Driving forces and current-voltage characteristics of amino acid transport in Riccia fluitans

The complete steady-state I–V relationship of α-aminoisobutyric acid transport across the plasmalemma of rhizoid cells from Riccia fluitans has been measured and analysed with special emphasis on α-aminoisobutyric acid equilibrium and saturation conditions. (A) The electrical data show that: (1) the amino acid-induced electrical current saturates after the addition of the amino acid, regardless of the concentration; (2) a steady state is reached 1–2 h after incubation in α-aminoisobutyric acid, but after less that 5 min in the presence of 1 mM CN⁻; (3) the steady-state I–V characteristic of α-aminoisobutyric acid transport is a sigmoid curve and fairly symmetric in current with respect to the voltage axis; and (4) the equilibrium potential is clearly a function of the amino acid accumulation ratio. It is suggested that the sigmoid curve represents the characteristic of carrier-mediated α-aminoisobutyric acid transport with a voltage-insensitive step, possibly the translocation of the unloaded carrier, rate-limiting. Since under normal conditions the voltage-sensitive rate constant k_oi is much greater than k_io, it is further suggested that the energy to drive this system is put into the transfer of positive charge from outside to the cytoplasm. (B) Accumulation ratios have been determined by inspection of current-voltage data, and additionally by compartmental analysis on green thalli from Riccia fluitans. Both methods give ratios far too low compared with the thermodynamically possible accumulation of about 10⁴. It is suggested that substantial leakages via different non-electrical pathways prevent equilibrium at steady state, and it is concluded that in such leaky systems the thermodynamic equilibrium condition is not suitable for estimating stoichiometries.     

Amino-acid absorption by developing herring eggs

¹⁴C-glycine absorption by eggs of the herringClupea harengus from a 2 µM solution at 15°C depends on the stage of embryonic development. Unidirectional¹⁴C-glycine influx rates are small at early stages: 0.6 ± 0.1 and 0.5 ± 0.1 pmoles egg^–1 h^–1 in embryos 5 h and 28 h after fertilization, respectively. They increase drastically about 51 h after fertilization (prior to blastopore closure) to 3.7 ± 0.9 pmoles egg^–1 h^–1. Glycine uptake steadily continues to increase almost until hatching (maximum values = 18.8 ± 2.7 pmoles egg^–1 h^–1), decreasing slightly prior to hatching. Distribution ratios (radioactivity µl^–1 of egg volume: radioactivity µl^–1 ambient medium) exceed the equilibrium ratio of 1 between 51 h and 78 h after fertilization, reaching values of 4.7 two days prior to hatching, thus suggesting the presence of a transport mechanism capable of transferring the amino acid against the concentration gradient. Curves for concentration-dependent¹⁴C-glycine and¹⁴C--aminoisobutyric acid absorption are very similar; they consist of a linear portion at higher concentrations and a saturable component, indicating a mediated uptake process. Calculations performed by means of aminoacid absorption rates and O₂ uptake data suggest that herring eggs scarcely obtain nutritional benefits from absorption of dissolved amino acids in natural spawning areas.     

Effects of chelating agents on the initial interaction of phytohemagglutinin with lymphocytes and the subsequent stimulation of amino acid uptake

The chelating agents, ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) and EDTA, had no effect on the initial interaction of phytohemagglutinin with lymphocytes at concentrations which have been shown previously to inhibit the development of the phytohemagglutinin response completely. However, they had a marked inhibitory effect on uptake of the amino acid analog, α-aminoisobutyric acid in both unstimulated and phytohemagglutinin-stimulated cells. The inhibition of amino acid uptake by EGTA could be reversed by adding Ca²⁺ but not Mg²⁺. These results demonstrated that Ca²⁺ is not essential to the initial interaction of phytohemagglutinin with the cell, but does influence amino acid transport which may be a critical preparatory event for later increased protein synthesis.     

Incorporation and Degradation of C and H-labeled Thymidine by Sugarcane Cells in Suspension Culture

Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to β-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-¹⁴C]thymidine is lost as ¹⁴CO₂, but the label of [³H]methylthymidine is found in the cell as [³H]β-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines.     

