Isolation, Purification and Cryopreservation of Cells from Neonatal Bovine Testis

The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the ceils were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli ceils were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while fxozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol. L^-1 trehalose had the best effect in sertoli cells crvooreservation.

Isolation and Purification of Unstable Iridoid Glucosides from Traditional Chinese Medicine by Preparative High Performance Liquid Chromatography Coupled with Solid-phase Extraction

An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matography(HPLC) and solid-phase extraction(SPE). The aim of this study was to obtain an effective method with high preparative efficiency and importantly to avoid the transformation of unstable compounds. The preparative HPLC system was based on an LC/MS controlled four-channel autopurification system. The SPE method was performed with a C18 packing material to trap the target compounds and to remove the acidic additive derived from the mobile phase. Using this method, the unstable iridoid glucosides(IGs) as model compounds were successfully isolated and purified from the extract of Hedyotis diffusa Willd. Six IGs(including one new minor IG) and one nucleotide compound were simultaneously obtained, each with a purity of 91% as determined by HPLC. The structures of the isolated compounds were identified by UPLC/Q-TOF MS, UV, 1D and/or 2D NMR. It was demonstrated that the combination of preparative HPLC with SPE is a versatile tool for preparative purification of unstable compounds from complex natural products.

Isolation and Purification of Bovine Colostrum sIgA and IgG

To further utilize bioactive substance such as bovine colostrum sIgA and IgG, sIgA and IgG were isolated and purified simultaneously by salting out, ultrafiltration and gel chromatography, etc. The analysis of results were showed quantitatively by nonhydrogenized SDS-PAGE, and quanlities of sIgA and IgG were respectively detected by Western Blot. The results showed that the purity and yield of bovine colostrum sIgA were 85.3% and 42.8%, respectively, while the purity and yield of bovine colostrum IgG were respectively 97.2% and 64.4%. This preparative method provides theoretical and experimental foundation for sIgA industrial production.

ISOLATION AND PURIFICATION OF METHANE IVf ONOOXYGENASE FROM METHYLOMONAS SPECIES GYJ3

A three—component enzyme system that catalyzes in vivo the oxidation of CH_4 to CH_3OH has been purified with high specific activity from an unusual type I methanotroph through the use of stabilizing reagents.

A Review on the Isolation and Purification of D-allulose

D-allulose has very little content in nature,and it needs to be synthesized artificially and meet the purity requirements of industrial grade.The basic physical and chemical properties of D-allulose,its preparation methods and many different ways of isolation and purification were described.In order to achieve the goal of industrial production of D-allulose as soon as possible,the research progress of D-allulose isolation and purification technologies at home and abroad in recent years was classified and discussed,so as to provide useful reference for the practical improvement of D-allulose isolation and purification process technologies.

Apoptosis and oxidative injury of donor islets during isolation and purification

Objective To observe the changes of islet cell apoptosis and oxidation - antioxidation before transplantation,and to explore pathways of islet protection. Methods Fifteen human pancreases were perfused with Hanks solution containing collagenase,then digested and isolated.

Study on purification and ultrastructure of a baculovirus in Penaeus chinensis

A kind of baculovirus was isolated from the cephalothorax homogenate of sick or morbid Penaeus chinensis by differential centrifugation and density gradient centrifugation. Electron microscopic examination of ultrathin section of the gills, stomach and mid-gut tissues also revealed the presence of rod-shaped baculoviral particles with the same size in the affected cell nuclei, where most of the virions arranging in cluster assembled and caused a series of cytopathic changes. The virion covered with bilaminal envelope was 320 ~ 400 nm × 100 ~ 130 nm in size, whereas the nucleocapsid ranged in size of 250~ 300 nm in length and 70 ~ 100 nm in breadth respectively. No nuclear polyhedron or granulin occlusion theies have been found in cells. According to the principle of viral classification, this newly found virus could probably belong to the non-occluded subgroup of insect baculoviridae, i. e., C subgroup baculovirus.

Purification and Characterization of a Novel Lipase from Antarctic Krill

Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.

