Penetration of Mannitol into the Intracellular Space of Chlorella sorokiniana

It was found that in the alga, Chlorella sorokiniana, mannitol penetrated the plasmalemma (normally thought to be impermeable to mannitol) into the intracellular space. The rate of penetration is exponential and relatively slow, having a half-time of 6 to 12 minutes and requiring over 60 minutes for complete penetration. This penetration was demonstrated both by the Millipore filtration of cells incubated with ¹⁴C-mannitol and by centrifugation of the cells through a silicon oil layer after incubation with tritiated water and ¹⁴C-mannitol. Further, mannitol caused an inhibition of both autotrophic (on CO₂) and heterotrophic (on glucose) growth. A low rate of mannitol metabolism was demonstrated, although this rate could not support heterotrophic growth.     

Osmotic behavior of liposomes of phosphatidylcholine and phosphatidylsulfocholine as a function of lipid concentration

A relationship between the initial rate of liposome swelling, d(1/A)/dt and the reciprocal of the lipid concentration of the liposomes has been derived and then utilized to describe the osmotic swelling behavior of serially diluted liposomes and chloroplasts exposed to hypertonic urea solutions. The slopes of plots of d(1/A)/dt vs. the reciprocal of the lipid concentration of liposomes were not affected by differences in the initial absorbance of phosphatidylcholine-sterol bilayers, and were used to assess the ability of sterols to reduce the initial rates of urea permeation through dimyristoylphosphatidylcholine (DMPC) bilayers in the liquid-crystalline state. Multilamellar liposomes and sonicated vesicles were prepared from dimyristoylphosphatidylsulfocholine (DMPSC), in which the quaternary ammonium group of choline is replaced by -S⁺(CH₃)₂. Cholesterol reduced the initial rate of osmotic urea penetration into liposomes and the rate of 6-carboxyfluorescein efflux from vesicles at 35°C. The effect of cholesterol on bilayers of phosphatidylsulfocholine and phosphatidylcholine was very similar, suggesting that no strict structural requirements need be met in the choline moiety for lecithin-cholesterol interaction. The sulfonium analog could thus functionally replace phosphatidylcholine in natural membranes.     

Glycerolipid formation and PGE2 and PGF2α production of rat renal papillae in vitro, the effects of urea

The glycerolipid production by rat renal papillary slices varied inversely with the urea concentration (0a–1660 mM) whether the production was measured as labelling of the glycerol backbone from glucose or as incorporation of labelled arachidonic acid and palmitic acid. The rate of phospholipid formation was most dependent on medium urea concentrations in the range between 0 and 1100 mM. The production of prostaglandins PGE₂ and PGF_2α, measured radioimmunologically or by an isotope derivative method was in the same range inversely related to the production of glycerolipids and chain elongations. The effect of urea on prostaglandin formation is probably indirectly caused by the inhibition of the phospholipid formation and chain elongation, since the effect was abolished by 1% defatted albumin in the medium. The data suggest that the level of free arachidonic acid within the cells is controlled to an important extent by glycerolipid formation and chain elongation.     

Measurement of the partial pressure of dissolved CO2 for estimating the concentration of Streptococcus thermophilus in coculture with Lactobacillus bulgaricus

Summary The use of mixed cultures in the food industry is hindered by the lack of rapid and specific measurement techniques. The coculture of Streptococcus thermophilus and Lactobacillus bulgaricus, used for producing starters for yoghurt production, is a simple model of a mixed culture.After verifying that Streptococcus thermophilus specifically degraded urea, we attempted to correlate the cell concentration of the species first with urease activity and then with the rate of CO₂ production from urea in the medium. The measurement was performed in real time with a specific electrode for dissolved CO₂. The results obtained with the method have the same uncertainy as those obtained by cell counts. The estimation is valid between 10⁷ and 10⁹ cells/ml.     

On simultaneous transport of water and solute through plant cell membranes: Evidence for the absence of solvent drag effect and insensitivity of the reflection coefficient

