Non‐invasive imaging of allogeneic transplanted skin graft by 131I‐anti‐TLR5 mAb

Although ¹⁸F‐fluorodeoxyglucose (¹⁸F‐FDG) uptake can be used for the non‐invasive detection and monitoring of allograft rejection by activated leucocytes, this non‐specific accumulation is easily impaired by immunosuppressants. Our aim was to evaluate a ¹³¹I‐radiolabelled anti‐Toll‐like receptor 5 (TLR5) mAb for non‐invasive in vivo graft visualization and quantification in allogeneic transplantation mice model, compared with the non‐specific radiotracer ¹⁸F‐FDG under using of immunosuppressant. Labelling, binding, and stability studies were performed. BALB/c mice transplanted with C57BL/6 skin grafts, with or without rapamycin treatment (named as allo‐treated group or allo‐rejection group), were injected with ¹³¹I‐anti‐TLR5 mAb, ¹⁸F‐FDG, or mouse isotype ¹³¹I‐IgG, respectively. Whole‐body phosphor‐autoradiography and ex vivo biodistribution studies were obtained. Whole‐body phosphor‐autoradiography showed ¹³¹I‐anti‐TLR5 mAb uptake into organs that were well perfused with blood at 1 hr and showed clear graft images from 12 hrs onwards. The ¹³¹I‐anti‐TLR5 mAb had significantly higher graft uptake and target‐to‐non‐target ratio in the allo‐treated group, as determined by semi‐quantification of phosphor‐autoradiography images; these results were consistent with ex vivo biodistribution studies. However, high ¹⁸F‐FDG uptake was not observed in the allo‐treated group. The highest allograft‐skin‐to‐native‐skin ratio (A:N) of ¹³¹I‐anti‐TLR5 mAb uptake was significantly higher than the ratio for ¹⁸F‐FDG (7.68 versus 1.16, respectively). ¹³¹I‐anti‐TLR5 mAb uptake in the grafts significantly correlated with TLR5 expression in the allograft area. The accumulation of ¹³¹I‐IgG was comparable in both groups. We conclude that radiolabelled anti‐TLR5 mAb is capable of detecting allograft with high target specificity after treatment with the immunosuppressive drug rapamycin.     

GABA uptake in embryonic palate mesenchymal cells of two mouse strains

To obtain further evidence that the inhibitory neurotransmitter GABA functions in palate development, the presence of an active GABA uptake mechanism was sought using primary cultures of embryonic palate mesenchymal cells. Uptake was compared from cells of two inbred mouse strains in which the SWV strain shows greater sensitivity than the AJ strain to effects of GABA on palate morphogenesis and of diazepam in producing cleft palate (1). Palate cells were capable of accumulating [³H]GABA by saturable uptake mechanisms characteristic of a high and a low affinity active transport as indicated by temperature, Na⁺ ion and carrier dependence as well asK _m andV _max values that were comparable to other biological systems. TheV _max of the high-affinity uptake system from cells of the SWV strain was 1.8 fold higher than that of the AJ. GABA uptake was also observed in fibroblasts from various sources including embryonic mouse limb cells, human skin fibroblasts and 3T3 cells When active GABA uptake was measured in skin fibroblasts from the mouse SWV and AJ strains, the rate of uptake from SWV cells under high affinity conditions was also 1.8 fold greater than in AJ cells. Thus active GABA uptake appears to be genetically regulated in non-neural cells which may contribute to differential resonses to GABA.     

Angiotensin II binding to tissues of the rainbow trout,Oncorhynchus mykiss,studied by autoradiography

Summary Tissue slices from seawater-adapted and freshwater-adapted rainbow trout, Oncorhynchus mykiss, were exposed to ¹²⁵I-angiotensin II (1.01·10^-9 M) and binding sites located by light-microscopic autoradiography. Binding/uptake was significantly inhibited by excess (10^-5 M) unlabelled angiotensin II, suggesting specific binding/uptake of angiotensin II to the ventral and dorsal aorta (smooth muscle), urinary bladder (smooth muscle and epithelial lining), glomeruli and proximal tubules, the gill (lamellae and central filament), skin (epithelium), intestine and oesophagus (mucosal epithelium), liver, heart (ventricular myocytes), adrenocortical tissue and brain (cerebellum and medulla oblongata). The specific binding/uptake of angiotensin II to tissues of freshwater- and seawater-adapted animals were generally similar. However, binding/uptake by the proximal tubules was significantly higher in freshwater-adapted trout than seawater-adapted trout. Specific binding/uptake of angiotensin II by the smooth muscle of the bladder was significantly higher in trout adapted to seawater than trout adapted to freshwater.     

