Digestion Optimization

Iczkowski, K. A., Cheng, L., Crawford, B. G., and Bostwick, D. G. 1999. Steam heat with an EDTA buffer and protease digestion optimizes immunohistochemical expression of basal cell-specific antikeratin 34pE12 to discriminate cancer in prostatic epithelium. Mod. Pathol. 72 1-4. 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

An initial solution was prepared by the hydrofluoride method, i.e. melting of a mixture of ammonium hydrofluoride and tantalite, followed by the digestion of soluble components with water and separation of the solution by filtration. The prepared initial solution contained no free HF or any other acid, and had a pH 3. In order to obtain an optimal acidity level, sulfuric acid was added to the solution. Concentrations of Ta2Os (50-60 g/1) and Nb205 ( 30 g/1) were kept approximately constant during the preparation of the solutions. Extraction was performed using a polypropylene beaker and a magnetic stirrer. 

Figure 48-12. Schematic illustration of some aspects of the role of the osteoclast in bone resorption. Lysosomal enzymes and hydrogen ions are released into the confined microenvironment created by the attachment between bone matrix and the peripheral clear zone of the osteoclast. The acidification of this confined space facilitates the dissolution of calcium phosphate from bone and is the optimal pH for the activity of lysosomal hydrolases. Bone matrix is thus removed, and the products of bone resorption are taken up into the cytoplasm of the osteoclast, probably digested further, and transferred into capillaries. The chemical equation shown in the figure refers to the action of carbonic anhydrase II, described in the text. (Reproduced, with permission, from Jun-queira LC, Carneiro J BasicHistology. Text Atlas, 10th ed. McGraw-Hill, 2003.)...

Munoz O, Velez D, Montoro R (1999) Optimization of the solubilization, extraction and determination of inorganic arsenic [As(III) i- As(V)] in seafood products by acid digestion, solvent extraction and hydride generation atomic absorption spectrometry. Analyst 124 601-607. 

Nielsen, R. G. and Rickard, E. C., Method optimization in capillary zone electrophoretic analysis of hGH tryptic digest fragments, /. Chromatogr, 516, 99 1990. 

Pepsinogen is produced by the chief cells. Within the lumen of the stomach, this precursor molecule is split by HCl to form the active enzyme pepsin. Optimally active at an acidic pH (pH = 2), pepsin begins protein digestion by fragmenting proteins into smaller peptide chains. 

Pancreatic juice neutralizes the acidic chyme entering the duodenum from the stomach. Neutralization not only prevents damage to the duodenal mucosa, but also creates a neutral or slightly alkaline environment optimal for the function of pancreatic enzymes. The pancreas also secretes several enzymes involved in the digestion of carbohydrates, proteins, and lipids. 

Hancock, W.S., Chloupek, R.C., Kirkland, J.J., Snyder, L.R. (1994). Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples. I. Optimizing the separation of a growth hormone tryptic digest. J. Chromatogr. A 686, 31 -3. 

Comments There are several suggested controls for this assay, including use of yeast total RNA as a negative control (check for probe species specificity) and a no RNAse control to determine probe stability. In Fig. 6.3A, the positive control marker lane was produced by addition of R-luc-4 sites or F-luc mRNA only to the assay. Also, optimal times for RNAse digestion will vary from probe to probe. In addition, for maximum sensitivity a probe with high specific activity is preferable (yet still in molar excess to the mRNA). 

Mix the gel suspension in a shaker at 37°C for 4-48 hours. Maintain the gel in suspension during mixing. The optimal time for complete digestion varies depending on the IgG subclass... 

Elemental or peptide-based formulations have partially hydrolyzed protein or fat components. Peptide-based formulations replace some of the protein with dipeptides and tripeptides, thereby optimizing absorption in patients with impaired digestive or absorptive capacity. 

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