Ribonuclease of Bacillus intermedius 7 P. Purification by chromatography on phosphocellulose and several characteristics of the homogeneous enzyme]

A new procedure for isolation of homogenous ribonuclease of Bac. intermedius from a commercial source is described. The yields of 140 mg of RNAse from 200 g of the enzymic powder were attained. The amino acid composition of the enzyme was determined. The RNAse contains neither the sulfhydryl groups nor the disulfide bonds and has only one histidine residue. At the same time the amount of aromatic amino acid residues is relatively high. The enzyme is highly resistant to heat and acid treatment but is less stable in an alkaline solution. The pH optimum of the RNAse for the RNA digestion is 8,5; the temperature optimum for this reaction is 37 degrees. A spectrophotometric method for the RNAse activity assay using polyA as a specific substrate was developed. The purified product provides a suitable starting material for structural studies.     

Increased yield of high purity recombinant human interferon-gamma utilizing reversed phase column chromatography

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-gamma), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-gamma from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-gamma monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100 microg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 x 10(7) to 4 x 10(7)IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-gamma in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.     

A simplified universal genomic DNA extraction protocol suitable for PCR

Conventional genomic DNA extraction protocols need expensive and hazardous reagents for decontamination of phenolic compounds from the extracts and are only suited for certain types of tissue. We developed a simple, time-saving and cost-efficient method for genomic DNA extraction from various types of organisms, using relatively innocuous reagents. The protocol employs a single purification step to remove contaminating compounds, using a silica column and a non-hazardous buffer, and a chaotropic-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from cell lysates. We used this system to extract genomic DNA from different tissues of various organisms, including algae (Dunaliella salina), human peripheral blood, mouse liver, Escherichia coli, and Chinese hamster ovary cells. Mean DNA yields were 20-30 μg/cm(3) from fresh tissues (comparable to yields given by commercial extraction kits), and the 260/280 nm absorbance ratio was 1.8-2.0, demonstrating a good degree of purity. The extracted DNA was successfully used in PCR, restriction enzyme digestion and for recombinant selection studies.     

A novel method for the rapid purification of human and rat fibrin(ogen) degradation products in high yields.

A novel method is described for the preparation and purification of fibrin(ogen) degradation products in high yields. The high yields are due to two factors: a) an improved preparation method in which the heterogeneity in the size of the degradation products D is greatly reduced by performing the digestion with plasmin at well-controlled calcium concentrations (see ref.[22]). b) a new purification method, which includes Sephadex G-200 filtration and separation of D and E fragments by preparative isoelectric focusing. The latter step gives a complete separation of D and E fragments, without any overlap, and with a nearly 100% recovery in a short period of time. The properties of human and rat fibrin(ogen) degradation products are very similar.     

牛成纤维细胞的分离与体外培养

研究了牛胎儿和成年牛皮肤组织成纤维细胞的分离、培养、纯化方法和生长特征。通过组织块贴壁培养和分离单细胞接种培养均能获得原代牛皮肤细胞。用2.5 g/L胰蛋白酶+1mmol/L EDTA和5 g/L胶原酶I联合消化牛皮肤组织较2.5 g/L胰蛋白酶+1 mmol/L EDTA消化,得到更多的单个细胞,两者之间差异极显著(P<0.01),但其死细胞比率却有较大升高;2.5 g/L胰蛋白酶+1 mmol/L EDTA消化牛胎儿组织得到的单细胞数显著高于皮肤组织消化后得到的细胞数(P<0.01),死细胞比率也高于同种酶消化的皮肤组织。分离纯化的胎儿和皮肤成纤维细胞的生长曲线都正常且相似。2.5 g/L胰蛋白酶+1 mmol/L EDTA消化贴壁细胞后死细胞率明显高于用0.5g/L胰蛋白酶+0.53 mmol/L EDTA消化的细胞(P<0.05);培养24 h后细胞贴壁率前者要明显低于后者(P<0.05)。用0.5 g/L胰蛋白酶轻度消化混杂生长的成纤维细胞和上皮样细胞,经过反复贴壁传代2～3代,可得到较纯的成纤维细胞。     

Isolation and purification of cyanogen bromide-derived peptides of type II collagen directly from tissue (Swarm chondrosarcoma)

