Tissue homogenates, comparison

Similarly, the 4-methoxy-2-naphthylamides of Leu, Ala, Arg, and Glu (6.1, R=side chain of amino acid, R =MeO) were used to assess the type and activity of aminopeptidase in homogenates of conjunctival, nasal, buccal, duodenal, ileal, rectal, and vaginal tissues from rabbits. This systematic comparison afforded a better understanding of the role of the aminopeptidase barrier in peptide absorption from oral vs. non-oral routes [18]. In a comparable manner, the y-glutamyltranspeptidase and dipeptidase activities were investigated in mammary tissue with the 4-nitroanilides of Leu, Met, Lys, Glu, and Asp (6.2, R=side chain of amino acid) [19]. 

Figure 25 Treatment of a tumor of the base of skull with a carbon-ion beam. Comparison of the (physical) absorbed dose and of the biological dose (i.e., the dose weighted for the RBE variation in the carbon beam). The treatment is carefully planned in order to obtain a homogeneous biological dose to the PTV. For the same biological dose to the PTV, the normal tissues in the initial plateau receive a lower dose. (From Ref. 45.)...

A comparison of ELISA, HPLC, and HPLC-ELISA methods was published for the determination of SAL in chicken liver tissue. Samples were homogenized with MeOH and extracted with methylenechloride. Some samples were analyzed by HPLC using the isocratic solvent system and postcolumn derivatization (vanillin in MeOH containing sulphuric acid), with the eluent monitoring at 520 nm. The HPLC-ELISA system was used to characterize nonspecific effects analyzing column fractions by ELISA, since this detection is over 1000 times more sensitive than the spectrophotometric one. This alleviated the need to derivatize the drug prior to the detection (104). 

A novel cleanup procedure was used for TMP residues in tissue and milk samples. Tis-sue/milk samples were homogenized/dissolved with phosphate buffer, and the supernatant was purified on a Cl8 SPE cartridge previously conditioned by MeOH, water, and phosphate buffer containing pentanesulphonic acid (PSA). The TMP was eluted with MeOH phosphate buffer and injected directly into the ion-pair chromatographic system followed by dual-wavelength UV detection. The comparison of both signals ratio-enhanced the identification of the TMP peak besides the monitored UV spectra. Recoveries ranging from 73% to 98% were presented, and an LOD below the MRL set for TMP residues was achieved (176,177). 

Figure 2.8. Comparison of average (a) desipramine and (b) fluoxetine concentrations in rat brain tissue extracted by enzymatic digestion or homogenization. Error bars represent standard deviation mean value and are symmetrical about the mean. (Reprinted with permission from Yu et ai 2004.)...

Fig. 3. Comparison of different enzyme-linked immuno sorbent assay (ELISA) methods adapted for immuno-polymerase chain reaction (IPCR). Dependent on the purification grade of the sample to be analyzed and the availability of specific and functionalized antibodies, several typical ELISA protocols were adapted to IPCR. In the direct approach (A), the pure antigen is immobilized to the microplate surface and subsequently detected by a labeled specific antibody. If no labeled antibody is available (e.g., because of unpurified ascites fluid containing the antibody or loss in activity following labeling), a standardized labeled secondary species-specific antibody is used for detection of the primary antigen-specific antibody (B). For the detection of the antigen from matrices such as serum, plasma, tissue homogenate, and so on, a capture antibody immobilized to the microplate surface was used either in a direct (C) or indirect (D) sandwich approach, with the latter one additionally including a secondary species-specific detection antibody. For different methods of coupling antibody and DNA, abbreviated by in this figure, compare Fig. 2. Note that protein A chimeras (Fig. 2A) are not compatible with capture antibodies (Fig. 3C, D).

A microsomal FAS was implicated in the biosynthesis of methyl-branched fatty acids and methyl-branched hydrocarbon precursors of the German cockroach contact sex pheromone (Juarez et al., 1992 Gu et al., 1993). A microsomal FAS present in the epidermal tissues of the housefly is responsible for methyl-branched fatty acid production (Blomquist et al., 1994). The housefly microsomal and soluble FASs were purified to homogeneity (Gu et al., 1997) and the microsomal FAS was shown to preferentially use methylmalonyl-CoA in comparison to the soluble FAS. GC-MS analyses showed that the methyl-branching positions of the methyl-branched fatty acids of the housefly were in positions consistent with their role as precursors of the methyl-branched hydrocarbons. 

Fig. 4. BEO enhances p-Akt levels in the brain cortical tissue from rats subjected to permanent focal cerebral ischemia. Western blot analysis ofphospho-Akt (Ser473) (p-Akt) and total Akt performed using brain cortical homogenates from rats sacrificed 24 h after MCAo shows a trend toward a decrease of p-Akt and total Akt in the ipsilateral (I), ischemic, cortex as compared to contralateral (C), nonischemic, side intraperitoneal administration of BEO (0.5 ml/kg) 1 h before MCAo enhances p-Akt immunoreactivity in the ischemic cortex without increasing total Akt expression. Histograms show the results of the densitometric analysis of the bands corresponding to p-Akt, total Akt, and i3-actin. p-Akt and Akt levels were normalized to the values yielded by /3-actin and Akt phosphorylation was expressed by the ratio of p-Akt/total Akt data are reported as mean S.E.M. (n = 3 per group). Denote P < 0.01 versus contralateral side and denote P < 0.05 and P < 0.01 versus MCAo, ipsilateral side (ANOVA followed by Tukey-Kramer test for multiple comparisons).