Isolation and identification of chemical constituents from the vinegar-prepared Corydalis yanhusuo

Phytochemical investigation of the vinegar-prepared Corydalis yanhusuo led to the isolation of one aristolactam derivative,1,2,8,9-tetramethoxy-5-methyldibenzo[cd,f]indol-4-(5H)-one(1),and seven aporphine alkaloids,including 2,9,10-trimethoxydibenz[de,g]quinolin-7-one(2),1-hydroxy-2,9,10-trimethoxy-7H-dibenzo(de,g)quinoline-7-one(3),oxoglaucine(4),N-methyloxoglaucine trifluoroacetate trifluoroacetate(5),corunine acetate(6),pontevedrine(7),and oxoglaucidaline trifluoroacetate(8).The structures of the isolated compounds were elucidated by extensive spectroscopic data analysis and comparison with the previous reports.Among them,compounds 1 and 2 were obtained as natural products for the first time,and their NMR data were unambiguously assigned.In addition,compound 1 exhibited moderate cytotoxic activity against HepG2 cells with an IC50 value of 16.0±6.4μM.

Prokaryotic expression and purification of fibronectin leucine rich transmembrane protein 3 C-terminal domain proteins in rats

BACKGROUND： Studies have suggested that fibronectin leucine-rich transmembrane protein 3 （FLRT3） is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE： To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins. DESIGN, TIME AND SETTING： An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008. MATERIALS： Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coil （E. coil） JM109 were purchased from Promega. E. coil BL21 was provided by Novagen. METHODS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. coil BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained. MAIN OUTCOME MEASURES： Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. The recombinant protein was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing. CONCLUSION： From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E. coil BL21. Moreover, highly purified GST fusion proteins were obtained.

Isolation, purification and structural characterization of a water-soluble polysaccharide HM41 from Halenia elliptica D.Don

A water-soluble polysaccharide, HM（41）, was obtained from Halenia elliptica D. Don by acidic ethanol fractionation and gel filtration. Its homogeneity was confirmed by chromatography using multiple systems. HM（41） was composed of rhamnose（Rha）, arabinose（Ara）, xylose（Xyl）, mannose（Man）,galactose（Gal）, glucose（Glc） with a molar ratio of 1.0：5.5：1.8：3.0：9.4：21. The average molecular weight of HM（41） was approximately 1.17 ＊ 10~4. Periodate oxidation, Smith degradation, methylation and GC, IR,NMR, XRD, GC–MS analysis were used for the structural analysis of HM（41）. Its main chain was composed mainly of β-（1→4）Gal, β-（1→4）Glc and β-（1→6）Glc. β-（1 →4）Gal were substituted at 6-O and on average there were 14 branches among 23 main chain residues;（1→ 4）Glc had no branch;（1→6）Glc were substituted at 3-O and on average there were 9 branches among 14 main chain residues. The side chain was composed of（1→3,6）-Rha,（1→4）/（1→5）-Ara,（1 →4）/（1→5）-Xyl,（1→4,6）-Man and（1→2）-Glc. The terminal residue was composed of Ara, Xyl, Man, Gal, and Glc. Then, we demonstrated that HM and HM（41） had strong scavenging activities in vitro hydroxyl. Overall, HM and HM（41） may have potential applications in the antioxidants for medical and food industry.

Study on Extraction and Separation of Dihydromyricetin from Vine Tea [Ampelopsis grossedentata(Hand-Mazz) W. T. wang]

Ampelopsis grossedentata(Hand-Mazz) W. T. wang is a typical edible plant with important physiological health functions. Dihydromyricetin is the main active ingredient of A. grossedentata, which has good pharmacological effects such as antibacterial, anti-inflammatory, anti-oxidation, anti-tumor and liver protecting effects. This paper summarized the research progress on the extraction materials, extraction methods and separation and purification processes of dihydromyricetin in A. grossedentata to understand the best experimental method of dihydromyricetin, aiming to provide an important theoretical basis for the comprehensive development and utilization of dihydromyricetin.

