stigmasterol are presented. The method also provided a convenient access to prepare 18O-labeled oxyphytosterols of high chemical and isotopic purity and can easily be extended to further phytosterols and -stanols. This enables the gas chromatography-mass spectrometry analysis of oxyphytosterols and the study of their biological effects.     

Synthesis and chromatographic purification of recombinant human pituitary hormones

Recombinant DNA-derived proteins and, in particular, human pituitary hormones, are increasingly used for research, diagnostic and therapeutic purposes. This trend has demanded new synthetic approaches and improved purification techniques. The type and sequence of the purification steps have to be selected in accordance with the cloning and protein expression strategy, the host organism and cellular localization of the protein of interest, with a view to producing the desired product at a required purity, biological activity and acceptable cost. This review article describes and analyzes the main synthetic and purification strategies that have been used for the production of recombinant human growth hormone, prolactin, thyrotropin, luteinizing hormone and follicle-stimulating hormone, giving special consideration to the few published downstream processes utilized by the biotechnology industry. Practically all types of prokaryotic and eukaryotic organisms utilized for this purpose are also reviewed.     

核酸提取方法进展

核酸提取是分子生物学的基本方法,也是核酸诊断中最关键的方法,它是下游诊断、分析和制备的前提。过去的核酸提取方法繁琐费时且有限。目前,核酸提取方法已经取得了较大进步。本文综述了核酸提取方法的进展情况,包括传统的基于溶液的抽提方法、现在常用的柱提取法、正兴起的多效生物分子抽提法和自动化抽提系统等。多效生物分子抽提法、自动抽提系统的微型化和完全自动化或者它们的组合是核酸提取技术未来发展的方向。     

Caulerpenyne from Caulerpa taxifolia: A comparative study between CPC and classical chromatographic techniques

Caulerpenyne (Cyn) is a cytotoxic compound firstly isolated in 1978 from Caulerpa prolifera. This metabolite, constituted by a highly reactive diacetoxybutadiene moiety, exhibited a wide range of biological properties with mainly antibacterial properties and antitumoral activities. Few structure–activity relationships (SAR) are available to design more potent bioactive derivatives by pharmacomodulation. Cyn can be produced by total synthesis or extracted from natural sources in particular the green alga Caulerpa taxifolia. Since conventional chromatographic procedures to isolate Cyn from C. taxifolia are time- and solvent-consuming, it was crucial to find a more efficient process to obtain pure Cyn. In our study, Cyn has been purified from C. taxifolia with two different techniques: Centrifugal partition chromatography (CPC) and a classical chromatographic process. The comparative study showed that CPC constitutes a very simple and efficient process to access Cyn.     

Purification and characterization of vitellogenin and lipovitellins of the sand crab Emerita asiatica: molecular aspects of crab yolk proteins.

In the mole crab Emerita asiatica, the main yolk proteins consist of two slow moving lipovitellins (Lv I and Lv II) of glycolipoprotein nature. Lv I cleaves into subunits (MW: 109,000 and 105,000) and Lv II gives rise to six subunits (MW: 65,000, 54,000, 50,000, 47,000, 44,000, and 42,000) in SDS-PAGE (with beta-mercaptoethanol). In order to observe the stability of Lv II as well as to achieve better resolution of the proteins, two different buffer systems (Phosphate buffered saline and tris-buffered saline), 40% sucrose, and glass distilled water were used as homogenizing media. Among them, better resolution was achieved with tris-buffered saline and 40% sucrose, and tris-buffered saline seems to be the ideal medium for elution of Lv II. The analysis of biochemical constituents of the major Lv II reveals a percentage composition of 69.325, 27.927, and 2.753 respectively for protein, lipid, and bound sugars. In the I stage embryo, protein comprises about 67.276%, lipid 29.65%, and bound sugars 3.015%. Vitellogenin (Vg) electrophoretically corresponding to the Lv I and Lv II was present in the female haemolymph during the entire period of embryogenesis. The number of subunits (8) of Vg in all stages remained unaltered and their approximate molecular weights were Vg1, 91,000; Vg2, 87,000; Vg3, 83,000; Vg4, 61,000; Vg5, 58,000; Vg6, 45,000; Vg7, 42,000; and Vg8, 38,000. Different proteins present in the embryos (I and IV stage) and the serum obtained from the animal carrying the I stage embryo were separated by gel-filtration in high performance liquid chromatography (HPLC). Sephadex (G-200) gel filtration chromatography was used to purify the Lv II in large quantity. Total lipid extracted from Lv II as well as the embryos belonging to different stages of development were separated into their constituent neutral, glycolipids, and phospholipids, using silicic acid column chromatography. Thin layer chromatography (TLC) was used to isolate the different phospholipids purified from various stages of embryos and Lv II. As many as seven different phospholipids were separated from Lv II and I and IX stage embryos; and whereas thin layer chromatogram of V and VI stage embryos showed six different phospholipids, embryos of VII and VIII stage contained four phospholipid species. Cholesterol, glycolipids, and individual phospholipids isolated from the Lv II and I stage embryo were quantified spectrophotometrically and the results were discussed.     