The simultaneous efflux of tritiated water and ¹⁴C labelled ethanol from inner epidermal cells of the bulb scale of Allium cepa was measured with a specially designed efflux chamber. It was found that water and ethanol moved essentially independently. Rates of efflux of tritiated water and ¹⁴C ethanol were essentially the same in the presence or absence of a simultaneous influx of water. Using the same technique the efflux of tritiated water from the epidermal cells was measured during a simultaneous flow of nonlabelled ethanol. When tritiated water and ethanol moved in opposite directions, the water permeability values became slightly reduced depending upon the concentration of ethanol. When ethanol and tritiated water moved in the same direction, however, no effect on water permeability values could be detected. These results are best explained by the molecular theory of diffusion across lipid bilayer membranes, and are consistent with the above findings of lack of interaction between water and ethanol as they are transported across the cell membrane. In another study, the solute permeability coefficients (K_s) for non-electrolytes such as urea and methyl urea were measured by plasmolyzing the epidermal cells and transferring them to equimolal solutions of urea and methyl urea. This method was also used to measure the reflection coefficient (σ) for these nonelectrolytes. The K_s values for methyl urea were 16 times greater than the ones for urea. The values of σ for both of these solutes, however, were very close to 1. Using the K_s data available in the literature for the subepidermal cells of the Pisum sativum stem basis, the σ values were calculated for malonamide, glycerol, methyl urea, ethyl urea, dimethyl urea, and formamide. Again the K_s values for these nonelectrolytes varied by several orders of magnitude, whereas all σ values were found to be close to 1. These findings point out that σ is an insensitive parameter and that K_s, the solute permeability constant, has to be used for characterizing solute transport through the membrane. The present study shows that fast (e.g. ethanol, formamide) as well as slowly permeating molecules do not interact with water as they are transported across the cell membrane. Aqueous pores for the simultaneous transport of water and solutes, therefore, are absent in the plant cell membranes investigated here.     

Urea degradation by picophytoplankton in the euphotic zone of Lake Biwa

The ability of photoautotrophic picoplankton Synechococcus to degrade urea was examined in the euphotic zone of Lake Biwa. Samples were divided into pico (0.2–2.0 μm) and larger (>2.0 μm) size fractions by filtration. The rates of urea degradation (the sum of the rates of incorporation of carbon into phytoplankton cells and of liberation of CO₂ into water) measured by radiocarbon urea were 8 and 17 μmol urea m⁻³ day⁻¹ in June and July, respectively, for the picophytoplankton in the surface water, and 196 and 96 μmol urea m⁻³ day⁻¹, respectively for the larger phytoplankton. The rates decreased with depth, somewhat similar to the vertical profiles of the photosynthetic rate. The urea degradation rates were obviously high under light conditions. In daylight, urea was degraded into two phases, carbon incorporation and CO₂ liberation, whereas in the dark it was degraded only into the CO₂ liberation phase. The contribution of picophytoplankton to total phytoplankton in urea degradation was high in the subsurface to lower euphotic layer. Urea degradation activity was higher in the picophytoplankton fraction than in the larger phytoplankton fraction. Shorter residence times of urea were obtained in the upper euphotic zone. The contribution of picophytoplankton to urea cycling was 4% to 35%. The present results suggest that the picophytoplankton Synechococcus is able to degrade urea and effectively makes use of regenerated urea as a nitrogen source in the euphotic layer, and that picophytoplankton play an important role in the biogeochemical nitrogen cycle in Lake Biwa. Received: June 25, 1998 / Accepted: February 10, 1999     

Effect of urea on the kinetics of ligand combination reactions of hemoglobins A and H, alphaSH chains and myoglobin.

In O₂ equilibrium studies of hemoglobin it was observed that the presence of urea increases f₅₀2 and decreases co-operativity. The results were interpreted on the basis of unfolding and swelling of the peptide chains. The results of the present kinetic investigation indicate that while the CO combination rate constants for α^SH chains, hemoglobin H and myoglobin do not change with increasing concentrations of urea, the corresponding rate constants for deoxy hemoglobin A increase continuously with urea concentration. At urea concentrations of 4 m or more, the reaction time course becomes biphasic. The fast component of the reaction time course yields CO combination rate constants which are in close agreement with the rate constants of dimeric and monomeric hemoglobins. These results indicate that up to 4 m-urea the kinetic and equilibria parameters increase due to weakened constraints imposed by intersubunit contacts and bonds. At higher urea concentrations Hb₄ is significantly dissociated into dimers and monomers, and hence the high ligand affinity and decreased co-operativity of the system. The implications of higher ligand combination rate constants of the deoxyhemoglobin tetramer in the presence of urea on the reaction mechanism are discussed.     

