Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea carboxylase, as well as other proteins including urease, across kingdoms.

Cocrystallization is commonly used for its ability to improve the physical properties of APIs, such as solubility, bioavailability, compressibility, etc. The pharmaceutical industry is particularly interested in those cocrystals comprising a GRAS former in connection with the target API. In this work, we focus on the potential of urea as a cocrystal former, identifying three novel pharmaceutical cocrystal systems with catechin, 3-hydroxyl-2-naphthoic and ellagic acid. Interestingly, the stability of catechin under high humidity or high temperature environment is improved upon cocrystallization with urea. Moreover, the solubility of ellagic acid is improved about 17 times. This work displays the latent possibility of urea in improving the physical property of drug molecules using a cocrystallization approach.

It remains unclear whether extreme levels of blood urea nitrogen (BUN) and BUN to creatinine ratio (BUN/Cr) can increase future risk of stroke. We conducted this study to investigate the associations of BUN and BUN/Cr with incident stroke and its subtypes.

Urea at post-dialysis levels induces increased ROS in a number of cell types. The aim of this study was to determine whether urea-induced production of ROS remains elevated after urea is no longer present, and, if it does, to characterize its origin and effects. Human arterial endothelial cells were incubated with 20 mM urea for two days, and then cells were incubated for an additional two days in medium alone. Maximal ROS levels induced by initial urea continued at the same level despite urea being absent. These effects were prevented by either MnSOD expression or by Nox1/4 inhibition with GKT13781. Sustained urea-induced ROS caused a persistent reduction in mtDNA copy number and electron transport chain transcripts, a reduction in transcription of mitochondrial fusion proteins, an increase in mitochondrial fission proteins, and persistent expression of endothelial inflammatory markers. The SOD-catalase mimetic MnTBAP reversed each of these. These results suggest that persistent increases in ROS after cells are no long exposed to urea may play a major role in continued kidney damage and functional decline despite reduction of urea levels after dialysis.

In plants, urea derives either from root uptake or protein degradation. Although large quantities of urea are released during senescence, urea is mainly seen as a short-lived nitrogen (N) catabolite serving urease-mediated hydrolysis to ammonium. Here, we investigated the roles of DUR3 and of urea in N remobilization. During natural leaf senescence urea concentrations and DUR3 transcript levels showed a parallel increase with senescence markers like ORE1 in a plant age- and leaf age-dependent manner. Deletion of DUR3 decreased urea accumulation in leaves, whereas the fraction of urea lost to the leaf apoplast was enhanced. Under natural and N deficiency-induced senescence DUR3 promoter activity was highest in the vasculature, but was also found in surrounding bundle sheath and mesophyll cells. An analysis of petiole exudates from wild-type leaves revealed that N from urea accounted for >13% of amino acid N. Urea export from senescent leaves further increased in ureG-2 deletion mutants lacking urease activity. In the dur3 ureG double insertion line the absence of DUR3 reduced urea export from leaf petioles. These results indicate that urea can serve as an early metabolic marker for leaf senescence, and that DUR3-mediated urea retrieval contributes to the retranslocation of N from urea during leaf senescence.

Urea is the degradation product of a wide range of nitrogen containing bio-molecules. Urea amidolyase (UA) catalyzes the conversion of urea to ammonium, the essential first step in utilizing urea as a nitrogen source. It is widely distributed in fungi, bacteria and other microorganisms, and plays an important role in nitrogen recycling in the biosphere. UA is composed of urea carboxylase (UC) and allophanate hydrolase (AH) domains, which catalyze sequential reactions. In some organisms UC and AH are encoded by separated genes. We present here structure of the Kluyveromyces lactis UA (KlUA). The structure revealed that KlUA forms a compact homo-dimer with a molecular weight of 400 kDa. Structure inspired biochemical experiments revealed the mechanism of its reaction intermediate translocation, and that the KlUA holo-enzyme formation is essential for its optimal activity. Interestingly, previous studies and ours suggest that UC and AH encoded by separated genes probably do not form a KlUA-like complex, consequently they might not catalyze the urea to ammonium conversion as efficiently.