A preparative procedure is described for isolating type II collagen-fragments directly from tissue. Swarm chondrosarcoma from rat, a cartilagenous tissue rich in type II collagen, was digested by cyanogen bromide in 70% formic acid. The resulting crude extract was desalted (G 25 column chromatography) and lyophylized. The yield of peptide mixture was about 1 250 mg obtained from 100 g tissue. The method of purification commonly used for type II collagen prior to cyanogen bromide-cleavage yielded 20 mg peptides from 100 g tissue. Separation of the cyanogen bromide-derived fragments was performed by gel filtration. The column was run at 43 degrees C (denaturing-temperature of collagens) to avoid fibril formation, and a volatile buffer was used (ammonium formate buffer, pH 7.5) so that the effluent fractions could be easily lyophylized. Two-dimensional gel electrophoresis of the main peaks of the column profile demonstrated that this purification step resulted in a good separation of the fragment mixture, although additional steps may be necessary for complete separation of the peptides. The most striking advantages of the method for direct digestion of tissue outlined here are the increase in yield (about 60-fold) and the reduction of purification steps (avoiding type II collagen purification).     

An evaluation of techniques for the extraction and amplification of DNA from naturally shed hairs

Hair can be a valuable source of DNA for the noninvasive study of human and nonhuman populations. However, hairs contain extremely small quantities of DNA, making the method used to extract the DNA of paramount importance. This study compares the effectiveness of 4 different methods of DNA extraction from shed chimpanzee hair, as measured by the ability to amplify mtDNA targets using PCR. The most successful method is also the simplest, requiring only digestion of the root end in a buffer compatible with subsequent PCR without a prior purification or extraction step. Strategies to non-specifically preamplify the template are not successful with DNA from stored shed hairs.     

Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.     

Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterization

Onconase, an RNAse extracted from embryos of the Northern leopard frog ( Rana pipiens ), is in a confirmatory phase IIIb clinical trial for the treatment of unresectable malignant mesothelioma. Because the current purification process for onconase is cumbersome and laborious, the development of more efficient and cost-effective alternative sources is imperative. In this study, we assessed the potential of Pichia pastoris as an expression host for the large-scale production of onconase. Because of its specific N-terminal structure, active onconase with a correct N-terminus could not be secreted by an α-mating factor (α-MF)-prepro secretion signal, and an α-MF-pre secretion signal should be used instead. Onconase accumulated to a high concentration (about 300 and 150 mg L⁻¹ for glycosylated onconase and aglycosylated mutein, respectively) in high cell density fermentation, and was purified to homogeneity with high yields (56% for glycosylated onconase and 67% for aglycosylated mutein) by a simple purification process consisting of cation exchange chromatography and size exclusion chromatography. In vitro activity assays revealed that glycosylation decreased both the RNAse activity and the cytotoxic activity of onconase. The high expression level and subsequent facile purification process make P. pastoris an efficient and cost-effective host for the large-scale production of onconase.     

海南产桶形芋螺线粒体基因组DNA提取条件的优化

提取海南产桶形芋螺线粒体基因组完整DNA (mtDNA),并对提取条件进行优化。以桶形芋螺腹足肌肉、毒腺和肝胰脏三个不同组织为材料,分别采用改进高盐沉淀法、细胞器/磁珠法和试剂盒提取三种方法,提取桶形芋螺mtDNA,并利用琼脂糖凝胶电泳和紫外分光光度计对提取mtDNA的纯度和浓度进行测定。以coxⅠ-rRNA小亚基基因和α-芋螺毒素基因设计引物,通过PCR反应来确证所提取的DNA确实是mtDNA。试剂盒法提取肝胰脏、高盐沉淀法提取肝胰脏和腹足肌肉组织这三种方法的产率很高,分别为44.4μg/mg、43.3μg/mg和32.6μg/mg。A260/280比值表明,改进高盐沉淀法提取毒腺和腹足肌肉组织,细胞器磁珠法提取腹足肌肉组织的mtDNA纯度很高。综合比较,采用改进高盐沉淀法,利用桶形芋螺腹足肌肉组织所提取的mtDNA产率高、质量好、纯度高。高质量芋螺mtDNA的获取为利用分子生物学方法对芋螺进行遗传进化分析和系统分类提供了基础。     