Effects of luminal osmolarity on gastric acid secretion in the little skate,Raja erinacea

Summary Isolated gastric mucosa of the skate shows marked changes in acid secretory rate (J _H ), electrical potential difference (PD), and transepithelial resistance (R) with changes in mucosal bathing solution composition and a constant serosal solution. Removal of the 350 mM urea usually present in the mucosal solution reduces acid secretory rate by 25%, while adding urea to 1 M has no significant effect. Complete removal of osmotic solutes (distilled water) inhibits secretion by 78%, isotonic urea (no salts) inhibits by 54%, while isotonic salts alone (no urea) gives control secretory rates. The changes in PD and R are consistent with acid secretory changes. Theory and experience with terrestrial organisms would not predict these changes. The most likely explanation is osmotic swelling and shrinking of the surface cells, and occlusion of the secretory tubules in the swollen condition. Since marine species never encounter hypo- or hyperosmotic conditions due to food ingestion, their surface cells may be water permeable, unlike the situation in terrestrial and fresh water animals.Abbreviations J _H acid secretory rate per square centimetre tissue area - OC oxyntic cell - PBC pit border cell - PD transepithelial electrical potential difference - R transepithelial electrical resistance per square centimetre tissue area - SEC Surface epithelial cell     

KINETICS OF PENETRATION : VII. MOLECULAR VERSUS IONIC TRANSPORT

In some living cells the order of penetration of certain cations corresponds to that of their mobilities in water. This has led to the idea that electrolytes pass chiefly as ions through the protoplasmic surface in which the order of ionic mobilities is supposed to correspond to that found in water. If this correspondence could be demonstrated it would not prove that electrolytes pass chiefly as ions through the protoplasmic surface for such a correspondence could exist if the movement were mostly in molecular form. This is clearly shown in the models here described. In these the protoplasmic surface is represented by a non-aqueous layer interposed between two aqueous phases, one representing the external solution, the other the cell sap. The order of penetration through the non-aqueous layer is Cs > Rb > K > Na > Li. This will be recognized as the order of ionic mobilities in water. Nevertheless the movement is mostly in molecular form in the nonaqueous layer (which is used in the model to represent the protoplasmic surface) since the salts are very weak electrolytes in this layer. The chief reason for this order of penetration lies in the fact that the partition coefficients exhibit the same order, that of cesium being greatest and that of lithium smallest. The partition coefficients largely control the rate of entrance since they determine the concentration gradient in the non-aqueous layer which in turn controls the process of penetration. The relative molecular mobilities (diffusion constants) in the non-aqueous layer do not differ greatly. The ionic mobilities are not known (except for K⁺ and Na⁺) but they are of negligible importance, since the movement in the non-aqueous layer is largely in molecular form. They may follow the same order as in water, in accordance with Walden''s rule. Ammonium appears to enter faster than its partition coefficient would lead us to expect, which may be due to rapid penetration of NH₃. This recalls the apparent rapid penetration of ammonium in living cells which has also been explained as due to the rapid penetration of NH₃. Both observation and calculation indicate that the rate of penetration is not directly proportional to the partition coefficient but increases somewhat less rapidly. Many of these considerations doubtless apply to living cells.     

Using urea phosphate to enhance the effect of gibberellin A3 on grape size

Gibberellic acid (GA₃) is widely used to enlarge the berries of seedless grapes (Vitis vinifera L). In cv. Sultana (Thompson Seedless) the addition of 1000 mg/L urea phosphate (UP) to GA₃ solutions after fruit set reduced the pH of the solutions to a stable pH 2.9 and enhanced the effect of GA₃ on berry size and delayed maturation. Addition of citrate buffer, pH 2.9, to GA₃ sprays did not affect berry size or maturation. The possibility of improved GA penetration due to the low pH is considered. The nutritional effect of UP and direct enhanced penetration by the urea ion are also discussed.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 1735-E, 1986 series.     

Urease-catalyzed urea synthesis.

The equilibrium of hydrolytic reactions can be shifted toward condensation by carrying out the reaction at low water concentration. The rate and yield of urease-catalyzed urea synthesis from (NH₄)₂CO₃ or NH₄HCO₃ has been examined as a function of water concentration (in mixtures with organic solvents), substrate and H⁺ concentration, and polarity of the nonaqueous component of the solvent. Similar effects of organic solvents are observed on the reaction rate in both directions; the results suggest that at least in some conditions the reaction proceeds through nonenzymically formed carbamate. The equilibrium concentration of urea, in 50% ($\text{v}\text{v}$) water, varies over 10-fold, depending on the nature of the nonaqueous component of the solvent; nonhydroxylic solvents such as acetone given the highest yield. Solubility measurements suggest that the interactions of the solvent mixtures with (NH₄)₂CO₃ (or carbamate), rather than urea, are responsible for the variations in urea yield. Activities of water and the ionic components of the equilibrium are strongly influenced by the nature of the nonaqueous component of the solvent, as well as its concentration.     

Arginase,an s-phase enzyme in a human cell line

Suspension cultures of ‘Chang liver’ cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S+G₂) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G₁/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4–6% per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in ‘Chang liver’ cells is discussed.     

