Receptors for Neuropeptides: Ligand Binding Studies
Edwin E. Daniel in Neuropeptide Function in the Gastrointestinal Tract, 2019
Markers for membranes from smooth muscle (5′-nucleotidase, EC 3.1.3.5), mitochondria (cytochrome C oxidase, EC 1.9.3.1), and endoplasmic reticulum (NADPH cytochrome C reductase, EC 1.6.99.3) are relatively good, so identification and reasonable separation of these membranes is possible. However, since the starting homogenate may contain membranes from several cell types, it is essential to assess and separate these membranes. Until recently, not much effort has been devoted to addressing this problem. Since many smooth muscles along the GI tract are highly innervated, one major hinderance to progress in this aspect has been the meager knowledge of contamination by neuronal membranes due to the lack of specific markers for neuronal membranes.
Microbiology of the Autopsy
Julian L Burton, Guy Rutty in The Hospital Autopsy, 2010
Quantification of bacterial growth may be a way of differentiating between infecting and colonising flora. Knapp and Kent (1968) analysed cultures obtained from homogenates of peripheral lung tissue. They found that large numbers of organisms (>106 colony-forming units per cubic centimetre of tissue) correlated with histological evidence of infection, whereas contaminating organisms were present in much lower numbers (<104/cm3). The problem that arises when tissue is homogenised for quantitative counts is that organisms contaminating the surface of the tissue become incorporated into the homogenate, giving false results. This is overcome if quantification is performed using impression cultures. In this instance, the cut surface of the tissue is pressed onto agar culture plates, allowing the quantity, types and distribution of bacteria to be determined. Surface contaminants are localised at the edges of the print, and friable tissues (e.g. lung) can be rapidly frozen to allow easier handling without interfering with the culture results. Using this technique, Zanen-Lim and Zanen (1980) found that large numbers of organisms, usually in pure culture, were associated with pulmonary infection in life. The techniques available in each microbiology laboratory will vary and will need to be discussed with the microbiologist.
Water Permeability of Amphibian Urinary Bladder
Gheorghe Benga in Water Transport in Biological Membranes, 1989
Some attempts were made at isolating membrane fractions, especially the apical membrane. First in 1966, Hays and Barland223 described a preparation of cellular envelope. Scraped cells were homogenized in a low-ionic strength medium, then homogenate was centrifuged to separate the cell envelope from nuclear and other cellular debris. Essentially, apical sheets were observed. This technique has found an interesting development — the possibility of isolating the plasma membrane with aggregates in place, when preparations were first stimulated by cAMP or vasopressin.245 Rodriguez and Edelman227 have used a separation on sucrose gradient to obtain different types of membrane. A labeling of the apical proteins by 125I iodination was previously performed in order to serve as a marker of the apical membrane. An endocytosis of 125I and lactoperoxidase was not completely excluded in their conditions, and the formation of a network of fibrous material was constricting.
The possible protective role of N-acetyl cysteine on duodenal mucosa of high fat diet and orlistat treated adult male albino rats and the active role of tumor necrosis factor α (TNFα) and Interleukin 6 (IL6) (histological and biochemical study)
Published in Ultrastructural Pathology, 2022
Laila Moustafa El-Shal, Alyaa A. Abd El-Star, Abeer M. Azmy, Heba M. Elnegris
Pieces from the first one-third of duodenum were processed according to Bandyopadhyay et al. 23 They were located on blotting paper and then positioned in a 1.5 mL microtube in a cold in 0.9% saline phosphate-buffered saline (diluted ratio 1:5 according to their respective weight; pH 7.2). Homogenization was achieved with a mechanic homogenizator, Ultra-Turrax (IKA T10 basic, Staufen, Germany); then, the homogenate was centrifuged at 3500 rpm for 15 min. After centrifugation of the homogenate, the supernatant was collected and diluted. The oxidative stress status in duodenal homogenate was assessed via determination of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) using commercially available colorimetric kits (Biodiagnostic, Giza, Egypt) according to manufacturer’s instructions.24
Recent advances in proteolytic stability for peptide, protein, and antibody drug discovery
Published in Expert Opinion on Drug Discovery, 2021
Xianyin Lai, Jason Tang, Mohamed E.H. ElSayed
In the last step, more complex peptidase mixtures highlighted in purple in Figure 2 are selected for testing the proteolytic stability. For peptide, protein, and antibody drugs that are absorbed in the stomach, stomach fluid should be selected since it has more complex enzymes than pepsin alone. For the drugs to be absorbed in the small intestine, small intestine fluid is a good choice since it is more complex than trypsin, chymotrypsin, and elastase. For both oral and subcutaneous drugs, peptidases in the blood and tissues are another set of challenges. Because there are too many carboxypeptidases and aminopeptidases to select for individual testing, blood, plasma, or serum is a great source to examine all potential cleavages from a broader group of peptidases. When the drugs are internalized into cells, they encounter peptidases in the endolysosome rather than the cytoplasm. Therefore, using subcellular fractions of the endolysosome is the primary choice. Whole crude tissue homogenates from the stomach, small intestine, liver, kidney, heart, and others is another resource to use. However, a risk of over degradation exists when the whole homogenate is used since all peptidases are released from all components in a cell. After the three steps, the proteolytic stability issue should be addressed well. If the drugs still have a proteolytic stability issue, an in vivo approach should be applied to identify metabolites from animal bodily fluids or tissues.
MiADMSA abrogates sodium tungstate-induced oxidative stress in rats
Published in Drug and Chemical Toxicology, 2022
Sherry Sachdeva, Ankita Sharma, S. J. S. Flora
MiADMSA was selected at 50 mg/kg on the basis of previous published literature which proved it to be the minimum effective dose with least side effects (Flora et al. 2012). MiADMSA was dissolved in normal saline and administered to rats, also, an equal volume of saline was given to normal control and tungstate control animals. The body weight of rats was recorded every alternate day to monitor any possible change in body weight during dosing. The animals wre sacrificed 48 h after the last dosing to allow sufficient time for complete absorption of the dose material. After the completion of the treatment schedule. Euthanasia was administered using Isoflurane inhalant followed by gentle cervical dislocation. Rats were then placed in a closed glass chamber where high levels of anesthetic gas (3%) were introduced. 1 ml of blood samples were collected from unconsciousness animals in ethylenediamine tetraacetic acid (EDTA) anticoagulant tubes from the retro-orbital vein by a glass capillary tube puncture. Liver, kidney and spleen were collected, washed with cold normal saline, blotted and all the extraneous materials were removed. The homogenate was used for the estimation of various biochemical parameters. Tissues were stored at −20 °C till further analysis.
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