A rapid procedure for the isolation of phycobilisomes from cyanobacteria

This paper describes a method for the rapid isolation of phycobilisomes using a cationic detergent, CTAB (cetyltrimethylammonium bromide). The method has distinct advantages over those currently in use in that (i) release of intact phycobilisomes from cells in the presence of CTAB occurs in 40 s (as compared to 40-60 min of incubation required with Triton X-100), thereby reducing the chances of proteolysis of the component phycobiliproteins; and (ii) these phycobilisome preparations have reduced chlorophyll contamination in the initial stages. In addition this method also helps retain the structural and functional properties, as evidenced by spectroscopy and sodium dodecyl sulfate-polyacrylamide gel analysis.     

Quantifying cholesterol synthesis in vivo using (2)H(2)O: enabling back-to-back studies in the same subject

The advantages of using (2)H(2)O to quantify cholesterol synthesis include i) homogeneous precursor labeling, ii) incorporation of (2)H via multiple pathways, and iii) the ability to perform long-term studies in free-living subjects. However, there are two concerns. First, the t(1/2) of tracer in body water presents a challenge when there is a need to acutely replicate measurements in the same subject. Second, assumptions are made regarding the number of hydrogens (n) that are incorporated during de novo synthesis. Our primary objective was to determine whether a step-based approach could be used to repeatedly study cholesterol synthesis a subject. We observed comparable changes in the (2)H-labeling of plasma water and total plasma cholesterol in African-Green monkeys that received five oral doses of (2)H(2)O, each dose separated by one week. Similar rates of cholesterol synthesis were estimated when comparing data in the group over the different weeks, but better reproducibility was observed when comparing replicate determinations of cholesterol synthesis in the same nonhuman primate during the respective dosing periods. Our secondary objective was to determine whether n depends on nutritional status in vivo; we observed n of ～25 and ～27 in mice fed a high-carbohydrate (HC) versus carbohydrate-free (CF) diet, respectively. We conclude that it is possible to acutely repeat studies of cholesterol synthesis using (2)H(2)O and that n is relatively constant.     

The radioreceptor assay: a simple, sensitive and rapid analytical procedure

Radioreceptor assays (RRAs) are analogous in concept to radioimmuno assays. Characteristic to both methods is the saturable, specific, competitive and reversible ligand/receptor interaction. The RRA is simple, sensitive and reproducible and provides a degree of precision comparable to more sophisticated analytical techniques. Since RRAs require little specialized equipment, they can be used routinely by any laboratory engaged in biochemical research or, as an inexpensive exercise to teach the fundamentals of ligand binding and analytical pharmacology.     

A rapid procedure for the isolation, identification and estimation of citrinin

A rapid procedure for the isolation, identification, and estimation of citrinin, the antibiotic cum nephrotoxin, has been proposed. The colorimetric procedure proposed for estimation makes use of the phenolic function of the compound. This method was found to be applicable to a wide variety of materials.     

Measurement of TG synthesis and turnover in vivo by 2H2O incorporation into the glycerol moiety and application of MIDA

A method is presented for measurement of triglyceride (TG) synthesis that can be applied to slow-turnover lipids. The glycerol moiety of TG is labeled from 2H2O, and mass isotopomer distribution analysis (MIDA) is applied. Mice and rats were given 4-8% 2H2O in drinking water; TG-glycerol was isolated from adipose and liver during < or =12-wk of 2H2O labeling. Mass isotopomer abundances in the glycerol moiety of TG were measured by GC-MS. The combinatorial pattern of isotopomers revealed the number of H atoms in glycerol incorporating label from 2H2O (n) to be 3.8-4.0 of a possible 5 for adipose tissue and 4.6-4.8 for liver TG. Hepatic TG-glycerol in fact reached 97% predicted maximal value of label incorporation (4.4-4.6 x body 2H2O enrichment), indicating near-complete replacement of the liver TG pool. Label incorporation into adipose tissue revealed turnover of mesenteric TG to be faster (k = 0.21 day-1) than other depots (k = 0.04-0.06 day-1) in mice. TG isolated from subcutaneous depots of growing adult rats plateaued at 85-90% of calculated maximal values at 12 wk (k = 0.05 day-1), excluding significant dilution by unlabeled alpha-glycerol phosphate. Turnover of plasma TG, modeled from 2H incorporation over 60 min, was 0.06 min-1 (half-life 11.5 min). In summary, use of 2H2O labeling with MIDA of TG-glycerol allows measurement of new alpha-glycerol phosphate-derived TG synthesis and turnover. The hypothesis that mesenteric TG is more lipolytically active than other depots, previously difficult to prove by isotope dilution techniques, was confirmed by this label incorporation approach.     

A rapid and mild procedure for the isolation of DNA from mammalian cells

A procedure is described for the isolation of DNA from mammalian cells using polycarbonate filters. This method results in high yields of large-molecular-weight DNA, which is essentially free from protein and RNA. The procedure is rapid (approximately 2 h), does not require organic solvents, and can isolate 1.5–140 μg DNA per filter without the addition of carrier. The isolation of DNA from aflatoxin B₁-treated cells by the filter method is described in order to illustrate its advantages for the preparation of DNA containing lesions of low chemical stability.     