Hemiascomycetes, including the pathogen Candida albicans, acquire nitrogen from urea using the urea amidolyase Dur1,2, whereas all other higher fungi use primarily the nickel-containing urease. Urea metabolism via Dur1,2 is important for resistance to innate host immunity in C. albicans infections. To further characterize urea metabolism in C. albicans we examined the function of seven putative urea transporters. Gene disruption established that Dur3, encoded by orf 19.781, is the predominant transporter. [(14)C]Urea uptake was energy-dependent and decreased approximately sevenfold in a dur3Δ mutant. DUR1,2 and DUR3 expression was strongly induced by urea, whereas the other putative transporter genes were induced less than twofold. Immediate induction of DUR3 by urea was independent of its metabolism via Dur1,2, but further slow induction of DUR3 required the Dur1,2 pathway. We investigated the role of the GATA transcription factors Gat1 and Gln3 in DUR1,2 and DUR3 expression. Urea induction of DUR1,2 was reduced in a gat1Δ mutant, strongly reduced in a gln3Δ mutant, and abolished in a gat1Δ gln3Δ double mutant. In contrast, DUR3 induction by urea was preserved in both single mutants but reduced in the double mutant, suggesting that additional signalling mechanisms regulate DUR3 expression. These results establish Dur3 as the major urea transporter in C. albicans and provide additional insights into the control of urea utilization by this pathogen.

To lay a theoretical basis for the preparation of peanut protein-based adhesives and promote the sustainable development of the adhesive industry, properties of peanut protein isolate (PPI), arachin and conarachin-based adhesives modified by urea and epichlorohydrin (ECH) were investigated under different urea concentrations. When the urea concentration was 2 mol l-1, the wet shear strength of the PPI-based adhesive was 1.24 MPa with the best water resistance. With the increase of urea concentration from 0 to 4 mol l-1, the apparent viscosity of the PPI-based adhesive increased from 3.87 to 136.80 Pa s and the solid content increased from 18.11% to 31.11%. Compared with conarachin-based adhesive, the properties of arachin-based adhesive were improved more obviously during the combined modification. Scanning electron microscopy images illustrated that when the urea concentration was 2 mol l-1, the surface of the PPI-based adhesive was more compact and smoother, which was beneficial to the improvement of water resistance and related to the structure changes of arachin and conarachin components. Fourier-transform infrared spectroscopy results indicated that different urea concentrations caused the change of ester and ether bonds in the PPI-based adhesive, which was mainly related to arachin component. Thermogravimetry results suggested that when the urea concentration was 2 mol l-1, the decomposition temperature of protein skeleton in the PPI-based adhesive reached a maximum of 314°C exhibiting the highest thermal stability. The improvement of the thermal stability of conarachin was greater than that of arachin during the combined modification.

The milk urea concentration (MUC) serves as indicator of urinary nitrogen emissions, but at comparable crude protein (CP) intake, cows with high (HMU) and low (LMU) MUC excrete equal urea amounts. We hypothesized that urea and uric acid transporters and sizes of the kidney, mammary gland, and rumen account for these phenotypes. Eighteen HMU and 18 LMU Holstein dairy cows fed a low (LP) and normal (NP) CP diet were studied. Milk, plasma and urinary urea concentrations were greater with NP feeding, while plasma and urinary urea concentrations were comparable between phenotypes. Milk and plasma uric acid concentrations were higher with LP feeding but not affected by phenotype. The milk-urine uric acid ratio was greater in HMU cows. The mRNA expressions of the ruminal urea transporter SLC14A1 and AQP10, the mammary gland and rumen AQP3, and the mammary gland uric acid transporter ABCG2 were not affected by group or diet. Renal AQP10, but not AQP3, AQP7, and SLC14A2 expressions, and the kidney weights were lower in HMU cows. These data indicate that renal size and AQP10 limit the urea transfer from blood to urine, and that MUC determines if uric acid is more released with milk or urine.

Urea sensors based on electrodes in direct contact with the medium have limited long-term stability when exposed to complex media. Here, we present a urea biosensor based on urease immobilized in an alginate polymer, buffered at pH 6, and placed in front of a newly developed fast and sensitive CO2 microsensor, where the electrodes are shielded by a gas-permeable membrane. The CO2 produced by the urease in the presence of urea diffuses into the microsensor and is reduced at a Ag cathode. Oxygen interference is prevented by a Cr2+ trap. The 95% response time to changes in urea concentration was 120 s with a linear calibration curve in the range 0-1000 μM and a detection limit of 1 μM. The Ni2+ cofactor to improve sensor performance was continuously supplied from a reservoir behind the sensor tip. The stability of the urea sensor was optimized by the addition of bovine serum albumin as a stabilizer to the urease/alginate mixture that was cross-linked with glutaraldehyde and Ca2+ ions. This immobilization strategy resulted in about 70% of the initial urea sensor sensitivity after two weeks of continuous operation. The sensor was successfully tested in blood serum.

