Characterization of urea transport in Bufo arenarum oocytes

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.     

Urea production and transport in teleost fishes

Teleosts appear to have retained the genes for the urea cycle enzymes. A few species express the full complement of enzymes and are ureotelic (e.g., Lake Magadi tilapia) or ammoniotelic (e.g., largemouth bass), whereas most species have low or non-detectable enzyme activities in liver tissue and excrete little urea (e.g., adult rainbow trout). It was surprising, therefore, to find the expression of four urea cycle enzymes during early life stages of rainbow trout. The urea cycle may play a role in ammonia detoxification during a critical time of development. Exposure to alkaline water (pH 9.0-9.5) or NH4Cl (0.2 mmol/l) increased urea excretion by several-fold in trout embryos, free embryos and alevin. Urea transport is either by passive simple diffusion or via carried-mediated transport proteins. Molecular studies have revealed that a specialised urea transport protein is present in kidney tissue of elasmobranchs, similar to the facilitated urea transporter found in the mammalian inner medulla of the kidney.     

Role of Co-Solute in Biomolecular Stability: Glucose, Urea and the Water Structure

We have studied the action of urea and glucose on the stability of DNAand micelles. We measured the melting temperature of aqueous solutionsof DNA with urea or glucose as a co-solute; we have also measured thechanges in the critical micelle concentrations (cmc) of Sodium DodecylSulfate and Triton X-100 by addition of urea and glucose. Ourexperimental results show that glucose increases the melting temperature ofDNA and decreases the cmc, while urea acts in the contrary direction. The effects of urea and glucose on the stability of DNA and micelles canbe explained by the weakening and enhancement of hydrophobicinteractions, respectively. These effects on hydrophobic interactions arediscussed in this paper.     

The Erythrocyte Urea Transporter UT-B

During the past decade significant progress has been made in our understanding of the role played by urea transporters in the production of concentrated urine by the kidney. Urea transporters have been cloned and characterized in a wide range of species. The genomic organization of the two major families of mammalian urea transporters, UT-A and UT-B, has been defined, providing new insight into the mechanisms that regulate their expression and function in physiological and pathological conditions. Beside the kidney, the presence of urea transporters has been documented in a variety of tissues, where their role is not fully known. Recently, mice with targeted deletion of the major urea transporters have been generated, which have shown variable impairment of urine concentrating ability, and have helped to clarify the physiological contribution of individual transporters to this process. This review focuses on the erythrocyte urea transporter UT-B.     

Interactions between lignin and urea researched by molecular simulation

Molecular simulations of interactions between urea molecules and lignin polymer have been carried out with the aim of understanding the mechanism of urea slow-release behaviours in lignin–urea materials. It has been found, by docking technology and natural bond orbital analysis, that H-bonds and π-electronic conjugation effect are the main driving forces to keep urea molecules adsorbed on the lignin. In the NPT (isothermal–isobaric ensemble) simulations, mean-squared displacement results show that water molecules can promote the urea molecules gradually away from the lignin. Furthermore, in NVT (canonical ensemble) molecular dynamic simulations, results on diffusion constants of urea molecules in lignin–urea system show that diffusion constant of urea molecules in a urea–water–lignin system increases with an increase in the water content. Conclusions gained from two different kinds of simulation are in agreement with each other and are consistent with the experimental observations.     

Organ specificity and lactate-dehydrogenase activity. Differential inhibition by urea and related compounds

1. The effect of urea on the lactate-dehydrogenase activities of human-heart and -liver tissue extracts and on crystalline ox-heart and rabbit-muscle enzyme have been determined. Similar studies on electrophoretically separated isoenzyme fractions have shown an inverse relationship between sensitivity to urea inhibition and electrophoretic mobility. 2. With pyruvate as substrate a sharp change in the nature of the inhibition of tissue lactate dehydrogenase with increasing concentrations of urea occurs at 1 m or 4 m with the electrophoretically slow and fast isoenzymes respectively. 3. At concentrations of urea less than 1 m, inhibition of the purified enzymes is competitive with respect to pyruvate and 2-oxobutyrate. 4. Similar studies have been carried out with methylurea and hydantoic acid, both of which are more potent inhibitors than urea.     