Uptake of sulphate,taurine, cysteine and methionine by symbiotic and free-living dinoflagellates

Sulphate uptake by Amphidinium carterae, Amphidinium klebsii and Gymnodinium microadriaticum grown on artificial seawater medium with sulphate, cysteine, methionine or taurine as sulphur source occurred via an active transport system which conformed to Michaelis-Menten type saturation kinetics. Values for K _m ranged from 0.18–2.13 mM and V _max ranged from 0.2–24.2 nmol · 10⁵ cells^–1 · h^–1. K _m for symbiotic G. microadriaticum was 0.48 mM and V _max was 0.2 nmol · 10⁵ cells^–1 · h^–1. Sulphate uptake was slightly inhibited by chromate and selenate, but not by tungstate, molybdate, sulphite or thiosulphate. Cysteine and methionine (0.1 mM), but not taurine, inhibited sulphate uptake by symbiotic G. microadriaticum, but not by the two species of Amphidinium. Uptake was inhibited 45–97% under both light and dark conditions by carbonylcyanide 3-chlorophenylhydrazone (CCCP); under dark conditions sulphate uptake was 40–60% of that observed under light conditions and was little affected by 3-(3,4-dichlorophenyl) 1,1-dimethylurea (DCMU).The uptake of taurine, cysteine and methionine by A. carterae, A. klebsii, cultured and symbiotic G. microadriaticum conformed to Michaelis-Menten type saturation kinetics. K _m values of taurine uptake ranged from 1.9–10 mM; for cysteine uptake from 0.6–3.2 mM and methionine from 0.001–0.021 mM. Cysteine induced a taurine uptake system with a K _m of 0.3–0.7 mM. Cysteine and methionine uptake by all organisms was largely unaffected by darkness or by DCMU in light or darkness. CCCP significantly inhibited uptake of these amino acids. Thus energy for cysteine and methionine uptake was supplied mainly by respiration. Taurine uptake by A. carterae was independent of light but was inhibited by CCCP, whereas uptake by A. klebsii and symbiotic G. microadriaticum was partially dependent on photosynthetic energy. Taurine uptake by cultured G. microadriaticum was more dependent on photosynthetic energy and was more sensitive to CCCP. Cysteine inhibited uptake of methionine and taurine by cultured and symbiotic G. microadriaticum to a greater extent than in the Amphidinium species. Methionine did not greatly affect taurine uptake, but did inhibit cysteine uptake. Taurine did not affect the uptake of cysteine or methionine.     

The relationship of sodium uptake,potassium rejection,and skin potential in isolated frog skin

1. Isolated surviving frog skin, when bathed with the same kind of diluted Ringer's solution on both sides, shows a negative correlation between net active salt uptake by the epithelium and spontaneous skin potential. Average values of 0.15 to 0.86 µeq. x hr.^–1 x cm.^–2 were measured and correlated with average skin potentials ranging from 107 to 25 mv. 2. Sodium uptake exceeded chloride uptake by about the same amount, irrespective of the height of the skin potential. 3. The same skins which exhibited a negative correlation between net uptake of sodium chloride and skin potential showed a positive correlation between net potassium rejection from the epithelium and skin potential, for voltages above 30 to 40 mv. In skins of voltages lower than this, potassium ions were taken up rather than rejected. Average values for rejection of +11.8 to –0.8 centi-µeq. x hr.^–1 x cm.^–2 were measured. 4. Net fluid uptake, associated with active uptake of sodium chloride, was small and occurred in the direction of the salt uptake. No dependence of net fluid uptake upon skin potential was observed. 5. Skins of winter frogs, pretreated with a commercial purified ACTH preparation, were less active than their respective controls with regard to uptake of sodium chloride. Rejection of potassium was the same in treated and untreated skins. Posterior pituitary factors, as possible contaminants, did not account for the effect of the ACTH preparation. 6. DOCA, DOC, and cortisone did not alter the normal correlation referred to under (1) and (3). 7. In interpreting the experimental results on theoretical grounds, it is suggested (a) that in normal skin, it is the variation in the electric conductance in skin of chloride ions which essentially, although not exclusively, determines the rate of net uptake of sodium chloride, (b) that a factor in the ACTH preparation used, possibly ACTH itself, may have lowered the electric conductance in skin of sodium ions either truly or apparently, (c) that potassium ions are treated by the skin primarily as passive ions. There is some indication that potassium ions are also actively taken up by the epithelium of skin.     

