DNA extraction by zinc.

A fast, very simple and efficient method of DNA extraction is described which takes advantage of DNA sedimentation induced by millimolar concentrations of ZnCl2. The zinc-induced sedimentation is furthermore strongly promoted by submillimolar phosphate anion concentrations. Within <30 min, the method recovers >90% of DNA irrespective of whether a plasmid DNA or short oligonucleotides are the extracted material. The method works with plasmid DNA and oligonucleotide concentrations as low as 100 ng/ml and 10 microg/ml, respectively, without using any expensive facilities or toxic chemicals.     

Affinity partitioning and extraction of proteins.

Affinity partitioning of enzymes and plasma proteins in aqueous two-phase systems has been reviewed. Besides basic theoretical considerations of the principle of affinity partitioning the chemistry of coupling ligands to the polymers, the nature and properties of selected biomimetic ligands like dye-ligands, immunoligands, metal chelate ligands and hydrophobic ligands are reported. The usefulness of affinity partitioning for studying the affinity of ligands and their specificity to proteins has been demonstrated by selected examples. The method proved also applicable to study the structural dynamics of proteins as exemplified with phosphofructokinase from baker's yeast and human alpha-2-macroglobulin. The current knowledge of metal chelate affinity partitioning is presented as well as the applicability of affinity partitioning for the purification of enzymes.     

Edge extraction by active defocusing.

A novel edge extraction method that employs an active defocusing technique is presented. The method is based on the principle that a Laplacian-of-Gaussian (LOG) operation can be approximated by a Difference-of-Gaussian (DOG) operation. While such an operation is usually done in digital image processing, it can also be very effective conducted in a combination of optical techniques and digital processing. In this edge extraction method, a focused image of an object in a scene is first acquired. The image of the scene is then slightly defocused by changing the focal length of the camera. A real time subtraction operation is applied to subtract the defocused image from the previously acquired image. It produces a residual image that emphasizes abrupt intensity variations. An objective evaluation, called an edge index, is performed on the resulting image. The amount of defocusing is carefully adjusted according to this measurement so that a desired edge image is generated. Boundaries of objects can then be obtained by further enhancement of the edge image. Since this edge detection method is an optical-based process aided by digital processing, it is fast and relatively inexpensive.     

Continuous counter-current two-phase aqueous extraction.

Continuous counter-current column operation provides operating convenience for contacting two-phase aqueous partitioning systems for protein extraction. We discuss in detail the important parameters for designing spray, packed, plate and York-Scheibel columns for protein recovery using both polymer-polymer and polymer-salt two-phase aqueous systems. We compare the various contractors for their operating and extraction efficiency. The work also provides a step-by-step design procedure and specific recommendations for future data needs.     

Rapid extraction of plasmids from Clostridium perfringens.

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.     

Acid-labilization of sterols for extraction from yeast.

A wild type strain of yeast, Saccharomyces cerevisiae, pretreated with a mild acid hydrolysis, exhibited a 4-fold increase in sterol yield upon saponification and extraction. This increased yield is reflected in both major and minor sterols (ergosterol; zymosterol) and sterol esters.     

Purification of herpesvirus nucleocapsids by fluorocarbon extraction.

Purified nucleocapsids were prepared from herpes simplex virus type-1 and herpes simplex virus type-2 infected cells, using a fluorocarbon extraction technique with Freon 113. Fifty to 85% of the total nucleocapsid population remained 'nucleated' or full in terms of the nucleic acid core making them useful as a source of DNA. CsCl banded DNA represented 30--40% of the theoretical yield of the total nucleocapsid population.     

Oral glucose decreases hepatic extraction of insulin.

Peripheral venous (plasma) insulin and C-peptide concentrations were measured in eight normal subjects given oral or intravenous glucose sufficient to produce similar plasma glucose concentrations. The expected increased insulin response to oral as compared with intravenous glucose was not matched by a comparable increase in C-peptide concentration. The ratio of insulin to C-peptide concentrations doubled 30 minutes after oral glucose was given; no comparable rise was seen with intravenous glucose (p = 0.01). This finding is interpreted as evidence for decreased hepatic extraction of insulin after administration of oral glucose. Such a decrease could account for at least half of the well known difference in peripheral insulin concentrations after administration of oral as compared with intravenous glucose.     

