A fast and versatile method for extraction and quantitation of long-chain acyl-CoA esters from tissue: content of individual long-chain acyl-CoA esters in various tissues from fed rat.

A method for the extraction of acyl-CoA esters from tissue, and their subsequent analysis by HPLC is described. The lipids are removed by a two-phase extraction in a chloroform/methanol/water system. The long-chain acyl-CoA esters are extracted using methanol and a high salt concentration (2 M ammonium acetate). Reextraction of the dry residue after evaporation of extraction solvent results in low overall recoveries (20%). By adding 1 mg/ml acyl-CoA-binding protein to the extraction solvent the overall recovery was increased to 55%. The method is easy and fast to perform and is thereby suitable for analysis of a large number of samples. The advantages of the method over previously published methods are discussed.     

An improved phycobilin extraction method

One unique feature in cyanoprokaryotes, rhodophytes, and cryptophytes is the presence of phycobilin pigments-these water soluble pigments can absorb red, orange, yellow, and green light enhancing the spectral range available for cellular conversion to chemical energy. The presence of phycobilin pigment complexes can be detected using fluorescence, or absorbance measures. Efficient detection of these compounds is essential for use in calibrating absorbance in remote sensing or in physiological studies. The standard procedure for phycobilin analysis involves sonication, extraction in buffer potentially coupled with additional digestion steps using enzymes, repeated freeze and thawing cycles, followed by filtration, and spectrophotometric analyses. An alternative method, using asolectin-CHAPS ((3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid – AC)) solution for extraction, and three cycles of freeze/thawing/sonication, was compared to the phosphate buffer (PB) standard procedure. Cultures of both coccoid and filamentous cyanoprokaryota had improved extraction efficiency (38–80%) using AC. After two complete extractions, no pigment was detectable in AC and near baseline fluorescence was observed in the cell pellet, whereas the PB extraction method removed <90% of the phycobilins after two extractions. Phycocyanin concentration measured by AC extraction was better correlated to lipophilic pigment concentration than using phosphate buffer extraction. AC buffered to pH 6.7 was more effective than AC 3.75. One potential source of experimental error was determined to be the use of a baseline correction for the extraction buffer, not the sample.     

An improved method for the extraction of prostaglandins

Present methods of extracting prostagalndins (PGs) give poor recoveries from synovial fluid, probably because the PGs bind to protein and are lost in the precipitation stage of extraction. Addition of the nonpolar detergent sodium lauryl sulphate prior to extraction improves recovery of PGs. It is suggested that sodium lauryl sulphate competes with PGs for the binding sites.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

An improved method for extraction and separation of photosynthetic pigments

The method for extracting and separating hydrophobic photosynthetic pigments proposed by Katayama et al. (Japanese Journal of Phycology, 42, 71-77, 1994) has been improved to introduce it to student laboratories at the senior high school level. Silica gel powder was used for removing water from fresh materials prior to extracting pigments by a mixture of organic solvents that was also used for chromatographic separation of the pigments. A small silica gel thin-layer plate or a paper strip was used for separating the pigments. The improved method may be applicable to all kinds of plant materials including algae, is easier than most other methods, and can lead to more successful results in separating these pigments by both thin-layer chromatography and paper chromatography. The method has been carried out in student laboratories in some senior high schools and universities in Japan. The results indicate that this laboratory exercise is effective for students to recognise the unity and diversity of plants. Therefore, this laboratory seems to be useful for teaching plant systematics as well as for teaching photosynthesis.     

An improved method for genomic DNA extraction from cyanobacteria

We present an improved method for genomic DNA extraction from cyanobacteria by updating the earlier method from our group (Sinha et al. 2001) that does not require lysozyme treatment or sonication to lyse the cells. This method use lysis buffer to lyse the cells and also skips the initial treatments to remove the exopolysaccharides or to break the clumps. To test the efficacy of the method DNA was extracted from the freshwater cyanobacteria Anabaena variabilis PCC 7937, Anabaena sp. PCC 7120, Synechocystis sp. PCC 6803, Synechococcus sp. PCC 6301 and Rivularia sp. HKAR-4 (Accession number: FJ939128). The spectrophotometric and gel electrophoresis analysis revealed high yield and high quality of genomic DNA extracted by this method. Furthermore, the RAPD resulted in the amplification of unidentified genomic regions of various lengths; however, rDNA amplification gave only one band of 1.5 kb in all studied cyanobacteria. Thymine dimer detection study revealed that thymine dimers are induced only by UV-B radiation in A. variabilis PCC 7937 and there is no effect of PAR and UV-A on its genome. Collectively, all these findings put forward the applicability of this method in different studies and purposes.     

Extraction of tissue long-chain acyl-CoA esters and measurement by reverse-phase high-performance liquid chromatography

Long-chain acyl-CoA esters were extracted from freeze-clamped livers of fed and fasted rats according to the method of Mancha et al. [M. Mancha, G. B. Stokes, and P. K. Stumpf (1975) Anal. Biochem. 68, 600-608] and analyzed on a radially compressed C18, 5 microns, reverse-phase column using a gradient system consisting of acetonitrile and 25 mM KH2PO4, pH 5.3, at 254 nm. Total analysis time was 25 min. Eight peaks in the extract with carbon chain lengths of 12 to 18, which subsequently disappeared on alkaline hydrolysis, were identified. The major acyl-CoA peaks in the extract in order of increasing retention times were 14:0, 16:1, 18:2, 16:0, 18:1, and 18:0. Total liver long-chain acyl-CoA esters were 108 +/- 11 and 248 +/- 19 nmol/g protein for fed and fasted rats, respectively. On fasting (48 h) the levels of 18:2, 16:0, and 18:1 increased two-to threefold and that of 18:0 sixfold. The advantages of this method are that it not only provides a more direct determination of total tissue long-chain acyl-CoA esters, in that no decomposition of the CoA ester is involved, but it also detects the constituent molecular species.     