THE GLUCOSE TRANSPORT SYSTEM OF AMPHORA COFFEAEFORMIS (BACILLARIOPHYCEAE)1

Amphora coffeaeformis (Ag.) Kütz. var. perpusilla (Grun.) Cleve took up glucose by an inducible transport system. The system was induced by d -fructose, d -mannose, as well as glucose. Some d -pentoses also induced a glucose uptake system but it may not be the same one as that induced by hexose. d -fructose, d -mannose and 2-deoxy-d -glucose inhibited 2 mM glucose uptake at equimolar concentration, but d -pentoses did not. The uptake system decayed in ca. 5 h in the absence of glucose. The half-saturation constant for uptake, K₈ was ca. 0.1 mM glucose with a maximum uptake rate, V_max= 0.4 nmol/10⁶ cells-min^?1.     

Factorial design based curcumin ethosomal nanocarriers for the skin cancer delivery: in vitro evaluation

Abstract

Melanoma is the most deadly and life-threatening form of skin cancer with progressively higher rates of incidence worldwide. The objective of the present investigation is to develop and to statistically optimize and characterize curcumin (CUR) loaded ethosomes for treatment of melanoma. A two factor, three level (3²) factorial design approach was employed for the optimization of ethosomes. The prepared ethosomes were evaluated for size, zeta potential, entrapment efficiency, in vitro skin permeation and deposition ability. The optimized ethosomal formulation was evaluated for in vitro cytotoxicity and cellular uptake studies using A375 human melanoma cells. The optimized formulation has imperfect round shaped unilamellar structures with a mean vesicle size of 247?±?5.25?nm and an entrapment efficiency of 92.24?±?0.20%. The in vitro skin permeation studies proved the superiority of ethosomes over the traditional liposomes in terms of the amount of drug permeated and deposited in skin layers. Fluorescence microscopy showed the enhanced penetration of ethosomes into the deeper layers of the skin. In vitro cytotoxicity and cellular uptake studies revealed that curcumin ethosomes have significantly improved cytotoxicity and cellular uptake in A375 human melanoma cell lines. The colony formation assay results showed that curcumin ethosomes have a superior antiproliferative effect as they effectively inhibit the clonogenic ability of A375 cells. The flow cytometry results indicate that curcumin ethosomes induce cell death in A375 cells by apoptosis mechanism. The present study provides a strong rationale and motivation for further investigation of newly developed curcumin ethosomes as a potential therapeutic strategy for melanoma treatment.     

Phosphate and calcium uptake in beech (Fagus sylvatica) in the presence of aluminium and natural fulvic acids

In the present study we examine the effects of Al on the uptake of Ca²⁺ and H₂PO^-₄ in beech (Fagus sylvatica L.) grown in inorganic nutrient solutions and nutrient solutions supplied with natural fulvic acids (FA). All the solutions used were chemically well characterized. The uptake of Al by roots of intact plants exposed to solutions containing 0, 0.15 or 0.3 mM AlCl₃ for 24 h, was significantly less if FA (300 mg l⁻¹) were also present in the solutions. The Ca²⁺(⁴⁵Ca²⁺) uptake was less affected by Al in solutions supplied with FA than in solutions without FA. There was a strong negative correlation between the Al and Ca²⁺ uptake (r²=0.98). When the Al and Ca²⁺ (⁴⁵Ca²⁺) uptake were plotted as a function of the Al³⁺ activity (or concentration of inorganic mononuclear Al), almost the same response curves were obtained for the -FA and +FA treatments. We conclude that FA-complexed Al was not available for root uptake and therefore could not affect the Ca²⁺ uptake. The competitive effect of Al on the Ca²⁺ uptake was also shown in a 5-week cultivation experiment, where the Ca concentration in shoots decreased at an AlCl₃ concentration of 0.3 mM. The effect of Al on H₂PO⁻₄ uptake was more complex. The P content in roots and shoots was not significantly affected, compared with the control, by cultivation for 5 weeks in a solution supplied with 0.3 mM AlCl₃, despite a reduction of the H₂PO⁻₄ concentration in the nutrient solution to about one-tenth. At this concentration Al obviously had a positive effect on H₂PO⁻₄ uptake. The presence of FA decreased ³²P-phosphate uptake by more than 60% during 24 h, and the addition of 0.15 or 0.3 mM AlCl₃ to these solutions did not alter the uptake of ³²P-phosphate.     