脲梯度电泳方法的技术关键

介绍在应用丙烯酸胺－脲梯度电泳技术进行蛋白质折叠、去折叠研究工作中的实验步骤和技术关键,并在文献方法的基础上作了改进。通过加入15％～0％的甘油,抵消在凝胶中由于脲浓度不同而引起的溶液粘度变化,保证在凝胶上脲浓度不同的部位对蛋白质保持同样的电泳阻力,防止前沿偏斜．采用核黄素光照催化合脲凝胶的聚合,以防止凝胶在梯度灌制完成前发生聚合．加浓缩胶和样品梳于脲梯度胶上可较好地克服边缘效应,获得好的样品迁移图谱．     

Regulation of urea-uptake in the cyanobacterium Anabaena doliolum

Abstract The utilization of urea was studied in the cyanobacterium Anabaena doliolum . The uptake of urea was unaltered in the presence of ammonium. The cells receiving ATP exogenously showed an induced level of urea-uptake as compared with the control cells. Urease inhibitor acetohydroxamic acid and hydroxyurea as well as glutamate analogue, MSO, did not affect the uptake of urea. These results suggest: (1) urea and ammonia have different uptake sites, (2) urea-uptake is an energy dependent process, and (3) during short-term experiments, urea uptake is not linked with the enzyme urease or the ammonium assimilating enzyme glutamine synthetase.     

LDH isoenzymes in the axial muscle of the sharks Galeus melastomus and Etmopterus spinax

The lactate dehydrogenase isoenzyme patterns have been studied in the axial muscles of the sharks Etmopterus and Galeus. Samples from red, intermediate and white muscle fibres were run separately on a polyacrylamide slab-gel. Both sharks have three isoenzymes; all three are present in the red and intermediate fibres, while the white fibres contain only the two slowest-moving isoenzymes. The red fibres of both sharks contain most of the fastest-moving isoenzyme.
The isoenzymes have a high tolerance towards urea; the slow moving isoenzyme is inhibited at about 2 m urea, the next isoenzyme at 4-6 M urea, and some activity of the fast-moving isoenzyme is still present at 10 M urea in the incubation medium. The LDH distribution in the fibre types is studied by histochemistry on frozen sections.     

Preliminary evidence for the hormonal control of urea concentration in the marine pulmonate,Onchidium verruculatum

Changes in blood, hepatopancreas and kidney urea concentrations in O. verruculatum have been assessed in response to endocrinological provocations. Two hr after pleurovisceral (PV) ganglion removal blood and nephridial urea levels were found to be significantly lowered and elevated respectively, whereas the hepatopancreas urea value remained unaltered, over the sham control. Unboiled and boiled macerates of PV (2 gn/animal) ganglion were effective in significantly inhibiting urea levels in blood and kidney of normal intact individuals. The possibility of a neurohormone from the PV, influencing tissue urea levels is discussed.     

Urea derivatives on the move: cytokinin-like activity and adventitious rooting enhancement depend on chemical structure

Urea derivatives are synthetic compounds, some of which have proved to be positive regulators of cell division and differentiation. N -phenyl- N '-(2-chloro-4-pyridyl)urea (forchlorofenuron, CPPU) and N -phenyl- N '-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ), well known urea cytokinin representatives, are extensively used in in vitro plant morphogenesis studies, as they show cytokinin-like activity often exceeding that of adenine compounds. In recent years, renewed interest in structure–activity relationship studies allowed identification of new urea cytokinins and other urea derivatives that specifically enhance adventitious root formation. In this review, we report the research history of urea derivatives, new insights into their biological activity, and recent progress on their mode of action.     