The intracellular localization of long-chain acyl-CoA synthetase in brown adipose tissue.

1. The acyl-CoA synthetase activity in brown adipose tissue of cold-exposed guinea pig has been studied by measuring the rate of palmitoylcarnitine formation in the presence of excess carnitine palmitoyltransferase. 2. The rate of palmitoylcarnitine formation in the mitochondria was found to be 161 plus or minus 64 nmol.mg-minus-1. min-minus-1 (n=9). 3. In the absence of added palmitate and bovine serum albumin a total of 35 plus or minus 1 nmol endogenous fatty acids.mg-minus-1 were activated with three different mitochondrial preparations. 4. Three different experimental approaches have been used to study the subcellular localization of the enzyme: (a) conventional differential centrifugation (De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Biochem. J. 60, 604-617) (B) the determination of the sediterm of different marker enzymes (Slinde, E. and Flatmark. T. (1973) Anal. Biochem. 56, 324-340) and (c) the determination of the stoichiometry between the activities of these enzymes sedimented at higher centrifugal effects. 5. Throughout all fractionation procedures, the long-chain acyl-CoA synthetase follows strictly the amine oxidase generally considered to be exclusively located on the mitochondrial outer membrane.     

Increase in mitochondrial content of long-chain acyl-CoA in brown adipose tissue during cold-acclimation

The mitochondrial content of long-chain acyl-CoA esters in the brown adipose tissue of guinea pigs increased 3.5-fold from a level of 92 +/- 17 pmol per mg protein (+/- S.E.; n = 7) in the control animals adapted at 22 degrees C to a new steady-state level of 328 +/- 20 pmol per mg protein (+/- S.E.; n = 46) after 10 days of cold-acclimation (5 degrees C). These low values of long-chain acyl-CoA species and the slow adaptive response for their increase do not support the proposal (Cannon, B., Sindin, U. and Romert, L. (1977) FEBS Lett. 4, 43-46) that the fatty acid CoA-esters have a physiological function in the regulation of the H+ (or OH-) permeability of the mitochondrial inner membrane. Experimental evidence is presented supporting the proposal that the long-chain acyl-CoA species are largely confined to the cytosolic side of the inner membrane. The activity of the adenine nucleotide translocase, as estimated at 25 degrees C in the reverse direction, was found to increase 5-fold upon depletion of the mitochondria of fatty acids (free and esterified) by preincubation with bovine serum albumin. The presence of potent inhibitors, i.e., long-chain acyl-CoA species, of adenine nucleotide translocation in brown adipose tissue of thermogenically active animals further supports the conclusion that ATP hydrolyzing mechanisms contribute insignificantly to long-term thermogenesis. The low values of long-chain acyl-CoA hydrolase (EC 3.1.2.1) activity, as measured in intact mitochondria and on a mitochondrial matrix fraction (i.e., 1.6 nmol X min-1 per mg protein), do not support the proposal that the hydrolase activity plays a significant role in the loose-coupling of brown adipose tissue mitochondria, either by a futile cycle mechanism or promoted by free fatty acid-induced uncoupling.     

An improved method for embedding hard tissue in polymethyl methacrylate

An improved routine method for embedding tissue, especially hard tissue, in polymethyl methacrylate (pMMA) is described. The improvements were: the final dehydration step before MMA infiltration was performed with methanol in a Soxhlet apparatus; the stabilizer hydroquinone was not extracted from the monomer (MMA), and more important, the commonly used polymerization initiator, benzoyl peroxide (bpo), was replaced by the initiator, bis (4-tert-butylcyclohexyl)peroxydicarbonate (bbpd). Bbpd is preferred to bpo because it is not explosive, far less is needed and it has a suitable half life. Moreover, bbpd, as obtained from the manufacturer, needs no further purification, in contrast to bpo. Temperatures during bbpd initiated polymerization did not exceed 48 C. In bbpd initiated pMMA, bubbles were almost never generated.     

An improved method for embedding brain tissue in albumin-gelatin.

A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described. Formalin fixed brains of various ages of rhesus monkey (Macaca mulatta) were sunk in 10% phosphate buffered formalin which contained 30% sucrose, and then embedded in a 3% gelatin, 30% egg albumin solution which had been centrifuged to ensure uniformity. The albumin-gelatin was hardened in formaldehyde fumes and blocks cut frozen at 10-40 micron. Sections thus prepared can be handled easily and mounted without damage to the tissue. Modifications of conventional cell and fiber stains produce high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.     

An improved method for the fluorometric determination of tissue catecholamines

Catecholamines extracted from tissues are readily measured by fluorescence following trihydroxyindole derivatization. A complicating factor however, has been the variably high background fluorescence levels obtained which adversely affect the precision of the assay. Employing EDTA is shown to reduce the high background fluorescence to a consistently low level, and to improve the accuracy of the trihydroxyindole method. It is suggested that high background fluorescence readings obtained by previous workers were due to metal ions extracted from tissue and carried over into the derivatization procedure.Measurement of tissue catecholamines by fluorescent methods is improved by the addition of EDTA which gives a consistently low background with a concommitant increase in accuracy.