Application of high-performance capillary electrophoresis to the purification process of Escherichia coli K4 polysaccharide

The high-performance capillary electrophoresis (HPCE) (electrokinetic chromatography with sodium dodecyl sulphate) technique was applied to the extraction and purification process of the K4 polysaccharide from cultured bacteria in several stages. HPCE proved to be a technique with high resolution and sensitivity in analyzing K4 polysaccharide during its purification, in particular by using a strong anion-exchange resin. This is of paramount importance to monitor the product during the extraction and purification process or to test the purity of the final product. Furthermore, HPCE is able to verify that the extraction and purification process adopted is not carried out under drastic conditions capable of inducing fructose removal from the polysaccharide backbone.     

Molecular mechanism of cyclodextrin mediated cholesterol extraction

The depletion of cholesterol from membranes, mediated by β-cyclodextrin (β-CD) is well known and documented, but the molecular details of this process are largely unknown. Using molecular dynamics simulations, we have been able to study the CD mediated extraction of cholesterol from model membranes, in particular from a pure cholesterol monolayer, at atomic resolution. Our results show that efficient cholesterol extraction depends on the structural distribution of the CDs on the surface of the monolayer. With a suitably oriented dimer, cholesterol is extracted spontaneously on a nanosecond time scale. Additional free energy calculations reveal that the CDs have a strong affinity to bind to the membrane surface, and, by doing so, destabilize the local packing of cholesterol molecules making their extraction favorable. Our results have implications for the interpretation of experimental measurements, and may help in the rational design of efficient CD based nano-carriers.     

Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis

This work was performed to compare three precipitation protocols of protein extraction for 2-DE proteomic analysis using Arabidopsis leaf tissue: TCA-acetone, phenol, and TCA-acetone-phenol. There were no statistically significant differences in protein yield between the three methods. Samples were subjected to 2-DE in the 5 to 8 pH and 14-80 kDa ranges. The TCA-acetone-phenol protocol provided the best results in terms of spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility. In all, 93 qualitative or quantitative statistically significant differential spots were found between the three protocols. The 2-DE map of TCA-acetone-phenol extracts presented more resolved spots above 40 kDa, with no pI-dependent differences observed between the three protocols. 54 spots were selected for trypsin digestion, and the peptides were analyzed by MALDI-TOF-TOF MS. After database search using peptide mass fingerprinting, and MS/MS combined search, 30 proteins were identified, the proteins from chloroplastic photosynthetic and carbohydrate metabolism being those most highly represented. From these data, we were able to conclude that each extraction protocol had its main features. Considering this, the workflow of any standard comparative proteomic experiment should include the optimization and adaptation of the protein extraction protocol to the plant tissue and to the particular objective pursued.     

Scoring docking models with evolutionary information

We have developed methods for the extraction of evolutionary information from multiple sequence alignments for use in the study of the evolution of protein interaction networks and in the prediction of protein interaction. For Rounds 3, 4, and 5 of the CAPRI experiment, we used scores derived from the analysis of multiple sequence alignments to submit predictions for 7 of the 12 targets. Our docking models were generated with Hex and GRAMM, but all our predictions were selected using methods based on multiple sequence alignments and on the available experimental evidence. With this approach, we were able to predict acceptable level models for 4 of the targets, and for a fifth target, we located the residues involved in the binding surface. Here we detail our successes and highlight several of the limitations and problems that we faced while dealing with particular docking cases.     

Bioconversion of phytosterols to androstanes by mycobacteria growing on sugar cane mud

Direct sterol conversion of sugar cane mud (residue) by Mycobacterium sp. was demonstrated to be possible technologically, thus avoiding sugar cane oil extraction and further processes of extraction and purification of phytosterols from this oil. Indeed, mycobacterial cells were able to convert phytosterols from sugar cane mud into 4-androstene-dione (AD) and 1,4 androsta-diene-3,17-dione (ADD). For the various concentrations assayed, concomitant higher yields for both androstanes were achieved at 20% (w/w) sugar cane mud in media. Furthermore, conversions were similar to those from other substrates, such as a mixture of phytosterols. The results suggest that the mycobacterial cell is able to easily access and bioconvert sugar cane mud phytosterols.     