Figure 5. Comparison of the potency of isoproterenol in eliciting physiological and biochemical responses from the rat IL. Substantially lower concentrations of isoproterenol stimulate the release of IR-aMSH (aMSH) than are required to enhance cAMP accumulation by intact IL cells (cAMP), stimulate adenylate cyclase activity in cell-free homogenates of IL tissue (cyclase), or occupy the specific binding sites defined with IHYP (binding) (19).

Figure 10. Comparison of the potency of apomorphine as a dopaminergic agonist upon intact 1L cells or cell-free homogenate of 1L tissue.

If metabolism in animal models is extensive or the generated metabolite(s) is shown to have toxic effects, in vitro metabolism studies using isolated P-450 isozymes, tissue homogenates containing the microsomal fraction, hepatocytes, and liver slices are commonly conducted to determine if the extent of metabolism and the metabolite profile is similar for animals and humans. The results from these in vitro metabolism comparison studies can be used to select the animal models for definitive development studies that have similar metabolism profiles to humans. 

Comparison of tissue susceptibility to oxidative protein damage, manifested as protein carbonylation in response to acute oxidative stress induced by exposure to X-rays, has shown that brain homogenates from 22-month-old rats were more susceptible to oxidative stress than those from 3-month-old rats. The brain was more susceptible to oxidative damage than the heart. A comparison of brain and heart homogenates susceptibility to acute oxidative stress in five different species (mouse, rat, rabbit, pig, and pigeon) indicated that maximum life-span potential of the species was related directly to their ability to control oxidative damage (Al). 

Homogenization is needed because whole plants are known to contain different amounts of compounds (drugs) in different tissues within the plant. The amount reported will depend upon the tissue being analysed. Since it is not always easy to determine from the drug sample what the tissue is, or it is not practical to dissect out the different tissues, the easiest way to address this problem is to homogenize the samples. This ensures that any comparison between samples is meaningful. 

The ability to focus molecular expression analysis on only a limited number of cell types depends on cell separation methods that minimize the opportunity for other cell types to contribute to gene expression in situ. Even the most carefully gathered biological samples contain many cell types, especially if the sample is from inflamed or necrotic tissue. More homogeneous samples are provided by laser capture microdissection (LCM), a method that isolates individual cells or sections of tissue from a fixed sample [144-148]. The use of LCM minimizes contributions by nontarget cell populations in comparisons of diseased and normal tissues, but also introduces handling and preparation steps that can affect detection accuracy. 

An in vitro study of 2,4-DNP metabolism using rat liver homogenates identified 2-amino-4-nitrophenol and 4-amino-2-nitrophenol as metabolic products, with the 4-amino-2-nitrophenol present in greater abundance (Parker 1952). An additional ether-insoluble metabolite was tentatively identified as 2,4-diaminophenol. When 2-amino-4-nitrophenol or 4-amino-2-nitrophenol was incubated with rat liver homogenates, the 2-amino-4-nitrophenol was slowly metabolized to the ether-insoluble compound, while 4-amino-2-nitrophenol rapidly disappeared but with very little accumulation of the ether- insoluble compound. The reduction of the aminonitrophenols to the ether-insoluble compound appeared to be catalyzed by the same nitroreductase that reduces 2,4-DNP to the aminonitrophenols. A comparison of the activity of homogenates of various tissues in the rat and rabbit revealed that liver homogenate metabolized... 

To analyze free amino acids in plasma or tissue homogenates, it is necessary to remove proteins and peptides present in solution. The most widely used deproteinization method is precipitation with 5-sulfosalicylic acid followed by centrifugation for separating the precipitate. In comparison to other precipitation agents such as trichloroacetic acid, perchloric acid, picrinic acid, or acetonitrile, the best results with respect to completeness of precipitation are obtained with 5-sulfosalicylic acid [39]. Other deproteinization methods comprise ultrafiltration and ultracentrifugation [40], which have only recently been considered as sample preparation methods for amino acid analysis. 

This raises the question of what in vitro system to use and which system wiii provide reai data on the reiative rates of metaboiism in different species. Shouid tissue siices, isoiated ceiis, homogenates, or subceiiuiar fractions be used One approach to this difficuity is to use the rate of metaboUsm per gram of tissue (or mg protein) derived from the perfused organ studies in rat as the yardstick by which to vaiidate the rate of metaboiism in in vitro systems. Often the rate of metaboiism in siices, isoiated ceiis, homogenates, or subceiiuiar fractions with an arbitrary incubation medium is weii beiow the expected iev-eis. However, by judicious manipuiation of the incubation medium, it shouid be possibie to improve the nature and rates of metaboiism in vitro so that the rates approach the whoie perfused organ. If the improved rates are 80% or more than that in the whole organ, then cross-species comparisons that use the same conditions should provide data that can be used for go/no-go decisions. 

Fig. 3 Diagram of Real Time QuIC RT-QuIC and comparison of end-point dilution titrations of scrapie brain homogenate by RT-QuIC with animal bioassay. (Top panel) RT-QuIC analysis of normal and scrapie brain homogenate (BH) dilutions using hamster (90-231) rPrPc as a substrate. (Bottom panel) Comparison of hamster brain homogenate end point dilution titrations by RT-QuIC and animal bioassay. The Spearman-Karber estimate of the SD50 (i.e., seeding dose giving sufficient Thioflavin T fluorescence in half of the replicate wells) per 2 pL of neat brain tissue is indicated. Graphs adapted from [55]...

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