Loss of membrane transport ability in leaf cells and release of protein as a result of osmotic shock

Osmotic shock severely reduces the ability of aged strips of Phaseolus vulgaris leaves to take up α-aminoisobutyric acid, an amino acid analogue which is known to be transported by a specific mechanism. Cold osmotic shock, i.e., transfer from 0.5 m sucrose at 25 C to H₂O at 2 C, decreases α-aminoisobutyric acid uptake almost to zero. Substitution of 10⁻³m ethylenediaminetetraacetate for the sucrose, i.e., treatment which does not involve plasmolysis, produces a similar, but less severe, effect.     

Spatial and temporal population dynamics of a phyllosphere colonizing Bacillus subtilis biological control agent of sugar beet cercospora leaf spot

This study examines the spatial and temporal variation of populations of the biological control agent (BCA) BacB, a Bacillus subtilis isolate, in the field and growth chamber in the presence of the fungus, Cercospora beticola, the causal agent of Cercospora leaf spot of sugarbeet. The use of the selective BCA support substrate β-glucan, applied at 0, 0.5, and 1.0% of the spray solution, did not influence differences in total population numbers (spores + vegetative cells) of a spontaneous rifampicin resistant isolate of BacB (Rif+) over a 14 day spray period. BacB Rif+, applied as a spore formulation, declined from 10,000 CFU/cm² on day 0.5–100 CFU/cm² on day 14 at the three levels of β-glucan tested. Distribution of BacB Rif+ populations was modeled on a leaf scale, with and without β-glucan. Higher populations of vegetative cells were more likely at 14 days with 1% β-glucan than with 0% β-glucan. BacB populations were more aggregated without β-glucan than with the nutrient substrate. There was no correlation between BacB density and Cercospora leaf spot disease severity, indicating that neither antibiosis nor parasitism is likely an important mechanism of disease control.     

Relationship of Transplasmalemma Redox Activity to Proton and Solute Transport by Roots of Zea mays

Transplasmalemma redox activity, monitored in the presence of exogenous ferricyanide stimulates net H⁺ excretion and inhibits the uptake of K⁺ and α-aminoisobutyric acid by freshly cut or washed, apical and subapical root segments of corn (Zea mays L. cv “Seneca Chief”). H⁺ excretion is seen only following a lag of about 5 minutes after ferricyanide addition, even though the reduction of ferricyanide occurs before 5 minutes and continues linearly. Once detected, the enhanced rate of H⁺ excretion is retarded by the ATPase inhibitors N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and vanadate. A model is presented in which plasmalemma redox activity in the presence of ferricyanide involves the transport only of electrons across the plasmalemma, resulting in a depolarization of the membrane potential and activation of an H⁺-ATPase. Such a model implies that this class of redox activity does not provide an additional and independent pathway for H⁺ transport, but that the activity may be an important regulator of H⁺ excretion. The 90% inhibition of K⁺ (⁸⁶Rb⁺) uptake within 2 minutes after ferricyanide addition can be contrasted with the 5 to 15% inhibition of uptake of α-aminoisobutyric acid. The possibility exists that a portion of the K⁺ and most of the α-aminoisobutyric acid uptake inhibitions are related to the ferricyanide-induced depolarization of the membrane potential, but that the redox state of some component of the K⁺ uptake system may also regulate K⁺ fluxes.     

