Functional testing, protein

Abstract. A smooth empirical potential is constructed for use in off-lattice protein folding studies. Our potential is a function of the amino acid labels and of the distances between the Ca atoms of a protein. The potential is a sum of smooth surface potential terms that model solvent interactions and of pair potentials that are functions of a distance, with a smooth cutoff at 12 Angstrom. Techniques include the use of a fully automatic and reliable estimator for smooth densities, of cluster analysis to group together amino acid pairs with similar distance distributions, and of quadratic progrmnming to find appropriate weights with which the various terms enter the total potential. For nine small test proteins, the new potential has local minima within 1.3-4.7A of the PDB geometry, with one exception that has an error of S.SA. 

Fig. 29. Rejection of test proteins as a function of molecular weight, in a series of ultrafiltration membranes with different weight cut-offs (69).

Female sexual development and behaviour in mammals occurs by default and requires no ovarian secretion, and it is only in genetic males that the testis can secrete hormones which destroy this female pattern and superimpose that of the male. Sexual differentiation is not so well defined in fish, and larval exposure to both synthetic estrogens and androgens is widely used in aquaculture to produce monosex cultures. Endocrine disruption of sexual differentiation in fish may therefore reflect both the complexity and diversity of such processes between different species. Some care is required in use of the terms hermaphrodite and sex-reversal since a true hermaphrodite has both functional testes and ovaries and a sex-reversed fish is fully functional as its final sex—both produce the appropriate viable gametes. Such functional sex-reversal is not possible in mammals, but in some species of fish it is the normal developmental pattern. In most of the cases of hermaphroditism or sex-reversal reported in the non-scientific press, there is evidence only for a few ovarian follicles within a functional testis. This may be considered as feminisation or a form of intersex, and is very clearly endocrine disruption, but it is certainly neither sex-reversal nor hermaphroditism. In some cases the terms have even been used to infer induction of a single female characteristic such as production of yolk-protein by males. 

The LB technique was chosen for covering the spheres because it was shown to provide enhanced thermal stability of many types of proteins in deposited layers (Nicolini et al. 1993, Erokhin et al. 1995, Antolini et al. 1995), which no other technique is able to achieve. Since only the upper protein layer is involved in the catalytic activity, no special attention was paid to check whether the deposited layer is a monolayer or multilayer. However, the samples were thoroughly washed to remove protein molecnles not bound covalently to the sphere surface, since during the functional test these molecules could contribute to the measured apparent catalytic activity. 

Laboratory monitoring is performed before initiating therapy and before each cycle of chemotherapy. A complete blood count should be obtained prior to each course of chemotherapy to ensure that hematologic values are adequate. In particular, white blood cell counts and absolute neutrophil counts can be decreased in patients receiving chemotherapy such as irinote-can and 5-FU and increase the risk of infection. Baseline liver function tests and an assessment of renal function should be done prior to and periodically during therapy. Other selected laboratory tests include checking for the presence of protein in the urine in patients receiving oxaliplatin and bevacizumab. 

Dahlback B. Resistance to activated protein C, the Arg506 to Gin mutation in the factor V gene and venous thrombosis. Functional tests and DNA based assays, pros and cons. Thromb Haemost, 1995 73,739-42. 

Renal Effects. Renal function tests (serum creatinine and urate, urinary hemoglobin, protein and glucose) completed in 11 hexachloroethane workers were within the normal range (Selden et al. 1994). Plasma hexachloroethane levels in these workers, who wore protective equipment, were 7.3 + 6.04 pg/L at the time of the tests (Selden et al. 1993). Mild skin and mucous membrane irritation were reported in the exposed group, suggesting that exposure may have been through either the inhalation or dermal routes of exposure. 

Renal Effects. No effects on renal function tests (serum creatinine and urate, urinary hemoglobin, protein and glucose) were noted in 11 hexachloroethane-exposed workers who wore protective clothing (Selden et al. 1994). 