A Re-examination of the Penetration of Nitella Cells by Non-electrolytes

The increase, followed by culmination and decrease, of biological activity often observed within a homologous series of toxic compounds has previously been explained by assuming that their rate of movement between the lipid cell membrane and the aqueous protoplasm passes through an optimum as the partition coefficients increase progressively. In contrast to this, it has been claimed that direct measurements of the penetration rate of compounds through individual Nitella cells show tbat penetration rates are proportional to partition coefficients. In this attempt to investigate these two opposed hypotheses an analysis of published penetration rate data, obtained with Nitella, showed that the direct penetration measurements were best described by an equation which included a quadratic function of partition coefficient. There is, therefore, no inconsistency between the measurements of penetration by biological activity and by direct measurement. The compounds used in the latter procedure were all relatively hydrophilic and although a maximum rate of penetration was observed tbere was no indication of a subsequent decrease as there were no compounds with high partition coefficients. The correlation of penetration with other physical properties of the compounds is discussed.     

UPTAKE OF NITRATE,AMMONIUM, AND UREA BY NITROGEN-STARVED CULTURES OF MICROMONAS PUSILLA (PRASINOPHYCEAE): TRANSIENT RESPONSES1

Nitrogen uptake rates were measured as a function of time following saturating additions (15 μMg-at N·^?1) of ¹⁵N-labelid ammonium, urea, and nitrate to N-starved cultures of the picoflagellate Micromonas pusilla Butcher. Uptake rates were estimated from both the accumulation of ¹⁵N into the cells and the disappearance of nitrogen from the medium. Transient elevated (surge) uptake rates of NH₄⁺ and urea were observed after enrichment. During the first 5 min the initial urea and NH₄⁺ uptake rates were 2- and 4-fold greater than the maximum growth rate (μM_max)observed prior to No₃^? depletion in the cultures. The elevated urea uptake rates declined quickly to a relatively constant value, whereas the initial rates of NH₄⁺ uptake declined rapidly but were followed by a subsequent increase prior to remaining roughly constant. Nitrate was not taken up as readily by N-starved M. pusilla as the reduced N forms. Although NO₃⁺ uptake commenced immediately after enrichment (i.e. no lag period) the N-Specific rate over the next 6 h averaged half the μM_max observed during NO₃^? replete conditions.     

THE UPTAKE OF UREA BY MARINE PHYTOPLANKTON1

Half-saturation constants for urea uptake by 4 clones of neritic diatoms capable of utilizing urea were determined from short-term uptake studies with ¹⁵N-labeled urea. K ₈ values obtained were similar to those determined, earlier for ammonium, and since ammonium and urea concentrations are similar in the marine environment, it was concluded that these species are capable of utilizing ecologically significant concentrations of urea. Two of 3 species unable to grow on urea showed patterns of short-term uptake not unlike those of species capable of utilizing urea, which implies that, their assimilatory rather than uptake processes are defective with, regard to urea utilization. The third species initially took ¹⁵N (supplied as urea) into the cells but subsequently released it back into the medium.     

The effect of antigen concentration,antibody valency and size,and tumor architecture on antibody binding in multicell spheroids

Intact IgG₁ and F(ab′)₂ anti-carcinoembryonic antigen antibodies penetrate human colon adenocarcinoma multicell spheroids much more slowly than Fab fragments and the molecular weight and the binding site valency appear to be the most important factor in determining the rate of penetration. The rate is also influenced considerably by the number of antigen binding sites per cell, with a high antigen concentration slowing penetration appreciably. The tumor cell architecture appears to have a minor effect on antibody penetration when compared to antibody size or antigen concentration.     

Excretory nitrogen metabolism in the Chinese fire-belly newt<Emphasis Type="Italic"> Cynops orientalis</Emphasis> in water,on land,or in high concentrations of environmental ammonia

The Chinese fire-belly newt Cynops orientalis reverts to an aquatic mode of living when sexually mature. Despite living in water, sexually mature C. orientalis maintained high capacity for hepatic urea synthesis. However, it had a lower rate of urea production than other terrestrial amphibians because endogenous ammonia could diffuse out to the external medium as NH₃. This conserves cellular energy because urea synthesis is energetically expensive. Simultaneously, C. orientalis also reduced the rate of urea excretion, and excreted 33% of the total nitrogenous waste as ammonia. Upon exposure to land, C. orientalis increased the rate of urea synthesis from accumulating endogenous ammonia. The increased rate of urea synthesis was within the inherent capacity of the hepatic ornithine–urea cycle; there was no induction of hepatic carbamoyl phosphate synthetase or ornithine transcarbamoylase activities and there was no reduction in ammonia production. When exposed to water containing 75 mmol.l^–1 NH₄Cl, the rates of both urea synthesis and urea excretion increased. Under such experimental conditions, the ornithine–urea cycle may be operating close to its limit; glutamine began to accumulate in the body, and endogenous ammonia production via amino acid catabolism was reduced.Abbreviations CPS carbamoyl phosphate synthetase - FAA free amino acid - OTC ornithine transcarbamoylase - OUC ornithine–urea cycle - TCA trichloroacetic acid Communicated by I.D. Hume     