Strontium-calcium discriminationin vitro by rat tissues

Uptake of⁸⁹Sr and⁴⁵Ca by 15 soft tissues of adult rat was studiedin vitro to assess the extent of discrimination between Sr and Ca. While brain, kidney, placenta and uterus have lower uptake of⁸⁹Sr and 45 Ca that of diaphragm, lactating mammary gland, skeletal muscle, skin, spleen and testes is higher. Tissues with medium range uptake are heart, small intestine, liver, lung, non-lactating mammary gland and ovary. The 6 tissues displaying discriminating ability, as expressed by⁸⁹Sr/⁴⁵Ca (tissue/medium), in the decreasing order are: small intestine, kidney, lactating mammary gland, placenta, diaphragm and heart. Non-lactating mammary gland and the other tissues did not differentiate between Sr and Ca. The efect of several enzyme inhibitors, compounds influencing Sr-Ca metabolism and other factors was studied in terms of the nature and mechanism of Sr-Ca discrimination.     

ATP-dependent strontium uptake by basolateral membrane vesicles from rat renal cortex in the absence or presence of calcium

ATP-dependent Sr²⁺ transport was examined in vitro using basolateral membrane (BLM) vesicles isolated from rat renal cortex to clarify the discrimination mechanisms between strontium (Sr) and calcium (Ca) in renal tubules during reabsorption. ATP-dependent Sr²⁺ uptake and Ca²⁺ uptake were observed in renal BLM vesicles and were inhibited by vanadate. Hill plots indicate similar kinetic behavior for Ca²⁺ and Sr²⁺ uptake. The apparentK _m andV _max of ATP-dependent Sr²⁺ uptake were both higher than those for Ca²⁺ uptake. ATP-dependent Sr²⁺ uptake by BLM vesicles diminished in the presence of 0.1 μM Ca²⁺ and was more markedly inhibited by 1 μM Ca²⁺. Hill plots of Sr²⁺ uptake data with and without 0.1 μM Ca²⁺ showed that the cooperative behavior of Sr²⁺ uptake was not changed by Ca²⁺. In the presence of 0.1 μM Ca²⁺, the affinity of the transport system for Sr²⁺ and the velocity of Sr²⁺ uptake in the BLM were both decreased. However, the rate of Ca²⁺ uptake was not diminished by Sr²⁺ concentrations of <1.6 μM. These results suggest that Ca²⁺ is preferentially transported in the renal cortex BLM when Ca²⁺ and Sr²⁺ are present at the same time.     

Iron and cadmium uptake by duodenum of hypotransferrinaemic mice

Absorption from food is an important route for entry of the toxic metal, cadmium, into the body. Both cadmium and iron are believed to be taken up by duodenal enterocytes via the iron regulated, proton-coupled transporter, DMT1. This means that cadmium uptake could be enhanced in conditions where iron absorption is increased. We measured pH dependent uptake of ¹⁰⁹Cd and ⁵⁹Fe by duodenum from mice with an in vitro method. Mice with experimental (hypoxia, iron deficiency) or hereditary (hypotransferrinaemia) increased iron absorption were studied. All three groups of mice showed increased ⁵⁹Fe uptake (p<0.05) compared to their respective controls. Hypotransferrinaemic and iron deficient mice exhibited an increase in ¹⁰⁹Cd uptake (p<0.05). Cadmium uptake was not, however, increased by lowering the medium pH from 7.4 to 6. In contrast, ⁵⁹Fe uptake (from ⁵⁹FeNTA₂) and ferric reductase activity was increased by lowering medium pH in control and iron deficient mice (p<0.05). The data show that duodenal cadmium uptake can be increased by hereditary iron overload conditions. The uptake is not, however, altered by lowering medium pH suggesting that DMT1-independent uptake pathways may operate.     

Halobetasol propionate-loaded solid lipid nanoparticles (SLN) for skin targeting by topical delivery

