The Scientific Basis of Urinary Stone Formation
Anthony R. Mundy, John M. Fitzpatrick, David E. Neal, Nicholas J. R. George in The Scientific Basis of Urology, 2010
Urinary tract infections involving urea-splitting organisms often lead to the formation of stones that consist of calcium phosphate often mixed with magnesium ammonium phosphate (92). Chemically, the process involves the breakdown of urea by the enzyme urease to form ammonium, bicarbonate and hydroxyl ions (Fig. 15) with an accompanying marked alkalinization of urine (pH > 7.2). Under these conditions, the supersaturation of urine with respect to both calcium phosphate and magnesium ammonium phosphate reaches values that cause spontaneous precipitation of these salts and massive crystalluria (Fig. 16) (92,93). In addition, two of the known inhibitors of calcium phosphate crystal growth, pyrophosphate and citrate, are often low in the urines of patients with urinary tract infections, and this may allow the formation of larger than normal crystals and aggregates of calcium phosphate, thereby increasing the risk of stone formation (8). A list of the organisms causing urinary tract infection and their ability to produce urease is contained in Table 4. The risk factor model of infected stone formation is shown in Figure 17.
Mechanisms of Fibril Formation and Cellular Response
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin in XIth International Symposium on Amyloidosis, 2007
The urinary light chains from patients were purified (1) using dialysis, Affi-Gel blue treatment and gel filtration chromatography. SDS-PAGE and Western blot analyses were performed to determine which fractions contained the light chain. Aliquots from samples were treated with Asp-N, Glu-C, Lys-C, or trypsin in ammonium bicarbonate buffer. The enzymatic digests were analyzed using a Waters CapLC capillary HPLC coupled with an Applied Biosystems QSTAR Pulsar i quadrupole/orthogonal acceleration TOF mass spectrometer. A Magic Michrom C18 column (0.320 x 150 mm) was used for the separation at a flow rate of 1 uL/min (500 nL/min into the MS). The gradient was 5-50% B in 50 min followed by 50-70% B in 15 min (buffers A: 0.1% formic acid in water, buffer B: 85% acetonitrile 10% isopropanol and 5% water with 0.1% formic acid). The mass spectrometer was run in the IDA (information-dependent acquisition) mode with rolling collision energies, enabling the automated acquisition of MS/MS data from selected precursor peptide ions.
Urologic procedures
J. Richard Smith, Giuseppe Del Priore, Robert L. Coleman, John M. Monaghan in An Atlas of Gynecologic Oncology, 2018
Beyond the possible metabolic acidosis, urinary diversion can be associated with a number of other metabolically related disorders including vitamin B12 deficiency and osteomalacia. The most common segment utilized for diversion is the terminal ileum. Absorption of vitamin B12 occurs primarily at this point. The rates of vitamin B12 are unknown among patients with urinary diversion, although some have reported they can be as high as 30% (Pfitzenmaier et al. 2003). Usually development of the deficiency requires 3 to 5 years after surgery for the body’s stores to have become depleted. However, serious neurological sequelae can occur as a result. Further neurological sequelae also occur as a result of magnesium deficiency, drug intoxication, and abnormalities of ammonium/bicarbonate metabolism in patients with urinary diversion. A clinician needs to keep these in mind in the long-term follow-up of their patients and be vigilant for signs of any of these metabolic derangements.
Evaluation of molecular brain changes associated with environmental stress in rodent models compared to human major depressive disorder: A proteomic systems approach
Published in The World Journal of Biological Psychiatry, 2018
David Alan Cox, Michael Gerd Gottschalk, Viktoria Stelzhammer, Hendrik Wesseling, Jason David Cooper, Sabine Bahn
MDD tissue storage, preparation and proteomic abundance comparisons were performed as previously defined (Gottschalk et al. 2014). Approximately 12–16 mg of mouse tissue per sample were used for the SD model and 22–28 mg of rat tissue per sample were used for the CMS and PNS models. A previously published protein tissue isolation protocol was applied to all rodent model tissue samples (Ernst et al. 2012). Samples were added to a fractionation buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% ASB14 and 70 mM DTT at a 5:1 (v/w) ratio (Martins-de-Souza et al. 2007). Sonification and vortexing (at 4 °C for 30 min) of the samples was carried out before centrifuging at 17,000 × g at 4 °C. A Bradford assay (Bio-Rad) was used to determine protein concentrations of the supernatants in triplicate, using acetone to precipitate proteins (approximately 100 μg) from each sample. One hundred microlitres of ammonium bicarbonate (50 mM) were used to dissolve the precipitates, before protein concentrations were determined. Reduction of protein sulfhydryl groups was carried out using 40 μg of proteins and 5 mM DTT at 60 °C for 30 min. Alkylation was carried out using 10 mM iodoacetamide and incubating in the dark at 37 °C for 30 min. Protein digestion was performed using porcine tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin at a 1:50 (w/v) ratio for 17 h at 37 °C. Reactions were stopped via the addition of 8.8 M HCl at a 1:60 (w/w) ratio.
Porous discoidal polymeric particles for effective drug delivery minimizing phagocytosis
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2021
Susmita Aryal, Sanghyo Park, Chaewon Park, Moon Jung Choi, Jaehong Key
About 70 mg PLGA was dissolved in 200 µl of acetone and vortexed. Thirty milligram of curcumin was dissolved in 50 µl of DMSO, and 4% of ammonium bicarbonate was dissolved in DI water. All three solutions were mixed together, and the mixture was sonicated and loaded on the PVA film. The film was dried at 37 °C for 5 min and dissolved in water. The solution was dissolved for 2 h and filtered and centrifuged. The particles were collected. For non-porous DPPs, ammonium bicarbonate was not used, and the method for fabrication was similar. For flow cytometry, FITC conjugated PLGA was used, as mentioned earlier [21]. Briefly, 100 mg of PLGA was dissolved in 5 ml DMSO and activated using DCC and NHS. The reaction was carried out for 4 h. FITC was dissolved in activated PLGA, and the reaction was carried out for another 24 h. The sample was dialyzed against DI water to remove unreacted FITC and was lyophilized. The conjugation was confirmed by fluorescence microscopy.
Development of an enzymatic method for the evaluation of protein deposition on contact lenses
Published in Biofouling, 2022
Lactoferrin from human milk, secretory immunoglobulin A (sIgA) from human colostrum, recombinant human lysozyme and bovine serum albumin (albumin), tetramethylrhodamine isothiocyanate labelled albumin (Alb-TRITC, with an extent of labelling of ≥0.5 mol TRITC mol−1 albumin), fluorescein 5(6)-isothiocyanate (FITC) (purity = 90%), Lucifer Yellow VS dilithium salt (LY) (purity of ∼85%) were purchased from Sigma-Aldrich (St Louis, MO, USA). The lenses used in this study included four types of silicone hydrogel contact lens materials (Lotrafilcon A, Senofilcon A, Balafilcon A and Comfilcon A) and one conventional poly HEMA-based hydrogel contact lens material (Etafilcon A). The properties of each material are described in Table 1, supplementary material. Sequencing grade modified trypsin was purchased from Promega (San Luis Obispo, CA). L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) trypsin immobilized on beaded agarose and C18 (200 μl) Stage Tips were purchased from ThermoFisher Scientific (NSW, Australia). Urea was obtained from VWR International Ltd (Poole, UK). Ammonium bicarbonate, heptafluorobanzile and formic acid were purchased from Merck (Darmstadt, Germany). Five milliliter polypropylene, sterile flat bottom tubes with screw cap were purchased from Sarstedt (NSW, Australia).
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