Urease is an enzyme containing a dinuclear nickel active center responsible for the hydrolysis of urea into carbon dioxide and ammonia. Interestingly, inorganic models of urease are unable to mimic its mechanism despite their similarities to the enzyme active site. The reason behind the discrepancy in urea decomposition mechanisms between inorganic models and urease is still unknown. To evaluate this factor, we synthesized two bis-nickel complexes, [Ni2L(OAc)] (1) and [Ni2L(Cl)(Et3N)2] (2), based on the Trost bis-Pro-Phenol ligand (L) and encompassing different ligand labilities with coordination geometries similar to the active site of jack bean urease. Both mimetic complexes produced ammonia from urea, (1) and (2), were ten- and four-fold slower than urease, respectively. The presence and importance of several reaction intermediates were evaluated both experimentally and theoretically, indicating the aquo intermediate as a key intermediate, coordinating urea in an outer-sphere manner. Both complexes produced isocyanate, revealing an activated water molecule acting as a base. In addition, the reaction with different substrates indicated the biomimetic complexes were able to hydrolyze isocyanate. Thus, our results indicate that the formation of an outer-sphere complex in the urease analogues might be the reason urease performs a different mechanism.

We have reported catch-up growth with hemodialysis (HD) of approximately 15 hours/week. Without an equilibrated post-treatment blood urea nitrogen, the variable-volume single-pool (VVSP) model will not account for urea rebound, inflating the estimated HD dose (K(d)t/V). A two-pool model (FVDP) predicts rebound, but requires fixed compartment volumes for the equations to be solvable in closed form, also inflating K(d)t/V.

High kidney and salivary gland uptake is a common feature of prostate-specific membrane antigen (PSMA)-targeted radioligands derived from the lysine-urea-glutamic acid (Lys-urea-Glu) pharmacophore. In this study we investigated if radioligands derived from lysine-urea-2-aminoadipic acid (Lys-urea-Aad), lysine-urea-S-carboxylmethylcysteine (Lys-urea-Cmc) and lysine-urea-O-carboxylmethylserine (Lys-urea-Cms) pharmacophores with/without an albumin binder could retain good PSMA-targeting capability but with minimized kidney and salivary gland uptake. Methods: HTK03177 and HTK03187 were obtained by replacing Aad in the previously reported Lys-urea-Aad-derived HTK03149 with Cmc and Cms, respectively. HTK03170, HTK04048 and HTK04028 were derived from HTK03149, HTK03177 and HTK03187, respectively, with the conjugation of an albumin-binding moiety, 4-(p-methoxyphenyl)butyric acid. In vitro competition binding assays were conducted using PSMA-expressing LNCaP prostate cancer cells and [18F]DCFPyL as the radioligand. Imaging and biodistribution studies of 68Ga-labeled HTK03177 and HTK03187, and 177Lu-labeled HTK03170, HTK04048 and HTK04028 were performed in LNCaP tumor-bearing mice. Radioligand therapy study of [177Lu]Lu-HTK03170 was carried out in LNCaP tumor-bearing mice and [177Lu]Lu-PSMA-617 was used for comparison. Results: The calculated Ki(PSMA) values of Ga-HTK03177, Ga-HTK03187, Lu-HTK03170, Lu-HTK04048 and Lu-HTK04028 were 5.0±2.4, 10.6±2.0, 1.6±0.4, 1.4±1.0 and 13.9±3.2 nM, respectively. PET Imaging and biodistribution studies at 1 h post-injection showed that both [68Ga]Ga-HTK03177 and [68Ga]Ga-HTK03187 had high uptake in LNCaP tumor xenografts (24.7±6.85 and 21.1±3.62 %ID/g, respectively) but minimal uptake in normal organs/tissues including kidneys (7.76±1.00 and 2.83±0.45 %ID/g, respectively) and salivary glands (0.22±0.02 and 0.16±0.02 %ID/g, respectively). SPECT imaging and biodistribution studies showed that the LNCaP tumor uptake of 177Lu-labeled HTK03170, HTK04048 and HTK04028 peaked at 4-24 h post-injection at ~43-65 %ID/g and was relatively sustained over time. Their peaked average uptake in kidneys (≤ 17.4 %ID/g) and salivary glands (≤ 2.92 %ID/g) was lower and continuously reduced over time. Radioligand therapy study showed that compared with [177Lu]Lu-PSMA-617 (37 MBq), a quarter dose of [177Lu]Lu-HTK03170 (9.3 MBq) led to a better median survival (63 vs 90 days). Conclusions: Our data demonstrate that that Lys-urea-Aad, Lys-urea-Cmc and Lys-urea-Cms are promising pharmacophores for the design of PSMA-targeted radioligands especially for radiotherapeutic applications to minimize toxicity to kidneys and salivary glands.