Urea is Necessary for the Culture of Embryos of the Marsupial Frog Gastrotheca riobambae, and is Tolerated by Embryos of the Aquatic Frog Xenopus laevis

Urea was found in the capsular fluid that bathes Gastrotheca riobambae embryos during incubation in the maternal pouch. The urea concentration in this fluid is higher than in blood from the mother, indicating that urea is accumulated by the embryo during the period of maternal incubation. Gastrotheca tadpoles tolerate up to 500 mM urea with 86% survival after 24 hours and die in solutions of 0.5 mM ammonia. These findings suggest that urea plays a role in the adaptation of G. riobambae embryos to the conditions of water stress within the maternal pouch. To improve the in vitro culture conditions of early embryos taken from the maternal pouch, a saline solution that contains urea was designed (GRS). GRS plus 30 mM urea was used for the culture of cleavage to the neurula stage embryos of G. riobambae. During organogenesis, the urea concentration was raised to 60 mM. Early embryos of Xenopus laevis tolerate urea, and in addition, no inducing effects of urea have been detected in animal cap explants of Xenopus .     

Aqueous two-phase systems containing urea: influence on phase separation and stabilization of protein conformation by phase components.

During recombinant Escherichia coli fermentation with high expression levels, inclusion bodies are often formed. Aqueous two-phase systems have been used in the presence of urea for the initial recovery steps. To investigate phase behavior of such systems we determined phase diagrams of poly(ethylene glycol) (PEG)/sodium sulfate/urea/water and PEG/dextran T-500 (DEX)/urea/phosphate buffer/water at different concentrations of urea and different molecular weight of PEG. PEG/Na2SO4 aqueous two-phase systems could be obtained including up to 30% w/w urea at 25 degrees C and PEG/dextran T-500 up to 35% w/w urea. The binodial was displaced toward higher concentrations with increasing urea concentrations. The partition coefficient of urea was near unity. An unstable mutant of T4-lysozyme with an amino acid replacement in the core (V149T) was used to analyze the effect of phase components on the conformation of the enzyme. We showed that partitioning of tryptophan was not dependent on the concentration of urea in the phase system.     

Correlation Between Urea and Other Chemical and Biological Parameters in Waters of Lake Suwa,Japan

The vertical profiles of chemical and biological parameters, including urea concentration, have been measured periodically since February of 1977 at a central station in Lake Suwa, which is one of the typical eutrophic lakes in Japan. The seasonal trend in the standing stock of urea in the central water column, together with the ratio of urea versus total inorganic nitrogen in the euphotic zone from 12 March, 1977 to 25 July, 1978, are presented. The possible importance of bacterial decomposition of dead phytoplankton as a urea source in natural waters is demonstrated by this study. At times, a highly significant correlation between the vertical profile of urea and vertical distributions of other chemical and biological parameters which were also measured was found. An apparent in situ utilization of urea by phytoplankton is suggested on the basis of vertical profiles of urea and other chemical and biological parameters.     

Ammonia excretion and urea handling by fish gills: present understanding and future research challenges

In fresh water fishes, ammonia is excreted across the branchial epithelium via passive NH(3) diffusion. This NH(3) is subsequently trapped as NH(4)(+) in an acidic unstirred boundary layer lying next to the gill, which maintains the blood-to-gill water NH(3) partial pressure gradient. Whole animal, in situ, ultrastructural and molecular approaches suggest that boundary layer acidification results from the hydration of CO(2) in the expired gill water, and to a lesser extent H(+) excretion mediated by apical H(+)-ATPases. Boundary layer acidification is insignificant in highly buffered sea water, where ammonia excretion proceeds via NH(3) diffusion, as well as passive NH(4)(+) diffusion due to the greater ionic permeability of marine fish gills. Although Na(+)/H(+) exchangers (NHE) have been isolated in marine fish gills, possible Na(+)/NH(4)(+) exchange via these proteins awaits evaluation using modern electrophysiological and molecular techniques. Although urea excretion (J(Urea)) was thought to be via passive diffusion, it is now clear that branchial urea handling requires specialized urea transporters. Four urea transporters have been cloned in fishes, including the shark kidney urea transporter (shUT), which is a facilitated urea transporter similar to the mammalian renal UT-A2 transporter. Another urea transporter, characterized but not yet cloned, is the basolateral, Na(+) dependent urea antiporter of the dogfish gill, which is essential for urea retention in ureosmotic elasmobranchs. In ureotelic teleosts such as the Lake Magadi tilapia and the gulf toadfish, the cloned mtUT and tUT are facilitated urea transporters involved in J(Urea). A basolateral urea transporter recently cloned from the gill of the Japanese eel (eUT) may actually be important for urea retention during salt water acclimation. A multi-faceted approach, incorporating whole animal, histological, biochemical, pharmacological, and molecular techniques is required to learn more about the location, mechanism of action, and functional significance of urea transporters in fishes.     