In vitro Study on Human Trophoblast Cells Infected with HCMV

Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient.Cells and purity were identified by using immunocytochemistry assay with anti-CK7,Vim and β-hCG antibodies.HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection(p.i.).The results showed tha...

Isolation and Structural Analysis of an Acidic Polysaccharide from Astragalus membranaceus （Fisch.） Bunge

A new water-soluble hetero-polysaccharlde, APSID3, was obtained from a hot-water extract of the roots of Astragalus membranaceus （Flsch.） Bunge by DEAE-Sepharose Fast Flow and Sephacryl S-300 chromatography. The molecular weight of APSID3 was estimated to be 5.79 × 10^5 Da. Based on a sugar composlUon analysis, methylatlon analysis, partial hydrolysis and 13C nuclear magnetic resonance experimentation, It was concluded that the minimal repeat unit of APSID3 was composed of one terminal arablnose, one 1,5-1Inked arabinose, one 1,3-1Inked rhamnose, one 1,3,4-1Inked rhamnose, five 1,4-1Inked methyl galacturonates and six 1,4-1inked methyl glucuronates.

Studies of Coltivirus in China

Objective The purpose of this article is to review the developments of studies of Coltivirus in ChinaData sources The data used in this review was obtained mainly from the studies of Coltivirus reported from 1990 to 2003 in China.Study selection Relevant articles on studies of Coltivirus in domestic and foreign literature were selected.Data extraction Data were maily extracted from the articles which are listed in the reference section of this review.Results Many Coltiviruses have been isolated not only from blood samples of patients with unknown fever or from cerebrospinal fluid of patients with encephalitis in Xishuangbanna area in Yunnan province, but also from mosquitoes collected in many areas in China. In some patients diagnosed as Japanese encephalitis or unknown fever, an increase of Coltivirus IgG antibody of fourfold, or more, has been detected using ELISA. Similarly, Coltivirus IgM antibody was positive in some patients with Japanese encephalitis or viral encephalitis. From most Chinese patients, except the northeastern, the isolates of Coltiviruses belong to subgroup B2, according to RT-PCR amplification of the ninth and twelfth segments of the isolates and sequence analysis of their amplicons. Some biological properties of Chinese Coltiviruses isolates are different from that of North American Coltiviruses.Conclusions The isolates of Coltiviruses from Chinese patients are one of the common agents causing viral encephalitis and unknown fever in summer-autumn season. It might be an important public health problem due to its high isolation rate and wide distribution in China. Mosquito is the main transmission vector of the virus.

Bacterial extracellular vesicles as bioactive nanocarriers for drug delivery:Advances and perspectives

Nanosized extracellular vesicles derived from bacteria contain diverse cargo and transfer intercellular bioactive molecules to cells.Due to their favorable intercellular interactions,cell membrane-derived bacterial extracellular vesicles(BEVs)have great potential to become novel drug delivery platforms.In this review,we summarize the biogenesis mechanism and compositions of various BEVs.In addition,an overview of effective isolation and purification techniques of BEVs is provided.In particular,we focus on the application of BEVs as bioactive nanocarriers for drug delivery.Finally,we summarize the advances and challenges of BEVs after providing a comprehensive discussion in each section.We believe that a deeper understanding of BEVs will open new avenues for their exploitation in drug delivery applications.

Three new ursane-type triterpenoids from Rosmarinus officinalis and their biological activities

Three new ursane-type triterpenoids,3-oxours-12-en-20,28-olide(1),3β-hydroxyurs-12-en-20,28-olide(2)and 3β-hydroxyurs-11,13(18)-dien-20,28-olide(3),were isolated from a potent anti-inflammatory and antibacterial fraction of the ethanolic extract of Rosmarinus officinalis.Their structures were elucidated by a combination of extensive 1D-and 2D-NMR experiments,MS data and comparisons with literature reports.Compounds 1-3 exhibited significantly inhibitory effects on nitric oxide production in lipopolysaccharide-activated mouse RAW264.7 macrophages,but no antibacterial activity was found at a concentration of 128μg·mL^(-1).