Urea

About: Urea is a research topic. Over the lifetime, 21394 publications have been published within this topic receiving 382444 citations. The topic is also known as: carbamide & carbonic acid diamide.

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

Abstract: Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function.

Counteracting effects of urea and betaine in mammalian cells in culture.

Abstract: Urea and methylamines, such as betaine, are among the major organic osmotic effectors accumulated by organisms under hyperosmotic (high NaCl) stress; the mammalian renal medulla also accumulates such compounds in antidiuresis. Studies on isolated proteins show that urea generally destabilizes protein structure, whereas methylamines are generally stabilizers capable of offsetting the effects of urea. The counteracting-osmolytes hypothesis predicts that cells exposed to high urea concentrations require methylamines for optimal function. In this study, urea, betaine, and other solutes (NaCl, glycerol, sorbitol) were added to growth medium of cultured mammalian cells under conditions in which most solutes entered the cells. For two renal [Madin-Darby canine kidney (MDCK) and PAP-HT25] and one nonrenal (Chinese hamster ovary) cell line, urea (greater than 100 mM) or betaine (greater than 50-100 mM) alone inhibited cell growth and survival, measured as colony-forming efficiency. However, the addition of betaine (up to 120 mM) to media with urea (50-300 mM) greatly increased colony-forming efficiency above that with urea alone. A similar, although less marked effect, was seen on colony sizes with MDCK cells. These results support the counteracting-osmolytes hypothesis.

Synthesis of microbial protein in the rumen. III.The effect of dietary protein

Abstract: Protein production in the rumen of sheep fed on a virtually protein-free diet supplemented with urea and higher volatile fatty acids (VFA) and yielding 600 g digestible organic matter per day amounted to 90 g/day. When gelatin was substituted for the higher VFA and 50% of the urea nitrogen, microbial protein production remained at a similar level (91 g/day); with casein, production increased to 101 g/day, and with zein to 104 g/day. Nitrogen balances increased from 4.1 g/day in sheep fed on the casein diet to 5.5 g/day in those fed on the zein diet (P < 0.05). These values were both significantly higher than those for the urea/VFA or gelatin-containing diets, reflecting the different levels of microbial protein production on the respective diets. In addition, 44 g un-degraded zein left the rumen daily, accounting for the increase in nitrogen balance on this diet above that on the casein diet. Negligible amounts of nitrogen were recycled on the urea/VFA, gelatin, and casein treatments, but at least 7.5 g recycled nitrogen was utilized in the rumen daily on the zein diet. This is equivalent to 47 g protein, sufficient in itself to satisfy the maintenance requirement of the sheep for protein. The yields of protein from ruminal fermentation on the three protein treatments suggest that the maximum possible yield may exceed 20 g/100 g organic matter digested in the rumen.

A Clean, More Efficient Method for In-Solution Digestion of Protein Mixtures without Detergent or Urea

Abstract: Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.