Measurement of in vivo nitric oxide synthesis in humans using stable isotopic methods: a systematic review

Stable isotopic methods are considered the “gold standard” for the measurement of rates of in vivo NO production. However, values reported for healthy human individuals differ by more than 1 order of magnitude. The reason for the apparent variability in NO production is unclear. The primary aim of this review was to evaluate and compare the rates of in vivo NO production in health and disease using stable isotope methods. Articles were retrieved using the PubMed electronic database. Information on concentrations, isotopic enrichments of fluxes, and conversion rates of molecules involved in the NO metabolic pathway was extracted from selected articles; 35 articles were included in the final analysis. Three protocols were identified, including the arginine-citrulline, the arginine-nitrate, and the oxygen-nitrate protocols. The arginine-citrulline protocol showed a wider variability compared to the arginine-nitrate and oxygen-nitrate protocols. The direction of the association between disease state and rate of NO production was essentially determined by the etiopathogenesis of the disorder (inflammatory, metabolic, vascular). Considerable variation in methodologies used to assess whole-body NO synthesis in humans exists. The precision of several aspects of the techniques and the validity of some assumptions made remain unknown, and there is a paucity of information about physiological rates of NO production from childhood over adolescence to old age.     

A simple, rapid and efficient isolation of erythrocyte Cu2Zn2-superoxide dismutase.

On the basis of the thermal stability of erythrocuprein (Cu2Zn2-superoxide dismutase) a rapid preparation technique was devised and successfully employed to isolate this protein. Partial heat-deterioration of the haemolysate and subsequent chromatography of the supernatant on DEAE-Sephacel and Sephadex G-75 yielded an electrophoretically homogeneous protein within a few days. The physicochemical properties and biochemical function were identical with those reported for Cu2Zn2-superoxide dismutases prepared by established methods.     

A rapid procedure for the isolation of plasmid DNA from environmental bacteria.

The INSTA-MINI-PREP method, a rapid protocol for plasmid DNA extraction, was originally developed to prepare plasmid DNA from 1 to 3 ml miniprep Escherichia coli cultures. Direct extraction of plasmid DNA is achieved by a two-phase solution which is separated by centrifugation in the presence of the INSTA-PREP gel barrier material. This method has been successfully tested on various environmental Salmonella strains, although it was not suitable for Pseudomonas aeruginosa and enterococci strains. The INSTA-MINI-PREP method is a new alternative procedure to screen plasmid contents of Salmonella and E. coli strains rapidly and easily.     

Mouse nerve growth factor: a rapid isolation procedure for the alpha and gamma subunits.

A rapid method for isolating the α and γ subunits of mouse submaxillary gland nerve growth factor, as by-products of the commonly used Bocchini-Angeletti (2.5 S) procedure, has been devised. Approximately 40 mg of each subunit is obtained from 400 pairs of adult glands. The subunits isolated in this fashion are indistinguishable from those obtained from the homogeneous 7 S complex as judged by gel electrophoresis or their association-dissociation behavior.     

Simple and rapid procedure for synthesis of deoxynucleoside 3'-2-cyanoethyl-N,N-diisopropyl-amino phosphites

2-Cyanoethyl-bis/N,N-diisopropyl/phosphoamidite can be prepared from PCl3 by a very fast and efficient procedure. Without purification it was used for the phosphitilation of suitably protected deoxynucleosides which after simple purification are obtained with a high yield, in a stable and easy to manage form.     

A simple, rapid, high yield isolation and purification procedure for chloroperoxidase isoenzymes

A simple four-step procedure has been developed for isolation of chloroperoxidase from the mold Caldariomyces fumago. Polyethyleneglycol precipitation of the contaminating pigment in the growth medium, followed by chromatography of the soluble enzyme fraction on a QAE-ZetaPrep-250 cartridge and ammonium sulfate precipitation affords isolation of the chloroperoxidase. Extensive dialysis and chromatography on DE-53 cellulose allows the separation and further purification of chloroperoxidase A and B isoenzymes.     

A rapid chemical procedure for isolation and purification of chromosomal DNA from gram-negative bacilli

A rapid and simple method for preparing chromosomal DNA from gram-negative bacilli is presented. It is based on the alkaline (NaOH 0.03 m) lysis of cell walls. The resulting emulsion is purified by proteinase K (0.625 mg/g of wet wt), SDS, and the deproteinizing agent (chloroform isoamyl alcohol). The purity, molecular nature, and yield of DNA obtained by the present method are compared with those of DNA extracted by Marmur's procedure and a Marmur's modified procedure. We have developed and standardized this original method to isolate double-stranded DNA, free of proteins and RNA contamination and with a significantly higher yield of DNA than the two other methods. This procedure is particularly useful for strains with low growth and can be applied in every field concerned with DNA analysis.