Urea removal from dialysate is the major obstacle in realization of a miniature dialysis device, based on continuous dialysate regeneration in a closed loop, used for the treatment of patients suffering from end-stage kidney disease. For the development of a polymeric urea sorbent, capable of removing urea from dialysate with high binding capacities and fast reaction kinetics, a systematic kinetic study was performed on the reactivity of urea with a library of low-molecular-weight carbonyl compounds in phosphate-buffered saline (pH 7.4) at 323 K. It was found that dialdehydes do not react with urea under these conditions but need to be activated under acidic conditions and require aldehyde groups in close proximity to each other to allow a reaction with urea. Among the 31 (hydrated) carbonyl compounds tested, triformylmethane, ninhydrin, and phenylglyoxaldehyde were the most reactive ones with urea. This is attributed to the low dehydration energies of these compounds, as calculated by Gibbs free energy differences between the hydrated and dehydrated carbonyl compounds, which are favorable for the reaction with urea. Therefore, future urea sorbents should contain such functional groups at the highest possible density.

In both prokaryotic and eukaryotic genomes, synonymous codons are unevenly used. Such differential usage of optimal or non-optimal codons has been suggested to play a role in the control of translation initiation and elongation, as well as at the level of transcription and mRNA stability. In the case of membrane proteins, codon usage has been proposed to assist in the establishment of a pause necessary for the correct targeting of the nascent chains to the translocon. By using as a model UreA, the Aspergillus nidulans urea transporter, we revealed that a pair of non-optimal codons encoding amino acids situated at the boundary between the N-terminus and the first transmembrane segment are necessary for proper biogenesis of the protein at 37°C. These codons presumably regulate the translation rate in a previously undescribed fashion, possibly contributing to the correct interaction of ureA-translating ribosome-nascent chain complexes with the signal recognition particle and/or other factors, while the polypeptide has not yet emerged from the ribosomal tunnel. Our results suggest that the presence of the pair of non-optimal codons would not be functionally important in all cellular conditions. Whether this mechanism would affect other proteins remains to be determined.

In humans, urea is excreted in sweat, largely through the eccrine sweat gland. The urea concentration in human sweat is elevated when compared to blood urea nitrogen. The sweat urea nitrogen (UN) of patients with end-stage kidney disease (ESRD) is increased when compared with healthy humans. The ability to produce sweat is maintained in the overwhelming majority of ESRD patients. A comprehensive literature review found no reports of sweat UN neither in healthy rodents nor in rodent models of chronic kidney disease (CKD). Therefore, this study measured sweat UN concentrations in healthy and uremic rats. Uninephrectomy followed by renal artery ligation was used to remove 5/6 of renal function. Rats were then fed a high-protein diet to induce uremia. Pilocarpine was used to induce sweating. Sweat droplets were collected under oil. Sweat UN was measured with a urease assay. Serum UN was measured using a fluorescent ortho-pthalaldehyde reaction. Immunohistochemistry (IHC) was accomplished with a horseradish peroxidase and diaminobenzidine technique. Sweat UN in uremic rats was elevated greater than two times compared to healthy pair-fed controls (220 ± 17 and 91 ± 15 mmol/L, respectively). Post hoc analysis showed a significant difference between male and female uremic sweat UN (279 ± 38 and 177 ± 11 mmol/L, respectively.) IHC shows, for the first time, the presence of the urea transporters UT-B and UT-A2 in both healthy and uremic rat cutaneous structures. Future studies will use this model to elucidate how rat sweat UN and other solute excretion is altered by commonly prescribed diuretics.

The acid mine drainage (AMD) in Carnoulès (France) is characterized by the presence of toxic metals such as arsenic. Several bacterial strains belonging to the Thiomonas genus, which were isolated from this AMD, are able to withstand these conditions. Their genomes carry several genomic islands (GEIs), which are known to be potentially advantageous in some particular ecological niches. This study focused on the role of the "urea island" present in the Thiomonas CB2 strain, which carry the genes involved in urea degradation processes. First, genomic comparisons showed that the genome of Thiomonas sp. CB2, which is able to degrade urea, contains a urea genomic island which is incomplete in the genome of other strains showing no urease activity. The urease activity of Thiomonas sp. CB2 enabled this bacterium to maintain a neutral pH in cell cultures in vitro and prevented the occurrence of cell death during the growth of the bacterium in a chemically defined medium. In AMD water supplemented with urea, the degradation of urea promotes iron, aluminum and arsenic precipitation. Our data show that ureC was expressed in situ, which suggests that the ability to degrade urea may be expressed in some Thiomonas strains in AMD, and that this urease activity may contribute to their survival in contaminated environments.