Evaluation of protein extraction methods for Vitis vinifera leaf and root proteome analysis by two-dimensional electrophoresis

An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis(2-DE)for recalcitrant plant species, in particular for grapevine(Vitis vinifera L.). Trichloroacetic acid-acetone(TCA-acetone)and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield,a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction.     

Evaluation of Protein Extraction Methods for Vitis vinifera Leaf and Root Proteome Analysis by Two-Dimensional Electrophoresis

An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis(2-DE)for recalcitrant plant species, in particular for grapevine(Vitis vinifera L.). Trichloroacetic acid-acetone(TCA-acetone)and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield,a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction.     

A Comparative Study of Four Viroids of Plants

The host-ranges and the reactions of particular plant hosts to inoculation with severe (s-PSTV) and mild (m-PSTV) strains of potato spindle tuber viroid, as well as with chrysanthemum stunt viroid (CSV) and cucumber pale fruit viroid (CPFV) were quite similar. Some minor differences did not exceed the limits of differences noted for the strains of the same plant viroid. Two-directional crossprotection was noted for each viroid pair when s-PSTV, m-PSTV, CSV and CPFV were tested on chrysanthemum cv., ‘Bonnie Jean’ plants. Finally, the relative mobility of RNAs of s-PSTV, m-PSTV, CSV and CPFV on 5% polyacrylamide gel was identical, no matter what extraction method from plant material was used. We postulate that these four plant viroids may be regardedas the strains of the same plant viroid “species”. On the other hand, chrysanthemum chlorotic mottle viroid (ChCMV) appeared to be a quite different plant pathogen. This viroid infected and caused symptoms only in chrysanthemum plants, and was able to infect and induce symptoms on plants which had already been infected with any other viroid studied, and it did not protect chrysanthemum cv. ‘Bonnie Jean’ plants against any of the other viroids. We were not able to locate a ChCMV-RNA band on polyacrylamide gel.     

桦褐孔菌三萜类物质的提取与含量测定

以桦褐孔菌发酵菌丝体为材料,通过对溶剂乙醇(95%)、甲醇、乙酸乙酯、丙酮、异丙醇、正己烷和氯仿的提取效果比较表明,提取三萜类化合物的最佳溶剂为异丙醇,提取时间为24h,每个样品所需溶剂量(6mL)和菌丝体样品量(100mg)较少,并可同时对大量样品进行有效提取。以白桦脂醇为标准品,对香草醛-冰醋酸-高氯酸分光光度法进行评价,证明该方法简单快速、准确度高、试验误差小、重复性好。利用此方法对桦褐孔菌的野生菌核和发酵菌丝体内三萜类化合物含量进行测定,结果表明野生菌核(59.86mg/g)和发酵菌丝体(94.92mg/g)中都含有很高的三萜类化合物,且发酵菌丝体中三萜类化合物含量高于野生菌核。因此在桦褐孔菌产品开发中,发酵菌丝体可代替野生菌核进行大工业化生产。     

Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli

The global pool of all metabolites in a cell, or metabolome, is a reflection of all the metabolic functions of an organism under any particular growth condition. In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction. In the past, a variety of cell lysis methods were adopted for assays of individual metabolites or groups of intermediates in pathways. In this study, metabolites were extracted from Escherichia coli using six different commonly used procedures including acid or alkaline treatments, permeabilization by freezing with methanol, high-temperature extraction in the presence of ethanol or methanol, and by lysis with chloroform-methanol. Metabolites were extracted by the six methods from cells grown under identical conditions and labeled with [14C]glucose. The metabolomes were compared after 2-dimensional thin-layer chromatography of labeled compounds. For global analysis, extraction with cold (-40 degrees C) methanol showed the greatest promise, allowing simultaneous resolution of more than 95 metabolite spots. In contrast, 80 or less spots were obtained with other extraction methods. Extraction also influenced quantitative analysis of particular compounds. Metabolites such as adenosine exhibited up to 20-fold higher abundance after cold methanol extraction than after extraction with acid, alkali, or chloroform. The simplicity, rapidity, and universality of cold methanol extraction offer great promise if a single method of lysis is to be adopted in metabolome analysis.     