Pilot plant scale extraction of alginates from Macrocystis pyrifera. 2. Studies on extraction conditions andmethods of separating the alkaline-insoluble residue

The effect of temperature (70, 80, 90 ^°C) and time (1–9 h) during the alkaline extraction step on alginate yield and quality were studied. The alginate yield increased with time and maximum yield was obtained after 3.5 h treatment, ranging from19.4% at 70 ^°C to 21.9% at 90 ^°C. The viscosity of the alginate produced was inversely correlated with the temperature and time. At70 ^°C the slope of the curve was almost zero(753 to 923 mPa s); at 90 ^°C the viscosity loss was 154 mPa s per hour during the first two hours, reducing from 523 to 86 mPa s after 5 h; 80 ^°C yielded values between those for 70 ^°C and90 ^°C. The best conditions for alkaline extraction were using pH 10 at 80 ^°C for two hours. The curves obtained gave useful information for controlling the viscosity of the alginate during production. It was found that viscosity of the paste formed during alkaline extraction (`process viscosity') was the best parameter to determine there action rate during extraction. Alginate yield increased during filtration time from 17.6% to 23.7%after 55 min at 70 ^°C. In this step the viscosity of the alginate obtained remained almost constant (522–610 mPa s), indicating no degradation of the products during filtration. The best dilution to filter the alginate extract was obtained at 45 mPa s. Diatomaceous earth (Celite) and expanded lava(Perlite) were tested as filter aids. Expanded lava was the best filter aid, using 1 kg per kilogram of alginate produced. Three methods were studied to separate the alkaline-insoluble residues after extraction: filtration, centrifugation, flocculation, and combinations of them. The best system found was filtration with flocculant in a rotary vacuum filter, with a knife advance of 0.1 mm every 3.5 seconds and drum rotation of 2 rpm, yielding an average filtration flow rate of 10.5 L min^-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

The extraction and characterization of bovine epidermal alpha-keratin.

1. The alpha-fibrous protein (alpha-keratin) component of bovine epidermis has been extracted and characterized. 2. Prekeratin, a multichain unit of the epidermal tonofilaments, was shown to consist of six different polypeptide chains on polyacrylamide-gel systems containing sodium dodecyl sulphate or sodium decyl sulphate with discontinuous gel buffers, but only three chains were seen when a gel system containing sodium dodecyl sulphate with a continuous gel buffer was used. 3. Extraction of the 'keratinized' stratum corneum and the living part of the epidermis with urea buffers at pH 7.6 or 9.0 released 60% of the total dry weight of the tissues in the form of alpha-helical polypeptides. 4. The numbers, relative amounts and properties of the extracted polypeptides were the same as the subunits of prekeratin and thus are derived from the tonofilaments in situ. 5. The subunits of prekeratin and the polypeptides extracted from the living cell layers contained an average of six cysteine residues, but those from the stratum corneum contained an average of three intrachain disulphide bonds. 6. The polypeptide chains aggregated through non-covalent interactions in vitro into filaments that were similar to the tonofilaments. 7. Since the polypeptides could be released from the stratum corneum without breaking covalent bonds, it is concluded that such bonds do not cross-link the tonofilaments and non-fibrous keratohyalin. It is suggested that the tonofilaments and keratohyalin of bovine epidermis are associated by secondary bonding forces.     

O2 extraction of right and left ventricles.

O2 extraction was measured simultaneously in right and left ventricles of dogs. Extraction was about 2.5 vol% higher in left ventricle. This figure, together with flow measurements of others (1-3), indicates that VO2/100g is at least twice as great in left ventricle as in right.     

Trends and future prospects of aqueous two-phase extraction.

Aqueous two-phase systems form the basis for an extraction technology of proteins. Trends in the development of the technology are reviewed and discussed with regard to future applications.     

Fluidized bed adsorption for whole broth extraction.

Fluidized bed adsorption using a high-density synthetic resin has proven to be an invaluable technique for separating novel compounds from unfiltered fermentation broths during the very early stages of fermentation development, where product concentrations are typically in the parts per million range. Previous initial downstream processing strategies consisted of cell separation from whole broth or direct extraction with water-immiscible solvents, both of which resulted in lengthy time cycles, conflicts with existing operations requiring the use of high-cost centrifugal separators, and environmental/solvent recovery concerns. Laboratory and subsequent pilot plant process development work along with concomitant improvements in yield, quality, and time cycles are presented for one of several fluidized bed processes piloted in Merck's Natural Product Isolation facility.