The clinical use of halobetasol propionate (HP) is related to some adverse effects like irritation, pruritus and stinging. The purpose of this work was to construct HP-loaded solid lipid nanoparticles (HP-SLN) formulation with skin targeting to minimizing the adverse side effects and providing a controlled release. HP-SLN were prepared by solvent injection method and formula was optimized by the application of 3² factorial design. The nanoparticulate dispersion was evaluated for particle size and entrapment efficiency (EE). Optimized batch was characterized for differential scanning calorimetry (DSC), scanning electron microscopy, X-ray diffraction study and finally incorporated into polymeric gels of carbopol for convenient application. The nanoparticulate gels were evaluated comparatively with the commercial product with respect to ex-vivo skin permeation and deposition study on human cadaver skins and finally skin irritation study. HP-SLN showed average size between 200?nm and 84–94% EE. DSC studies revealed no drug-excipient incompatibility and amorphous dispersed of HP in SLN. Ex vivo study of HP-SLN loaded gel exhibited prolonged drug release up to 12?h where as in vitro drug deposition and skin irritation studies showed that HP-SLN formulation can avoid the systemic uptake, better accumulative uptake of the drug and nonirritant to the skin compared to marketed formulation. These results indicate that the studied HP-SLN formulation represent a promising carrier for topical delivery of HP, having controlled drug release, and potential of skin targeting with no skin irritation.     

Rapid Effects of Abscisic Acid on Ion Uptake in Sunflower Roots

Short-term effects of ABA, ABA + kinetin and kinetin on ion (⁸⁶Rb-potassium and phosphate) and water uptake in sunflower plants (Helianthus annuus var. californicus) were examined with a continuous-recording technique. Ion uptake in the roots and transport to the shoots were also investigated by conventional tracer uptake experiments and by sap bleeding experiments with excised roots. After addition of 5 × 10^?6-4 × 10^?5M ABA to the root medium there was an immediate decrease (30–70%) in the rate of ion uptake which lasted 30–70 min. The rate of water uptake was not significantly affected as measured with this method. Ion transport to the shoots and to the bleeding sap of excised roots was decreased by ABA. ABA-induced inhibition of ion uptake was abolished by the presence of kinetin, and uptake was slightly stimulated by 2 × 10^?5M kinetin alone. We suggest that concentration gradients of ABA or rapid changes in the ABA-kinetin balance in the roots affect ion uptake and transport.     

Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy

In the present study a new luminescent dye 3‐N‐(2‐pyrrolidinylacetamido)benzanthrone (AZR) was synthesized. Spectroscopic measurements of the novel benzanthrone 3‐aminoderivative were performed in seven organic solvents showing strong fluorescence. The capability of the prepared dye for visualization has been tested on flax, red clover and alfalfa to determinate the embryo in plant callus tissue cultures. Callus cells were stained with AZR and further analysed utilizing confocal laser scanning fluorescence microscopy. Performed experiments show high visualization effectiveness of newly synthesized fluorescent dye AZR that is efficient in fast and relatively inexpensive diagnostics of callus embryos that are problematic due to in vitro culture specificity.     

Potassium and Sodium Fluxes in the PhytopathogenicFungus Fusarium oxysporum var. lini

Rubidium uptake in potassium-starved cells followed biphasic kinetics in the micromolar and millimolar range and was independent of the temperature. In contrast, Rb⁺ uptake in normal-K⁺ cells followed a monophasic kinetics in the millimolar range and increased at temperatures higher than 30°C. Differences in the K _m values and in the Arrhenius plots of Rb⁺ uptake suggest different uptake systems in K⁺-starved and in normal-K⁺ cells. In addition, the substantial inhibition of Rb⁺ uptake caused by carbonyl cyanide-m-chlorophenyl hydrazone indicates that these systems are strongly dependent on membrane voltage. Lithium (sodium) tolerance, influx, and efflux were separately studied. F. oxysporum was shown to be very tolerant to sodium, while lithium caused a specific toxic effect. Li⁺ uptake in K⁺-starved cells exhibits a monophasic kinetics with low affinity. Li⁺ efflux was not affected by external pH or addition of potassium to the medium, suggesting that a Na⁺/cation antiporter is not involved in this process. Received: 14 March 2000 / Accepted: 5 June 2000     

Modeling of Cellular Arginine Uptake by More Than One Transporter

Determining the kinetic constants of arginine uptake by endothelial cells mediated by more than one transporter from linearization of data as Eadie-Hofstee plots or modeling which does not include the concentration of trace radiolabeled amino acid used to measure uptake may not be correct. The initial rate of uptake of trace [³H]l-arginine by HUVECs and ECV₃₀₄ cells in the presence of a range of unlabeled arginine and modifiers was used in nonlinear models to calculate the constants of arginine uptake using GraphPad Prism. Theoretical plots of uptake derived from constants determined from Eadie-Hofstee graphs overestimated uptake, whereas those from the nonlinear modeling approach agreed with experimental data. The contribution of uptake by individual transporters could be modeled and showed that leucine inhibited the individual transporters differently and not necessarily competitively. N-Ethylmaleimide inhibited only y⁺ transport, and BCH may be a selective inhibitor of y⁺L transport. The absence of sodium reduced arginine uptake by y⁺L transport and reduced the K _m′, whereas reducing sodium decreased arginine uptake by y⁺ transport without affecting the K _m′. The nonlinear modeling approach using raw data avoided the errors inherent in methods deriving constants from the linearization of the uptake processes following Michaelian kinetics. This study provides explanations for discrepancies in the literature and suggests that a nonlinear modeling approach better characterizes the kinetics of amino acid uptake into cells by more than one transporter.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