Methyl-β-cyclodextrin is a useful compound for extraction and purification of prenylated enzymes from the retinal disc membrane

cGMP phosphodiesterase 6 (PDE6) and rhodopsin kinase (GRK1) are quantitatively minor prenylated proteins involved in vertebrate phototransduction. Here, we report that methyl-β-cyclodextrin (MCD), a torus-shaped oligosaccharide with a hydrophobic pore, can be used as a selective extractant for such prenylated proteins from frog retinal disc membranes, and that MCD makes it possible to purify frog PDE6 holoenzyme with very simple procedure. The EC50s of MCD for the extraction of GRK1 and PDE6 from the cytoplasmic surface of the disc membrane were 0.17 and 5.1 mM, respectively. By successive extraction of the membrane by 1 mM and then 20 mM MCD, we obtained crude GRK1 and PDE6, respectively. From the 20mM extract, we were able to purify the PDE6 holoenzyme using one-step anion-exchange column chromatography. From 1mM MCD extract, GRK1 was further purified by an affinity column. Following the removal of MCD by ultrafiltration, we were able to confirm integrity of these enzymes by reconstituting phototransduction system in vitro. We have therefore demonstrated that MCD is a useful compound for selective extraction and purification of prenylated peripheral membrane proteins from the cytoplasmic surface of biological membranes.     

Purification of normal cellular prion protein from human platelets and the formation of a high molecular weight prion protein complex following platelet activation

A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.     

桦褐孔菌三萜类物质的提取与含量测定

以桦褐孔菌发酵菌丝体为材料,通过对溶剂乙醇（95%）、甲醇、乙酸乙酯、丙酮、异丙醇、正己烷和氯仿的提取效果比较表明,提取三萜类化合物的最佳溶剂为异丙醇,提取时间为24h,每个样品所需溶剂量（6mL）和菌丝体样品量（100mg）较少,并可同时对大量样品进行有效提取。以白桦脂醇为标准品,对香草醛-冰醋酸-高氯酸分光光度法进行评价,证明该方法简单快速、准确度高、试验误差小、重复性好。利用此方法对桦褐孔菌的野生菌核和发酵菌丝体内三萜类化合物含量进行测定,结果表明野生菌核（59.86mg/g）和发酵菌丝体（94.92mg/g）中都含有很高的三萜类化合物,且发酵菌丝体中三萜类化合物含量高于野生菌核。因此在桦褐孔菌产品开发中,发酵菌丝体可代替野生菌核进行大工业化生产。     

Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks

In general, it is believed that the extraction of proteins from formalin-fixed paraffin embedded samples is not feasible. However, recently a new technique was developed, presenting the extraction of non-degraded, full length proteins from formalin fixed tissues, usable for western blotting and protein arrays. In the study presented here, we applied this technique to punch biopsies of formalin fixed tissues embedded in paraffin to reduce heterogeneity of the tissue represented in sections, and to ensure analysing mainly defined cellular material. Successful extraction was achieved even from very small samples (0.7 mm(3)). Additionally, we were able to detect highly glycosylated proteins and protein modification, such as phosphorylation. Interestingly, with this technique it is feasible to extract high quality proteins from 14 year old samples. In summary, the new technique makes a great pool of material now usable for molecular analysis with high throughput tools.     