Maturation of membrane function: Transport of amino acid by rat erythroid cells

The membrane changes which occur during cellular maturation of erythroid cells have been investigated. The transport of α-aminoisobutyric acid, alanine, and N-methylated-α-aminoisobutyric acid have been studied in the erythroblastic leukemic cell, the reticulocyte, and the erythrocyte of the Long-Evans rat. The dependence of amino acid transport on extracellular sodium concentration was investigated. Erythrocytes were found to transport these amino acids only by Na-independent systems. The steady state distribution ratio was less than 1. Reticulocytes were found to transport α-aminoisobutyric acid and alanine by Na-dependent systems, but only small amounts of N-methylated-α-aminoisobutyric acid. Small amounts of these amino acids were transported by Na-independent systems. The steady state distribution ratio was greater than one for Na-dependent transport. The erythroblastic leukemia cell, a model immature erythroid cell, showed marked Na-dependence (>90%) for α-aminoisobutyric acid and alanine transport, and >80% for the Na-dependent transport of N-methyl-α-aminoisobutyric acid. The steady state distribution ratio for the Na-dependent transport was >4. In the erythroblastic leukemic cell, at least three Na-dependent systems are present: one includes alanine and α-aminoisobutyric acid, but excludes N-methyl-α-aminoisobutyric acid; one is for α-aminoisobutyric acid, alanine and also N-methyl-α-aminoisobutyric acid; and one is for N-methyl-α-aminoisobutyric acid alone. In the reticulocyte, the number of Na-dependent systems are reduced to two: one for α-aminoisobutyric acid and alanine; one for N-methyl-α-aminoisobutyric acid. In the erythrocytes, no Na-dependent transport was found. Therefore, maturation of the blast cell to the mature erythrocyte is characterized by a systematic loss in the specificity and number of transport systems for amino acids.     

Technical communication: Determination of cuprous ions in bacterial leachates and for environmental monitoring

A sensitive and precise spectrophotometric method has been developed for the determination of copper(I) in bacterial leach liquors produced by the action of Thiobacillus ferrooxidans and T. thiooxidans on copper ores. In this method bicinchoninic acid (BCA) has been used as the chromogenic reagent which produces a stable purple complex with Cu(I) which was found to obey Beer's Law and with _max at 560 nm. The coloured complex has a molar extinction coefficient () value of 6.6 × 10³ l mol^–1 cm^–1; specific absorptivity () value of 0.104 ml^–1 g cm^–1 and the Sandell sensitivity (S) value was 0.0096 g cm². Optimal conditions for development of coloration/sensitivity were determined. Interferences due to cations and anions were investigated and various masking agents for alleviating their inhibition were studied. The method has been found very useful in determining ratios of Cu(I) to Cu(II) in bacterial leach liquors and should play a significant role in determining the reaction mechanisms of biological leaching and for environmental monitoring.     

The γ-irradiation of aqueous solutions of urea. Implications for chemical evolution

0.05 mole dm^–3, O₂-free aqueous solutions of urea were studies after receiving various doses of⁶⁰Co gamma rays (0.14–600 kGy). Urea was found to be relatively stable under radiation; its radiation chemical yield of decomposition was 0.47. Hydrogen (G=0.50), carbon dioxide (G=0.44), ammonia (G=0.22), oxalic acid (G=0.0054), malonic acid (G=0.000064) and three unidentified oligomers were found to be the main radiolytic products. The origin of these products is explained by free radical reactions initiated by the transients from water radiolysis (H·,·OH,e _aq ^– ).     

Acid hydrolysis of sugarcane bagasse for lactic acid production

In order to use sugarcane bagasse as a substrate for lactic acid production, optimum conditions for acid hydrolysis of the bagasse were investigated. After lignin extraction, the conditions were varied in terms of hydrochloric (HCl) or sulfuric (H₂SO₄) concentration (0.5–5%, v/v), reaction time (1–5 h) and incubation temperature (90–120 °C). The maximum catalytic efficiency (E) was 10.85 under the conditions of 0.5% of HCl at 100 °C for 5 h, which the main components (in g l⁻¹) in the hydrolysate were glucose, 1.50; xylose, 22.59; arabinose, 1.29; acetic acid, 0.15 and furfural, 1.19. To increase yield of lactic acid production from the hydrolysate by Lactococcus lactis IO-1, the hydrolysate was detoxified through amberlite and supplemented with 7 g l⁻¹ of xylose and 7 g l⁻¹ of yeast extract. The main products (in g l⁻¹) of the fermentation were lactic acid, 10.85; acetic acid, 7.87; formic acid, 6.04 and ethanol, 5.24.     