核酸提取方法进展

核酸提取是分子生物学的基本方法,也是核酸诊断中最关键的方法,它是下游诊断、分析和制备的前提。过去的核酸提取方法繁琐费时且有限。目前,核酸提取方法已经取得了较大进步。本文综述了核酸提取方法的进展情况,包括传统的基于溶液的抽提方法、现在常用的柱提取法、正兴起的多效生物分子抽提法和自动化抽提系统等。多效生物分子抽提法、自动抽提系统的微型化和完全自动化或者它们的组合是核酸提取技术未来发展的方向。     

Preparation and characterization of chitosans carrying aldehyde functions generated by nitrogen oxides

In this work, aldehyde-functionalized chitosan is produced by the reaction of chitosan with nitrogen oxides generated in situ from a HNO₃/H₃PO₄-NaNO₂ mixture. This method is more advantageous than the existing approaches, since the depolymerization is slower and the purification process is straightforward. The appearance of characteristic peaks in the Fourier transform infrared and carbon-13 nuclear magnetic resonance spectra (1733 cm⁻¹ and 183.4 ppm, respectively) of the product confirms the presence of the aldehyde functionality in the modified chitosans. The ¹H NMR spectra also revealed the presence of aldehyde groups. Furthermore, the gradual disappearance of the peaks due to aldehyde protons and a concomitant appearance of a new resonance at ∼8.05 ppm with increasing pH indicate the formation of Schiff's base between the aldehyde and the free amine groups. The aldehyde-functionalized chitosan prepared with 6 h of reaction time (chitosan-6h) forms a gel in situ without any added external crosslinker and it may potentially be useful as a vehicle for drug delivery.     

Improved chromatographic method for purification of lactoperoxidase from different milk sources

Our previous studies showed that sulfanilamide is a new competitive inhibitor of and can be used in the purification of lactoperoxidase (LPO, EC1.11.1.7) from milk. However, this method has some disadvantages like a lower purification factor. The aim of the present study is to improve the purification process of milk LPO from different sources. For this purpose, 16 commercial sulfanilamide derivatives were selected for inhibition studies to determine the best inhibitor of bovine LPO by calculating kinetic parameters. A cyanogen bromide-activated Sepharose 4B affinity matrix was synthesized by coupling with each competitive inhibitor. Among the inhibitors, 5-amino-2-methylbenzenesulfonamide and 2-chloro-4-sulfamoylaniline were used as ligands for the purification of LPO from bovine, buffalo, cow, and goat milks with 1059.37, 509.09, 232.55, and 161.90, and 453.12-, 151.86-, 869.00-, and 447.57-fold, respectively. Our results show that 5-amino-2-methylbenzenesulfonamide, 2-chloro-4-sulfamoylaniline, and 5-amino-1-naphthalenesulfonamide are the best inhibitors for one-step purification of the enzyme.     

Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liposome-inserting conjugate. 1. Gram-scale production and purification

A GMP-compliant process is described for producing F5cys-PEG-lipid conjugate. This material fuses with preformed, drug-loaded liposomes, to form "immunoliposomes" that bind to HER2/neu overexpressing carcinomas, stimulates drug internalization, and ideally improves the encapsulated drug's therapeutic index. The soluble, single-chain, variable region antibody fragment, designated F5cys, was produced in E. coli strain RV308 using high-density cultures. Affinity adsorption onto horizontally tumbled Streamline rProtein-A resin robustly recovered F5cys from high-pressure-disrupted, whole-cell homogenates. Two product-related impurity classes were identified: F5cys with mid-sequence discontinuities and F5cys with remnants of a pelB leader peptide. Low-pressure cation exchange chromatography, conducted at elevated pH under reducing conditions, enriched target F5cys relative to these impurities and prepared a C-terminal cysteine for conjugation. Site-directed conjugation, conducted at pH 5.9 +/- 0.1 with reaction monitoring and cysteine quenching, yielded F5cys-MP-PEG(2000)-DSPE. Low-pressure size exclusion chromatography separated spontaneously formed, high-molecular-weight conjugate micelles from low-molecular-weight impurities. When formulated at 1-2 mg/mL in 10 mM trisodium citrate, 10% sucrose (w/v), at pH 6.4 (HCl), the conjugate was stable when stored below -70 degrees C. Six scale-up lots were compared. The largest 40-L culture produced enough F5cys to manufacture 2,085 mg of conjugate, enough to support planned preclinical and future clinical trials. The conjugate was 93% pure, as measured by polyacrylamide gel electrophoresis. Impurities were primarily identified as product-related. Residual endotoxin, rProtein A, and genomic DNA, were at acceptable levels. This study successfully addressed a necessary step in the scale-up of immunoliposome-encapsulated therapeutics.     

Synthesis and characterization of hexa-n-propylcyclotriphosphazene

Dynamic vacuum thermolysis of (CH₃)₃SiN = P(n-Pr)₂(OPh) yielded hexa-n-propylcyclotriphosphazene, [N = P(n-Pr)₂]₃. It was found that pure [N = P(n-Pr)₂]₃is insoluble in most solvents, however, [N = P(n-Pr)₂]₃ can be crystallized directly from the crude reaction mixture using hot hexanes. The molecular structure of hexa-n-propylcyclotriphosphazene has been determined by single-crystal X-ray diffraction and multinuclear NMR studies.     

Hydrophobic chromatographic purification of ethylene-enhanced chlorophyllase from Citrus unshiu fruits

Ethylene-enhanced chlorophyllase from Citrus unshiu fruits was purified to a homogeneous state after solubilization with sodium cholate, using acetone precipitation and hydrophobic chromatography. The enzyme adhered to phenyl Sepharose CL-4B in 3M KCl and was eluted with a linear gradient of Triton X-100 (0–0.5%). Its MW (SDS-PAGE) was 27 000. The enzyme behaved as a protein of MW 110 000 on Sephacryl S-200 gel filtration. The enzyme showed a specific activity of 0.069 μamol chlorophyllide a produced/min/ mg protein. This purification procedure is a rapid method for obtaining pure chlorophyllase.     

Efficient one-step chromatographic purification and functional characterization of recombinant human Saposin C

Saposin (Sap) C is a small lysosomal disulfide bridge-containing glycoprotein required for glucosylceramide (GC) hydrolysis by glucosylceramidase (GCase). Sap C deficiency causes a variant form of Gaucher disease (GD), a rare genetic disorder characterized by GC accumulation in lysosomes of monocyte/macrophage lineage. Efforts to develop fast and efficient methodologies to express and purify Sap C have been made in the last years. Here, human Sap C was expressed in a bacterial strain that greatly enhances disulfide bond formation, and the recombinant protein was purified in a single chromatographic step using an affinity tag-based protein purification system. Mass spectrometry analysis demonstrated that disulfide bridges required for Sap C stability and functionality were retained. Consistently, the recombinant protein was shown to interact with anionic phospholipids-containing vesicles, and reconstitute GCase activity in vitro. Recombinant Sap C was efficiently endocytosed by Sap C-deficient fibroblasts, and targeted to lysosomes. These findings document that the bacterially purified Sap C exerts biological properties functionally equivalent to those observed for the native protein, indicating its potential use in the development of therapeutic intervention.     

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.