Tissues extracts

Parallel to the activities in the treatment of pernicious anemia were observations in the 1930s that most farm animals had a requirement for an unknown factor beyond the vitamins then known. The lack of this factor became apparent, eg, when chicks or pigs fed a diet with only vegetable protein evidenced slow growth rate and high mortahty. It became apparent that the requited factor, termed animal protein factor, was present in animal sources such as meat and tissue extracts, milk whey, and cow manure. Subsequent to its isolation, it was rapidly shown that vitamin B 2 is the same as animal protein factor. 

Extrinsic Pathway. Coagulation is initiated when tissue extracts with Hpid—protein properties are released from the membranes of endothehal cells following injury or insult. These substances, collectively designated tissue thromboplastin, complex with circulating Factor VII and in the presence of calcium ions subsequentiy activate Factor X (Fig. 1). In vitro evidence suggests that Factor X can be activated less rapidly through the interaction of kaUikrein [9001-01-8] with Factor VII. 

The tissue to be analyzed is placed directiy onto the gel. Using the tissue itself and not tissue extracts has advanced the study of proteins that are difficult to extract from tissue, or are damaged by the extraction procedure. Dtif is an important advancement in the area of sample handling and appHcation where direct appHcation of a soHd to a gel matrix may actually enhance resolution. 

Rcnsl dipcptidflsc (from porcine kidney cortex) [9031-96-3] Mr 47,000 [EC 3.4.13.11]. Purified by homogenising the tissue, extracting with Triton X-100, elimination of insoluble material, and ion-exchange, size exclusion and affinity chromatography. [Hitchcock et al. Anal Biochem 163 219 7957.]... 

A 17 amino acid long peptide sequentially related to opioid peptides in particular dynorphin A. OFQ/N is inactive at the 5, k, and p opioid receptors, but binds to its own NOP receptor (formerly ORL-1, for opioid receptor like-1). In contrast to opioid peptides, OFQ/N has no direct analgesic properties. OFQ/N is the first example for the discovery of a novel neurotransmitter from tissue extracts by using an orphan receptor as bait. Centrally administered in rodents, OFQ/N exerts anxiolytic properties. OFQ/N agonists and antagonists... 

Lindstrom and Schubert63 applied GC-MS, GC-MS-MS and direct inlet MS-MS to determine 1,1-dichlorodimethyl sulfone (201, DDS) in aquatic organisms outside a pulp mill bleach plant. Both GC-MS-MS and direct inlet MS-MS of tissue extracts of fish and mussel appeared to be sensitive, selective and fast techniques for the determination of DDS. 

Historically, the development of animal cell culture systems has been dependent upon the development of new types of tissue culture media. Mouse L cells and HeLa cells were developed using a balanced salt solution supplemented with blood plasma, an embryonic tissue extract, and/or serum. In 1955 Eagle developed a nutritionally defined medium, containing all of the essential amino acids, vitamins, cofactors, carbohydrates, salts, and small amounts of dialyzed serum (Table 1). He demonstrated that this minimal essential medium (MEM) supported the long-term growth of mouse L and HeLa ceils. Eagle s MEM was so well defined that the omission of a single essential nutrient eventually resulted in the death of these animal cells in culture. 

Water, plant tissue Extract with acetonitrile filter if necessary HPLC/UV/EC No data (water) 50 pg/kg (plants) 95-99 Clark et al. 1985... 

Plant tissue Extract with ethyl acetate and sodium sulfate filter through silanized glass wool GC/TID No data No data AOAC 1984... 

The method of choice for the determination of a- and P-endosulfan in blood, urine, liver, kidney, brain, and adipose tissue is gas chromatography equipped with an electron capture detector (GC/ECD) (Coutselinis et al. 1976 Demeter and Heyndrickx 1979 Demeter et al. 1977 Le Bel and Williams 1986). This is because GC/ECD is relatively inexpensive, simple to operate, and offers a high sensitivity for halogens (Griffith and Blanke 1974). After fractionation of adipose tissue extracts using gel permeation chromatography, detection limits of low-ppb (1.2 ng/g) were achieved for endosulfan and other chlorinated pesticides using GC/ECD (Le Bel and Williams 1986). 