Acceptor reactions of a novel transfructosylating enzyme from Bacillus sp.

Many different oligosaccharides were produced by transferring the fructose residue of sucrose to maltose, cellobiose, lactose and sucrose (self-transfer), where their yields of fructosylated acceptor products accounted for 26–30% (w/w). The maximum conversion yield (30%) was obtained in fructosyl cellobioside formation with 500 g sucrose l^–1 (substrate) and 200 g cellobiose l^–1 (acceptor). These four acceptors gave various products having DP (degree of polymerization) 2–7 by successive transfer reactions.     

BCA2/Rabring7 Promotes Tetherin-Dependent HIV-1 Restriction

Host cell factors can either positively or negatively regulate the assembly and egress of HIV-1 particles from infected cells. Recent reports have identified a previously uncharacterized transmembrane protein, tetherin/CD317/BST-2, as a crucial host restriction factor that acts during a late budding step in HIV-1 replication by inhibiting viral particle release. Although tetherin has been shown to promote the retention of nascent viral particles on the host cell surface, the precise molecular mechanisms that occur during and after these tethering events remain largely unknown. We here report that a RING-type E3 ubiquitin ligase, BCA2 (Breast cancer-associated gene 2; also called Rabring7, ZNF364 or RNF115), is a novel tetherin-interacting host protein that facilitates the restriction of HIV-1 particle production in tetherin-positive cells. The expression of human BCA2 in “tetherin-positive” HeLa, but not in “tetherin-negative” HOS cells, resulted in a strong restriction of HIV-1 particle production. Upon the expression of tetherin in HOS cells, BCA2 was capable of inhibiting viral particle production as in HeLa cells. The targeted depletion of endogenous BCA2 by RNA interference (RNAi) in HeLa cells reduced the intracellular accumulation of viral particles, which were nevertheless retained on the plasma membrane. BCA2 was also found to facilitate the internalization of HIV-1 virions into CD63⁺ intracellular vesicles leading to their lysosomal degradation. These results indicate that BCA2 accelerates the internalization and degradation of viral particles following their tethering to the cell surface and is a co-factor or enhancer for the tetherin-dependent restriction of HIV-1 release from infected cells.     

Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na⁺ ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O₂-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na⁺ to Na⁺-deficient cells, showing that Na⁺ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N,N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na⁺ chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na⁺ gradient is also required.     

Determination of free radical reaction products and metabolites of salicylic acid using capillary electrophoresis and micellar electrokinetic chromatography

Hydroxylated radical products of salicylic acid are often used as a relative measurement in free radical research. Several analytical methods exist to determine the amount of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. In this study we use capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) in order to determine these free radical products. The CZE experiment was optimized with a CZE simulation program in order to achieve an optimal pH. Calibration curves were recorded in the range 10⁻⁶–10⁻⁴ M and the detection limit was determined. For both CZE and MECC it was 2·10⁻⁷ M. Both methods resulted in a reproducible analysis of salicylate and its hydroxylated free radical products in 6 min.     

Amino acid auxotrophs from protoplast cultures of Nicotiana plumbaginifolia, Viviani

Summary Several amino acid requiring auxotrophs have been isolated from unsupplemented protoplast cultures of haploid Nicotiana plumbaginifolia following incubation with BUdR (1-5x10^-5, 2 days) and recovery on complete medium. The auxotrophic lines required the following amino acid(s) for growth: his, ile, leu, ile+val, met or try. Met^– is a new type isolated in higher plants. The same absolute amino acid requirement was observed in plants regenerated from auxotrophic cultures. Precursor feeding tests, enzyme assays, and/or metabolic complementation through protoplast fusion were used to identify the genetic lesion leading to auxotrophy. Mutant seeds were obtained from supplemented Met^– plants. Seeds were also collected from selfed plants regenerated from various complementing fusion products, and a His^– revertant. Genetic analysis indicated that under natural conditions of seed formation amino acid auxotrophy-in contrast to NR deficiency-failed to segregate in progeny tests.Abbreviations and definitions BUdR and FUdR 5-bromo- and fluoro-deoxyuridine respectively - AP imidazole acetol phosphate - IGP imidazole glycerol phosphate - NR nitrate reductase - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - TIP total isolation procedure - ER Escape rate—the proportion of the selected cell population surviving the BUdR treatment - BR Recovery rate—the proportion of clones identified as amino acid auxotrophs from total escaping clones - TS Total surviving colonies—the number of inoculated protoplasts/variant x plating efficiency - TST Total starvation time—the number of days on minimal medium (preincubation time+BUdR incubation time). The relationship days vs. number of divisions is as follows: 3- to 4-day-old protoplasts, 1 division; 5–6 days, 2 divisions; 7–8 days, 3 divisions Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Factors affecting the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation

The effect of medium composition on the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation has been studied. Four types of mycelial pellets were obtained under the conditions used and may be classified as (a) frayed and loose with 0.1–0.5 mm diameter (b) compact with 0.1–0.5 mm diameter (c) loose with 0.5–2.0 mm diameter and (d) compact with 0.5–2.0 mm diameter. Their respective maximum specific rates of formation and yields of itaconic acid, based on 100 g sucrose supplied, were (a) 1.25 mol mg^–1h^–1 and 55–59 g, (b) 0.27–0.43 mol mg^–1 h^–1 and 26–38 g, (c) 0.75–0.90 mol mg^–1 h^–1 and 45–51 g and (d) 0.12 mol mg^–1 h^–1 and 10 g. The presence of Ca²⁺, Zn²⁺ and Fe²⁺ in the basal medium at concentrations of 23.3 mg/100 ml, 0.01 mg/100 ml and 0.006 mg/100 ml respectively were found to be adequate and crucial in obtaining the desired outgrowth for both high production rates and consistent yields of itaconic acid. The further addition of either commercial plaster of Paris or analytical-reagent-grade CaSO₄, especially when activated by heating to 530°C and present in excess of solubility, results in small and frayed pellets, which lead to itaconic acid yields of 55–59 g acid/100 g sugar supplied.     

Effects of Ca on Amino Acid Transport and Accumulation in Roots of Phaseolus vulgaris

Ca²⁺ stimulates the uptake of α-aminoisobutyric acid (AIB) into excised or intact Phaseolus vulgaris L. roots by a factor of two. In roots depleted of Ca²⁺ by preincubation with ethylenediaminetetraacetate, ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid, or streptomycin, the stimulatory effect is 7- to 10-fold. In the presence of Ca²⁺, roots accumulate AIB more than 100-fold; Ca²⁺-depleted roots only equilibrate with AIB. Radioautography shows [¹⁴C]AIB to be present in all cells after 90 min. Although Ca²⁺-depleted roots lose accumulated [¹⁴C]AIB about 10 times faster than roots supplied with Ca²⁺, this increased efflux is not the main cause for the decrease in net uptake observed. The latter is rather due to a less negative membrane potential Δψ in Ca²⁺ depleted roots (−120 mV → −50 mV). The basic feature explaining all the results of Ca²⁺ deficiency is an increase in general membrane permeability. No indication of a specific regulatory function of Ca²⁺ in membrane transport of roots has been obtained.     

The Bioutilization of Thiodiglycol (a Detoxication Product of Mustard Gas): Isolation of Degrading Strains and Investigation of Degradation Conditions

The Alcaligenes xylosoxydans subsp.denitrificans strain TD1 capable of degrading thiodiglycol (TDG), a product of mustard gas hydrolysis, was isolated from soil contaminated with breakdown products of this chemical warfare agent. The selected stable variant of TD1 (strain TD2) can grow on TDG with a lag phase of 4–8 h and a specific growth rate of 0.04–0.045 h^–1. Optimal conditions for the biodegradation of TDG (pH, the concentration of TDG in the medium, and specific substrate loading) were determined. TDG was found to be degraded with the formation of diglycolsulfoxide and thiodiglycolic acid as intermediate products. The data obtained can be used to develop approaches to the bioremediation of mustard gas–contaminated soils.