Effect of nickel deficiency in soybeans on the phytotoxicity of foliar-applied urea

The leaf-tip necrosis commonly observed after foliar fertilization of soybean [Glycine max (L.) Merr.] plants with urea is usually attributed to ammonia formed through hydrolysis of urea by plant urease. We recently found, however, that although addition of a urease inhibitor (phenylphosphorodiamidate) to foliar-applied urea increased the urea content and decreased the ammonia content and urease activity of soybean leaves, it increased the leaf-tip necrosis observed after foliar fertilization. We concluded that this necrosis was due to accumulation of toxic amounts of urea rather than formation of toxic amounts of ammonia. To confirm this conclusion, we measured the urea content, urease activity, and leaf-tep necrosis of leaves of soybean plants treated with urea after growth of the plants in nutrient solutions containing different amounts of nickel (Ni), which is an essential component of urease. We found that the urease activity of these leaves decreased, and that their urea content and leaf-tip necrosis increased, with decrease in the Ni content of the nutrient solution. Besides supporting the conclusion that the leaf-tip necrosis observed after foliar fertilization of soybean with urea is due to accumulation of toxic amounts of urea in the soybean leaves, these observations indicate that Ni-deficient plants may have a lower urease activity than plants that are not deficient in Ni and may therefore be more susceptible to leaf burn when foliar-fertilized with urea.     

Permeability of rabbit polymorphonuclear leukocyte membranes to urea and other nonelectrolytes.

The diffusional permeability coefficients of rabbit polymorphonuclear leukocyte membranes to urea, methylurea and thiourea have been measured. It was found that the permeability coefficient of these membranes to urea is very low and that thiourea was more permeable than methylurea which was, in turn, more permeable than urea. These results suggest that there is no need to postulate a carrier-mediated mechanism for urea transport across biological membranes and that the concept of "aqueous pores" is not a general property of biological membranes but restricted only to certain cases.     

Correlation between in vivo and in vitro metabolic measurements. Maximum capacity for urea synthesis.

Estimations of enzyme activity in vivo have been or can often only be done at unphysiological conditions. A main biochemical goal is to correlate in vivo and in vitro measurements. A possible approach to this problem is presented based on forcing metabolic activity in vivo to the maximum for a certain metabolic sequence. Since the urea synthesis system, including maximal rates of enzyme activities, is well known, we have compared in vitro maximum rates for the individual enzymes of urea synthesis with in vivo rates as judged by urea levels in blood of rats given large amounts of protein. The excellent agreement found between the calculated maximum activities from in vitro measurements to the time needed to metabolize a protein overload is presented and comments made on its significance and on the importance of maintaining protein intake at moderate levels, for the capacity of the urea system is limited. Since the intake of large quantities of protein increases the urea level in blood and in other tissues and since high urea levels are somewhat deleterious "per se" and particularly due to equilibrium with cyanate, ingestion of excessive amounts of protein is at best expensive and possibly hazardous.     

Chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy

We have developed chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy. Chemically modified RNAs possessing urea/thiourea-bridged aromatic compounds instead of naturally occurring dinucleotides at the 3′-overhang region were easily prepared in good yields and were more resistant to nucleolytic hydrolysis than unmodified RNA. siRNAs containing urea or thiourea derivatives showed the desired knockdown effect. Furthermore, modified miR-143 duplexes carrying the urea/thiourea compounds in the 3′-end of each strand were able to inhibit the growth of human bladder cancer T24 cells.