Tissue extracts from 19 bald eagle Haliaectas leucocephalus) carcasses were examined to determine if biomagnification of TCDD had occurred in a manner similar to DDT. These carcasses came from the states of Alaska, Maine, North Dakota, Wisconsin, Michigan, Minnesota, Arkansas, Illinois, Missouri, Maryland, Virginia, Iowa, New York, New Jersey, and Florida between 1966 and 1971 and were collected and furnished by scientists at the Patuxent Wildlife Center, U.S. Department of the Interior, Laurel, Md. The samples were selected from these states to provide a widely dispersed sampling population. 

Standard curves performed under our defined radioimmunoassay conditions ([ H]PbTx-3 = 1 nM, antiserum dilution = 1 2000, assay volume = 1 ml) demonstrated the ability of this antiserum to bind equally to PbTx-2 and PbTx-3, suggesting specificity for the cyclic polyether backbone region of the molecule (Figure 8). The linear portion of the curve indicated a lower detection limit of 0.2-0.5 ng in saline buffer under these conditions. Evaluation of this assay for use with biological fluids and tissue extracts is underway. 

Until this point, the sample preparation techniques under discussion have relied upon differences in polarity to separate the analyte and the sample matrix in contrast, ultraflltration and on-line dialysis rely upon differences in molecular size between the analyte and matrix components to effect a separation. In ultrafiltration, a centrifugal force is applied across a membrane filter which has a molecular weight cut-off intended to isolate the analyte from larger matrix components. Furusawa incorporated an ultrafiltration step into his separation of sulfadimethoxine from chicken tissue extracts. Some cleanup of the sample extract may be necessary prior to ultrafiltration, or the ultrafiltration membranes can become clogged and ineffective. Also, one must ensure that the choice of membrane filter for ultrafiltration is appropriate in terms of both the molecular weight cut-off and compatibility with the extraction solvent used. 

Rosengren S, Firestein GS, Boyle DL. Measurement of inflammatory biomarkers in synovial tissue extracts by enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol 2003 10(6) 1002-1010. 

The final yield and purity of a heparin preparation depend largely on the use of appropriate, analytical methods at different stages of extraction and purification. Heparin in tissue extracts is still most commonly determined biologically, by such assays as the U.S.P. assay for anticoagulant activity. It is now recognized10 that the anticoagulant activity does not measure the actual concentration of heparin (see also Sections XII and XIII). 

The inner surface of a vial is treated with the insecticidal coating by introducing a measured volume, usually 5 or 10 ml., of standard solution or tissue extract and placing the vial in an air bath oven at 70° C. with care to avoid local overheating until the volume is reduced to about 1 ml. The vial is then removed and rolled by hand while the remaining solvent evaporates, care being taken to secure an even distribution over the sides and bottom. As soon as the vial is cool, the cover is put on with the open hole over the vial. Flies or other test insects may then be introduced as desired. 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

Polymer Labs. PLRP-S Gradient A 0.001 M oxalic acid, 0.5% formic acid + 3% THF in water, B = THF Positive ESF MS/MS OTC, CTC, TC, DC, and its 4-epimer in pig tissues Extraction with sodium succinate solution, protein removal with TCA, SPE cleanup on polymeric RP column DL = 0.5-4.2 ng/g [57]... 

Polymer Labs. PLRP-S 0.01M oxalic acid-ACN (75 25, v/v) UV 360 nm Animal tissues Extraction with oxalic buffer followed by chelation and deproteination, cleanup with styrene-divinylbenzene cartridge Rec 76-87% [77]... 

Egg, animal tissues Extraction with EA, evaporated, reconstituted in MeOH, online MCAC cleanup DL = 3 pg/kg 91-94% (egg) 101-104% (animal tissues) [40]... 

The tissue surrogates described here clearly represent a simplification of real FFPE tissues. However, they represent a useful and efficient construct for the evaluation and optimization of tissue extraction conditions for proteomic studies. More informative studies will likely be realized by using more complex tissue surrogates, which can be created by incorporating additional proteins into lysozyme solutions. Tissue surrogates comprised of up to five proteins have been successfully analyzed by MS (Fowler, unpublished data). Additionally, RNA, DNA, lipids, or carbohydrates can be added at nanomolar to millimolar concentrations to increase the complexity of the model system to better mimic whole tissue. The use of these more complex tissue surrogates should facilitate the development of protein recovery protocols optimal for proteomic investigation. 

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