Evidence for the role of intracellular glutamine level in the regulation of glutamine uptake in the cyanobacteriumAnabaena 7120

The role of intracellular glutamine concentration in the regulation of¹⁴C-glutamine uptake was studied in a diazotrophic cyanobacteriumAnabaena 7120. The uptake pattern was found to be biphasic, consisting of a rapid first phase lasting up to 60 s followed by a slower second phase. Azaserine, which could not inhibit in vitro and in vivo glutamine synthetase (GS) activity effectively, inhibited the¹⁴C-glutamine uptake. Glutamine uptake was also not significantly affected when glutamate, methylglutamate, aspartate, arginine, lysine, hydroxylysine, ornithine, and GS inhibitor,L-methionine-DL-sulfoximine (MSX) were simultaneously available during uptake assay, suggesting that glutamine uptake takes place via a general amino acid permease which does not, however, transport basic and acidic amino acids. The azaserine-treated cells had increased and decreased levels of glutamine and glutamate, respectively, suggesting that the increased intracellular glutamine level is responsible for the inhibition of¹⁴C-glutamine uptake and provides evidence here for the role of an intracellular glutamine pool in the regulation of¹⁴C-glutamine uptake inAnabaena 7120.     

Sodium-dependent uptake of taurine in encapsulated Staphylococcus aureus strain M

A novel uptake system for the unusual sulfonated amino acid taurine was discovered in the prokaryote, encapsulated Staphylococcus aureus strain M. This strain has been shown previously to contain taurine in its capsular polysaccharide. Taurine uptake by whole cells incubated in buffer showed a saturable dependency upon Na⁺ and taurine uptake was itself a saturable process, stimulated by glucose, and markedly affected by temperature. No evidence was found for the inducibility of taurine uptake. In the presence of 10 mM NaCl Lineweaver-Burk plots revealed a K_m of 42 μM and V_max of 4.6 nmol/min per mg dry weight for taurine uptake at 37°C. Increasing concentrations of Na⁺ decreased the K_m of the system and appeared to increase the V_max. Of various other cations tested only Li⁺ supported marked taurine uptake. Excess unlabelled taurine did not cause efflux of radioactivity taken up. Taurine was taken up into cold trichloroacetic acid-soluble material and did not chromatograph as taurine, indicating rapid metabolism during or closely following uptake. Taurine uptake appeared to occur via a highly specific system because amino acids representing the major known groups of amino acid transport systems in S. aureus did not inhibit taurine uptake, and uptake was only slightly diminished by the structurally closely related compounds hypotaurine and 3-amino-1-propane sulfonic acid. Sulfhydryl group reagents, electron transport inhibitors, an uncoupler and inhibitors of Na⁺-linked transport processes inhibited taurine uptake. A variety of other metabolic inhibitors had little effect on taurine uptake.     

Developmental changes in the45Ca2+ uptake byTrichoderma viride mycelium

The rate of the⁴⁵Ca²⁺ uptake by the submergedTrichoderma viride mycelium increased with the age of the culture from 6 h until a maximum which was reached at about 30 h, and then decreased until the uptake was virtually zero. The decrease in the rate of the⁴⁵Ca²⁺ uptake was accompanied by an increase of mycelial mass. The uptake rate could not be reactivated upon substituting the medium for a fresh one, without or with dilution of the mycelium. The results suggest that the rate of⁴⁵Ca²⁺ uptake reflects the biological age of the submerged culture. The surface-cultivated mycelium took up⁴⁵Ca²⁺ proportionally with time. The autoradiography of colonies showed that⁴⁵Ca²⁺ was distributed homogeneously throughout the mycelium during vegetative growth while conidiation was accompanied by a massive accumulation of⁴⁵Ca²⁺ in conidia. This work was supported by theSwiss National Science Foundation (joint Swiss-Slovak project 75LPJ041485) andSlovak Grant Agency (grant no. 1/1158/93).