Pressurized hot water extraction of anthraquinones from the roots of Morinda citrifolia

This study examines pressurized hot water extraction of anthraquinones from dried roots of Morinda citrifolia (Noni). The effects of various operating conditions such as water temperature (110, 170, and 220 degrees C), and water flow rate (2, 4, and 6 mL min(-1)) on extraction yield and extraction rate were determined. At 220 degrees C, the extraction yield was the highest and was approximately 43.6 mg g(-1). Subcritical water extraction at 4 mL min(-1) or higher was found to be able to recover all the antraquinones present in the roots within 3 h of extraction, even at 170 degrees C. Pressure had no significant effect on the results for the range of temperatures studied. The flow rate of 6 mL min(-1) resulted in the highest extraction rate, but the extraction efficiency, as measured by the amount of anthraquinones extracted per unit volume of water was lower than that of 4 mL min(-1). Anthraquinones solubility in pressurized hot water was determined. The results indicate a presence of a mass transfer limitation in the extraction process from solid matrix.     

Extraction of tetracycline from fermentation broth using aqueous two-phase systems composed of polyethylene glycol and cholinium-based salts

Aiming at developing not only cheaper but also biocompatible and sustainable extraction and purification processes for antibiotics, in this work it was evaluated the ability of aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and cholinium-based salts to extract tetracycline from the fermented broth of Streptomyces aureofaciens. Conventional polymer/salt and salt/salt ATPS were also studied for comparison purposes. The novel systems here proposed are able to extract tetracycline directly from the fermentation broth with extraction efficiencies higher than 80%. A tailored extraction ability of these systems can also be achieved, with preferential extractions either for the polymer- or salt-rich phases, and which further depend on the cholinium-based salt employed. The gathered results support the applicability of biocompatible ATPS in the extraction of antibiotics from complex matrices and can be envisaged as valuable platforms to be applied at the industrial level by pharmaceutical companies.     

Recent advances in microbial mining

Microbial mining of copper sulphide ores, has been practiced on an industrial scale since the late 1950s. Since then, advances in microbial mining and the role of microorganisms involved in solubilization of metals have assumed commerical importance. The fact that bioleaching processes save energy, have a minimum pollution potential and are able to yield value-added by-products make these processes invaluable. The metal extraction processes using microorganisms, which are currently in active use, concern copper and uranium bioleaching. Biobeneficiation is also applied at an industrial scale for recovery of gold from arsenopyrites. The developments in these processes during the last 15 years, with particular reference to developing nations, are reviewed. Information gathered on molecular genetics of these microorganisms should lead to a better understanding and control of microbial leaching processes. Areas still needing research to sustain economic expansion of microbial mining techniques are indicated.The author is with the Agharkar Research Institute, Agarkar Road, Pune 411 004, India     

Automated extraction improves multiplex molecular detection of infection in septic patients

Sepsis is one of the leading causes of morbidity and mortality in hospitalized patients worldwide. Molecular technologies for rapid detection of microorganisms in patients with sepsis have only recently become available. LightCycler SeptiFast test M(grade) (Roche Diagnostics GmbH) is a multiplex PCR analysis able to detect DNA of the 25 most frequent pathogens in bloodstream infections. The time and labor saved while avoiding excessive laboratory manipulation is the rationale for selecting the automated MagNA Pure compact nucleic acid isolation kit-I (Roche Applied Science, GmbH) as an alternative to conventional SeptiFast extraction. For the purposes of this study, we evaluate extraction in order to demonstrate the feasibility of automation. Finally, a prospective observational study was done using 106 clinical samples obtained from 76 patients in our ICU. Both extraction methods were used in parallel to test the samples. When molecular detection test results using both manual and automated extraction were compared with the data from blood cultures obtained at the same time, the results show that SeptiFast with the alternative MagNA Pure compact extraction not only shortens the complete workflow to 3.57 hrs., but also increases sensitivity of the molecular assay for detecting infection as defined by positive blood culture confirmation.