THE PENETRATION OF AMMONIA INTO FROG MUSCLE

1. A study was made of the electrolyte changes which occur when frog muscles are immersed in a Ringer solution with 1/5 of the Na replaced by NH₄Cl. Analyses were made in the solution and in the muscles for K and NH₃, and the muscles were also analyzed for Cl, HCO₃, and Na. Control muscles were immersed in normal Ringer''s solution and similarly analyzed. 2. The amount of ammonia taken up was about equal to the K and Na lost. There was also a small increase in chloride content. The bicarbonate content was the same in both experimental and control muscles, indicating no change in the muscle pH due to the NH₃ which penetrated. An increased loss of K due to the penetration of NH₃ was also demonstrated by the use of radioactive K. 3. After 5 hours, the concentration of ammonia per gram of muscle is about the same as the concentration in the solution. After 4 or 5 days, the concentration in the muscle is about 1.5 times that in the solution. The inside to outside NH₃ ratio is about equal to the corresponding H ion ratio, but is much less than the K ratio. 4. The rate of penetration of the NH₃ is increased by a rise of temperature, by stirring the solution, and by decrease in the concentration of Na, K, Ca, or Mg in the solution; it is decreased by increasing the size of the muscles or by killing them with chloroform or boiling. 5. Liver, smooth muscle, skin, and kidney, in a few experiments, behaved much like muscle except that there was a formation of urea in the case of liver. 6. The injection of NH₄Cl into anesthetized cats causes an increase in the level of K in the blood plasma.     

Short-term effects of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide on perennial ryegrass

The current study investigated the short-term physiological implications of plant nitrogen uptake of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide (nBTPT) under both greenhouse and field conditions. ¹⁵N labelled urea amended with 0.0, 0.01, 0.1 and 0.5% nBTPT (w/w) was surface applied at a rate equivalent to 100 kg N ha^–1 to perennial ryegrass in a greenhouse pot experiment. Root, shoot and soil fractions were destructively harvested 0.75, 1.75, 4, 7 and 10 days after fertilizer application. Urease activity was determined in each fraction together with ¹⁵N recovery and a range of chemical analyses. The effect of nBTPT amended urea on leaf tip scorch was evaluated together with the effect of the inhibitor applied on its own on plant urease activity.nBTPT-amended urea dramatically reduced shoot urease activity for the first few days after application compared to unamended urea. The higher the nBTPT concentration the longer the time required for shoot activity to return to that in the unamended treatment. At the highest inhibitor concentration of 0.5% shoot urease activity had returned to that of unamended urea by 10 days. Root urease activity was unaffected by nBTPT in the presence of urea but was affected by nBTPT in the absence of urea.Transient leaf tip scorch was observed approximately 7–15 days after nBTPT + urea application and was greatest with high concentrations of nBTPT and high urea-N application rates. New developing leaves showed no visual sign of tip necrosis.Urea hydrolysis of unamended urea was rapid with only 1.3% urea-N remaining in the soil after 1.75 days. N uptake and metabolism by ryegrass was rapid with ¹⁵N recovery from unamended urea, in the plant (shoot + root) being 33% after 1.75 days. Most of the ¹⁵N in the soil following the urea+0.5% nBTPT application was still as urea after 1.75 days, yet ¹⁵N plant recovery at this time was 25% (root+shoot). This together with other evidence, suggests that if urea hydrolysis in soil is delayed by nBTPT then urea can be taken up by ryegrass as the intact molecule, albeit at a significantly slower initial rate of uptake than NH₄ ⁺-N. Protein and water soluble carbohydrate content of the plant were not significantly affected by amending urea with nBTPT however, there was a significant effect on the composition of amino acids in the roots and shoots, suggesting a difference in metabolism.Although nBTPT-amended urea affected plant urease activity and caused some leaf-tip scorch the effects were transient and short-lived. The previously reported benefit of nBTPT in reducing NH₃ volatilization of urea would appear to far outweigh any of the observed short-term effects, as dry-matter production of ryegrass is increased.