Caffeine reduces TNFα up-regulation in human adipose tissue primary culture

Adipose tissue secretions play an important role in the development of obesity-related pathologies such as diabetes. Through inflammatory cytokines production, adipose tissue stromavascular fraction cells (SVF), and essentially macrophages, promote adipocyte insulin resistance by a paracrine way. Since xanthine family compounds such as caffeine were shown to decrease inflammatory production by human blood cells, we investigated the possible effect of caffeine on Tumor Necrosis Factor alpha (TNFalpha) and Interleukin-6 (IL-6) expression by human adipose tissue primary culture. For that purpose, human subcutaneous adipose tissue obtained from healthy non-obese women (BMI: 26.7 +/- 2.2 kg/m2) after abdominal dermolipectomy, was split into explants and cultured for 6 hours with or without caffeine. Three different concentrations of caffeine were tested (0.5 microg/mL, 5 microg/mL and 50 microg/mL). After 6 hours of treatment, explants were subjected to collagenase digestion in order to isolate adipocytes and SVF cells. Then, TNFalpha and IL-6 mRNA were analysed by real-time PCR alternatively in adipocytes and SVF cells. In parallel, we checked gene expression of markers involved in adipocyte differenciation and in SVF cells inflammation and proliferation. Our findings show a strong and dose dependent down-regulation of TNF-alpha gene expression in both adipocyte and SVF cells whereas IL-6 was only down regulated in SVF cells. No effect of caffeine was noticed on the other genes studied. Thus, caffeine, by decreasing TNFalpha expression, could improve adipose tissue inflammation during obesity.     

Hydrophobic properties of alkaline phosphatases

The butanol extraction method of Morton (1950), a routine step in enzyme purification, is discussed with special reference to a hydrophobic form of alkaline phosphatase from human liver tissue. This form slowly precipitates from butanol-extracted liver tissue homogenates stored at 4 degrees C. Furthermore, it is lost when acetone precipitation is applied as a purification procedure. The soluble form in liver tissue is shown to have a higher relative hydrophobicity than the serum liver/bone isoenzyme. The use of sodium cholate in the isolation of the hydrophobic form produces an artefact in isoelectric focusing, which can be abolished by dialysis prior to focusing.     

Purification of human kidney angiotensin I converting enzyme using reverse-immunoadsorption chromatography

A rapid and highly efficient procedure for purification of angiotensin I converting enzyme from human kidney has been developed. Following tryptic solubilization, the enzyme was partially purified by DEAE-cellulose and hydroxylapatite chromatography. The final step consisted of “reverse immunoadsorption” on a column prepared by coupling antisera raised against contaminating proteins to CNBr-activated Sepharose CL-6B. Starting with 600 g kidney tissue, 6.1 mg of enzyme was obtained with a specific activity of 108 U/mg using Hip-His-Leu as substrate, a 3400-fold purification with an overall yield of 26%. The preparation gave a single band on 7.5% SDS-urea gels and a single arc against antisera to impure enzyme in crossed immunoelectrophoresis. A single N-terminal amino acid (leucine) was detected by dansylation. This procedure has allowed the initiation of structural studies with the human enzyme. “Reverse immunoadsorption” may be a generally useful method for protein purification.     

Quantification of 8-iso-prostaglandin-F(2alpha) and 2,3-dinor-8-iso-prostaglandin-F(2alpha) in human urine using liquid chromatography-tandem mass spectrometry

Quantification of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) has been suggested to be a reliable indicator of lipid peroxidation that may be related to in vivo free radical generation, oxidative damage, and antioxidant deficiency. We have developed a LC-MS/MS method to quantify 8-iso- PGF(2alpha) and its dinor metabolite, 2,3-dinor-8-iso-prostaglandin F(2alpha) (2,3-dinor-8-iso-PGF(2alpha)), in human urine samples. After an initial purification step using an automated C18 solid phase extraction procedure, the urine sample was injected directly into a liquid chromatography (LC) system and detected with tandem mass spectrometry. The detection limit of the assay was 9 pg for 8-iso-PGF(2alpha) and 3 pg for 2,3-dinor-8-iso-PGF(2alpha) with both inter- and intraday variations of less than 12%. The inaccuracies were less than 3% for both analytes at three different levels. The urinary excretion rate of 2,3-dinor-8-iso-PGF(2alpha) was higher than that of 8-iso-PGF(2alpha), and changed in proportion to the parent compound (R = 0.70, n = 60). Values obtained with this method showed good linear correlation to duplicate 8-iso-PGF(2alpha) measurements performed with GCMS (R = 0.97, n = 15). The mean excretion rates of 8-iso-PGF(2alpha) and 2,3-dinor-8-iso-PGF(2alpha) were significantly higher in smokers than in nonsmokers (0.53 +/- 0.37 vs. 0.25 +/- 0.15 microg/g creatinine, p = 0.002 for 8-iso-PGF(2alpha) and 8.9 +/- 3.8 vs. 4.6 +/- 2.6 microg/g creatinine, p = 0.003 for 2,3-dinor-8-iso-PGF(2alpha), respectively). The excellent accuracy, reproducibility, and high throughput of this method should permit it to be used in large clinical studies and standard clinical laboratories.     

Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography

C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin.     

RNAdigest: A Web-Based Tool for the Analysis and Prediction of Structure - Specific RNAse Digestion Results

Despite recent developments in analyzing RNA secondary structures, relatively few RNA structures have been determined. To date, many investigators have relied on the traditional method of using structure-specific RNAse enzymes to probe RNA secondary structures. However, if these data were combined with novel computational approaches, investigators would have an informative and valuable tool for RNA structural analysis. To this end, we created the web server “RNAdigest.” RNAdigest uses mfold RNA structural models in order to predict the results of RNAse digestion experiments. Furthermore, RNAdigest also utilizes both RNA sequence and the experimental digestion patterns to formulate the constraints for predicting secondary structures of the RNA. Thus, RNAdigest allows for the structural interpretation of RNAse digestion experiments. Overall, RNAdigest simplifies RNAse digestion result analyses while allowing for the identification of unique fragments. These unique fragments can then be used for testing predicted mfold structures and for designing structural-specific DNA/RNA probes.     

A rapid protocol for purification of total RNA for tissues collected from pigs at a slaughterhouse

Since RNA extraction is a crucial step in many molecular techniques, the protocols for sample collection and RNA purification need to be adapted to optimize their performance when samples are collected from animals at commercial facilities. Here we provide an RNA purification protocol for animal tissues collected from slaughterhouses. This protocol, modified from other techniques, uses TRIzol Reagent. Sample collection was performed wearing sterile gloves and facemasks, using sterile surgical instruments, and no longer than 8 min spent for each sample. A 0.9% sterile sodium chloride solution was used to wash the tissue before each sample collection. The whole process of RNA extraction was performed under cold environment and sterile conditions. This protocol produced good RNA yields (50 μg RNA per 100 mg tissue), good integrity and purity (Abs(260/280) from 1.8 to 2.0), from tissues such as liver, muscle, hypophysis, adipose tissue, and intestinal mucosa, in less than 2 h.     

Determination of testosterone and its tissue metabolites (DHT and 3 alpha-diol) in human plasma and prostatic tissue by isotopic dilution mass spectrometry

5 alpha-Dihydrotestosterone has been widely measured in human prostatic tissue using RIA since it is involved in the pathogenesis of human prostatic hyperplasia and seems to be the best index for the follow-up of patients affected by prostatic cancer under endocrine treatment. A GC-MS method for the simultaneous determination of testosterone (T), 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol) in prostatic tissue based on the isotopic dilution technique was developed. Tri-deuterated internal standards of each compound were previously synthetized in our laboratory. After extraction and purification on Sep-Pak C18 and Sephadex LH-20, T and its metabolites were measured as heptafluorobutyric ester (HFB) derivatives. Quantitative analysis was performed on a VG 7070 EQ mass spectromer equipped with a fused silica capillary column using the Selected Ion Monitoring technique. Steroid values (mean +/- SD; ng/g tissue) found in nine human hypertrophic prostates were: T: 0.71 +/- 0.43; DHT: 4.46 +/- 1.41; 3 alpha-diol: 0.34 +/- 0.23. Preliminary results obtained from the detection of the three androgens in human prostatic hyperplasia treated for 3 months with GnRH before surgery seem to indicate that DHT concentration decreases more than 10 times. Values obtained (n = 1; ng/g tissue) were: T: 0.194; DHT: 0.255; 3 alpha-diol: 0.015.     

A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells

Here, we describe a simple and efficient method for the expression and purification of active recombinant proteins in mammalian cells. This method uses the expression of T7 epitope-tagged proteins in transiently transfected 293T cells grown in monolayer, followed by anti-T7-agarose affinity chromatography. This procedure yields approximately between 75 and 100 microg of biologically active protein/150 cm(2) flask that can be used for biochemical studies. We have tested this protocol for the expression of the prototype SR protein, SF2/ASF, which is a member of the SR protein family with a role in constitutive and alternative splicing. We show that SF2/ASF purified using this protocol is able to complement an S100 HeLa extract, demonstrating that is biologically active. Moreover, expression of a novel SR-related protein that it is required for the second step of pre-mRNA splicing also rendered an active protein. In summary, we present a protocol based on transient transfection of mammalian cells that results in easy purification of significant amounts of biologically active proteins.