Laboratory procedures specific to galactosemia are dealt with below, but a number of other tests are also of value. Liver function tests, though sometimes helpful, are not always informative (see above, Section 2.2). Protein is usually present in the urine in untreated cases, but this occurs in other diseases. Aminoaciduria is a very common finding in galactosemia, though Holzel (H3) states it is absent in some older children with a mild form of galactosemia aminoaciduria occurs in other diseases and is frequently accompanied by proteinuria and glucosuria. 

The final approach is to use a combination of microarray techniques, antisense oligonucleotides, functional tests and the use of monoclonal antibodies to identify matrix proteins and families of matrix proteins involved in the biomineralization process, and ultimately to establish their function. The recent publication of the genome of the sea urchin... 

By far, the most widely used application of the yeast two-hybrid system as intimated in Introduction is the identification of protein partners for a test protein of either known or unknown function. Here, the DNA encoding the test protein or the domain of a test protein is cloned in frame into the bait vector. The fish vector contains cDNA and it is constructed so that there is one cDNA molecule per vector. Fish and bait vectors are cotransformed into the appropriate strain of competent yeast, and the resultant transformed yeast cells are screened for growth on SD media and for reporter gene activities. Putative positive clones are then isolated and characterized further. In the next section, each of these stages is discussed in detail. 

A number of clinical tests are available to detect kidney damage. The clinician examining a patient or the toxicologist monitoring an animal toxicity stndy collects urine and blood samples. Indications of kidney damage (which, of course, for the human patient could be related to many factors other then chemical toxicity) include urinary excretion of excessive amonnts of proteins and glucose, and excessive levels in the blood of unexcreted waste products such as urea and creatine. A number of additional kidney function tests are available to help pin down the location of kidney dysfunction. 

The viability and function tests described above are used to evaluate the hepatocytes within the slice. Up to now, tests to measure the viability of the non-parenchymal cells have not been reported. The presence of the latter cell types is one of the conceptual advantages of slices as compared to isolated hepatocytes. As some drug targeting devices are designed to target non-parenchymal cells in the liver, the development of tests for the sinusoidal cell types deserves more attention. For example, the uptake of substrates such as succinylated human serum albumin (Suc-HSA,which is specifically endocytosed by endothelial cells [79]), or hyaluronic acid [80], can be used to assess the functionality of endocytotic pathways in the endothelial cells in the liver [81]. Other modified proteins that are specifically taken up by Kupffer cells such as mannosylated HSA, may be used to assess the functionality of the endocytotic pathway in Kupffer cells [79]. Another parameter which can be used to assess the functionality of these non-parenchymal liver cells, is the excretion of cytokines in response to pro-inflammatory stimuli. Non-parench5mal cell function in liver slices will be described in more detail in the Section 12.7. 

Oral contraceptives have also been reported to produce increases in sulfobromophthalein retention and other liver function tests, as well as in prothrombin time, clotting factors VII, VIII, IX, serum thyroxine, and protein-bound iodine (B8). In a group of 48 women the mean cholesterol value was 206 41 mg/100 ml while they were receiving a variety of oral contraceptives and 179 28 mg/100 ml when they were not receiving the drugs (W19). 

Oral contraceptives have their most significant effect on endocrine parameters. Blood cortisol, thyroxine, protein-bound iodine, T3 uptake, and urinary free cortisol are elevated. Urinary 17,21-dihydroxy steroids, 17-ketosteroids, and estrogens are decreased. There is no effect on urinary catecholamines or VMA (Table 10) (LIO). The effect of thyroid functions tests is due to the administered hormone stimulating an increase in the production of thyroid-binding globulin which in turn binds 1-thyroxine. The lowering of free thyroxine stimulates the anterior pituitary to produce thyrotropin, which in turn stimulates the thyroid to produce more thyroxine. Since the additional thyroxine is bound to the extra protein, there is an equilibrium and the patient remains clinically euthyroid, but the protein-bound iodine and the thyroxine are elevated. 

Testing A Protein Product For Functional Properties. Chemical analyses for general composition are essentialto adequately do future functional testing. The percentage of protein and other components will invariably supply clues as to the expected functional properties. Physical analyses for size of... 

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