We present the first account of the structure-function relationships of a protein of the subfamily of urea/H(+) membrane transporters of fungi and plants, using Aspergillus nidulans UreA as a study model. Based on the crystal structures of the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT) and of the Nucleobase-Cation-Symport-1 benzylhydantoin transporter from Microbacterium liquefaciens (Mhp1), we constructed a three-dimensional model of UreA which, combined with site-directed and classical random mutagenesis, led to the identification of amino acids important for UreA function. Our approach allowed us to suggest roles for these residues in the binding, recognition and translocation of urea, and in the sorting of UreA to the membrane. Residues W82, Y106, A110, T133, N275, D286, Y388, Y437 and S446, located in transmembrane helixes 2, 3, 7 and 11, were found to be involved in the binding, recognition and/or translocation of urea and the sorting of UreA to the membrane. Y106, A110, T133 and Y437 seem to play a role in substrate selectivity, while S446 is necessary for proper sorting of UreA to the membrane. Other amino acids identified by random classical mutagenesis (G99, R141, A163, G168 and P639) may be important for the basic transporter's structure, its proper folding or its correct traffic to the membrane.

Urea is the most used fertilizer around the world as the main source of nitrogen to soil and plants. However, the administration of nitrogen dosage is critical, as its excess can be harmful to the environment. Therefore, the encapsulation of urea to achieve control on its release rates has been considered in several areas. In this work, encapsulation of urea by biodegradable polymer poly(3-hydroxybutyrate) (PHB) and its nanocomposites, namely PHB/MMT and PHB/OMMT, producing microcapsules by emulsion method is carried out. MMT and OMMT refer to Brazilian clays in a natural state and organophilized, respectively. In addition, the microcapsules are thus prepared to have their physicochemical characteristics investigated, then tested for biodegradation. Increment of microcapsules' crystallinity due to the increased amount of poly(vinylacetate) (PVA), as emulsifier agent in the continuous phase, was confirmed by X-ray diffractometry (XRD) and atomic force microscopy (AFM). The presence of urea within microcapsules was verified by XRD, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The soil biodegradation assessments showed that PHB/OMMT microcapsules present higher degradation rates in sandy soils. The overall results suggest that the composites performed better than neat PHB and are very promising; moreover, PHB/OMMT microcapsules proved to be the best candidate for the controlled-release of urea in soils.

The urea cycle generates arginine that is one of the major precursors for creatine biosynthesis. Here we evaluate levels of creatine and guanidinoacetate (the precursor in the synthesis of creatine) in plasma samples (ns = 207) of patients (np = 73) with different types of urea cycle disorders (ornithine transcarbamylase deficiency (ns = 22; np = 7), citrullinemia type 1 (ns = 60; np = 22), argininosuccinic aciduria (ns = 81; np = 31), arginase deficiency (ns = 44; np = 13)). The concentration of plasma guanidinoacetate positively correlated (p < 0.001, R2 = 0.64) with levels of arginine, but not with glycine in all patients with urea cycle defects, rising to levels above normal in most samples (34 out of 44) of patients with arginase deficiency. In contrast to patients with guanidinoacetate methyltransferase deficiency (a disorder of creatine synthesis characterized by elevated guanidinoacetate concentrations), creatine levels were normal (32 out of 44) or above normal (12 out of 44) in samples from patients with arginase deficiency. Creatine levels correlated significantly, but poorly (p < 0.01, R2 = 0.1) with guanidinoacetate levels and, despite being overall in the normal range in patients with all other urea cycle disorders, were occasionally below normal in some patients with argininosuccinic acid synthase and lyase deficiency. Creatine levels positively correlated with levels of methionine (p < 0.001, R2 = 0.16), the donor of the methyl group for creatine synthesis. The direct correlation of arginine levels with guanidinoacetate in patients with urea cycle disorders explains the increased concentration of guanidino compounds in arginase deficiency. Low creatine levels in some patients with other urea cycle defects might be explained by low protein intake (creatine is naturally present in meat) and relative or absolute intracellular arginine deficiency.