High-sensitivity cytofluorometric quantitation of lectin and hormone binding to surfaces of living cells.

With a specially equipped flow cytofluorometer it is possible to determine quickly and accurately binding constants and the maximum number of binding sites for ligands such as peptide hormones and lectins on surfaces of intact living cells, with incubation concentrations as low as 10^?11 M. Since the measurement is confined to cell-bound material the cells can be kept in their physiological environment, including free ligand molecules, even at the very moment of the assay. Thus there is no additional risk of perturbing the integrity of the membrane or of interfering with ligand-receptor interactions by washing or similar procedures. It was found that damaged cells, inevitably present in any population, are able to grossly distort binding patterns. Suggestions are given how such cells may be excluded from the measurement.     

Binding of chondroitin sulfate, dermatan sulfate and fat-storing cell-derived proteoglycans to rat hepatocytes

1. The interaction of isolated rat hepatocytes with exogenous 3H-labeled chondroitin-4-sulfate and dermatan sulfate and with biosynthetically 35S-labeled proteoglycans secreted by cultured rat liver fat-storing cells has been studied. 2. All ligands are bound by hepatocytes in a concentration-dependent manner. Scatchard-plot analysis of the data revealed the existence of high- and low-affinity binding modes. 3. The cell-bound exogenous [3H]glycosaminoglycans could be displaced by each unlabeled ligand and by heparin, whereas displacement of the endogenous material was less effective. 4. Binding of all ligands to hepatocytes increased with time. For the exogenous glycosaminoglycans the two- to threefold amount was retained at 37 degrees C as compared to 4 degrees C; it was markedly reduced by pretreatment of the cells with trypsin. 5. Degradation of the exogenous ligands could be detected neither for the cell-bound fraction nor for the free glycosaminoglycans in the culture medium. 6. The binding of the ligands to hepatocytes is viewed as a cell-matrix interaction. Its possible pathobiochemical relevance in liver fibrosis or neoplasia is discussed.     

The fate of IgE bound to rat basophilic leukemia cells. II. Endocytosis of IgE oligomers and effect on receptor turnover

We have assessed the internalization of variously sized oligomers of IgE bound to rat basophilic leukemia (RBL) cells by measuring their accessibility to the extracellular environment, and by direct visualization of the radiolabeled ligands. We also followed the fate of the internalized ligands and their receptors, as well as the fate of the free receptor on cells internalizing oligomers. In contrast to monomeric IgE, surface-bound oligomeric IgE was internalized. Notably, dimers provided an effective signal for internalization, although larger oligomers seem to be internalized more efficiently. In our experiments, 48% of the cell-bound dimers and 67% of the trimers were eliminated from the cell surface in 180 min. One-half of the maximal internalization observed with dimers and trimers occurred in 25 and 11 min, respectively. Release of radioactivity into the supernatant followed internalization; the released radioactivity did not bind to fresh cells and was only partially TCA-precipitable. Radioactive ligands remaining associated with the cells were unchanged as judged by m.w; they also were shown to remain receptor-bound. During either internalization or release of substantial amounts of the originally cell-bound oligomers, there was no increase in IgE-binding activity. In contrast, there was a transient drop (25%) in the number of free surface receptors suggesting internalization of the free receptors together with the oligomer-occupied receptor. Cells that failed to release histamine (RBL-I) processed dimeric and trimeric IgE similarly to histamine-releasing (RBL-2H3) cells. We conclude that dimeric and trimeric IgE are internalized by RBL cells and later are released to the medium in a partially degraded form. The ligand-bound receptor seems to be internalized with the ligand, along with some free receptor, and does not appear to be reusable or to recycle rapidly to the cell surface.     

Re-evaluating the effects of organic ligands on copper toxicity to barley root elongation in culture solution

Abstract

In the presence of weak ligands, both free ion activity and organic complexes of Cu should b considered when predicting Cu toxicity in aquatic and soil-plant systems. However, there is littl information about the quantitative contribution of Cu that is organically complexed to Cu toxicity. In thi study, a bioassay using barley root elongation in culture solution was used to investigate the effects o organic ligands with different conditional stability constants on Cu toxicity and the quantitativ contribution of the organically complexed Cu to the Cu toxicity. The results indicated that a significan decrease (p<0.05) in Cu toxicity, assessed by barley root elongation, was observed in response to th addition of organic ligands. The decrease differed, to some extent, with different organic ligands o disodium ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), oxalate and malate at low and constant free Cu²⁺ activity. Addition of EDTA or NTA resulted in strong reduction of Cu toxicity while modest reduction of Cu toxicity was observed for the addition of malate as the relatively wea ligand. Furthermore, the results of the present study revealed that the CuNTA^? and CuEDTA^2? complexes were not toxic, while the Cu–malate complexes were mildly toxic to barley root elongation More importantly, it was found that the toxicity of Cu–malate complexes were nearly 0.5-fold less than that of free Cu²⁺ ions.     

Harvesting Solar Energy by Means of Charge-Separating Nanocrystals and Their Solids

Conjoining different semiconductor materials in a single nano-composite provides synthetic means for the development of novel optoelectronic materials offering a superior control over the spatial distribution of charge carriers across material interfaces. As this study demonstrates, a combination of donor-acceptor nanocrystal (NC) domains in a single nanoparticle can lead to the realization of efficient photocatalytic^1-5 materials, while a layered assembly of donor- and acceptor-like nanocrystals films gives rise to photovoltaic materials.Initially the paper focuses on the synthesis of composite inorganic nanocrystals, comprising linearly stacked ZnSe, CdS, and Pt domains, which jointly promote photoinduced charge separation. These structures are used in aqueous solutions for the photocatalysis of water under solar radiation, resulting in the production of H₂ gas. To enhance the photoinduced separation of charges, a nanorod morphology with a linear gradient originating from an intrinsic electric field is used⁵. The inter-domain energetics are then optimized to drive photogenerated electrons toward the Pt catalytic site while expelling the holes to the surface of ZnSe domains for sacrificial regeneration (via methanol). Here we show that the only efficient way to produce hydrogen is to use electron-donating ligands to passivate the surface states by tuning the energy level alignment at the semiconductor-ligand interface. Stable and efficient reduction of water is allowed by these ligands due to the fact that they fill vacancies in the valence band of the semiconductor domain, preventing energetic holes from degrading it. Specifically, we show that the energy of the hole is transferred to the ligand moiety, leaving the semiconductor domain functional. This enables us to return the entire nanocrystal-ligand system to a functional state, when the ligands are degraded, by simply adding fresh ligands to the system⁴.To promote a photovoltaic charge separation, we use a composite two-layer solid of PbS and TiO₂ films. In this configuration, photoinduced electrons are injected into TiO₂ and are subsequently picked up by an FTO electrode, while holes are channeled to a Au electrode via PbS layer⁶. To develop the latter we introduce a Semiconductor Matrix Encapsulated Nanocrystal Arrays (SMENA) strategy, which allows bonding PbS NCs into the surrounding matrix of CdS semiconductor. As a result, fabricated solids exhibit excellent thermal stability, attributed to the heteroepitaxial structure of nanocrystal-matrix interfaces, and show compelling light-harvesting performance in prototype solar cells⁷.     

Properties of free and bound Citrobacter freundii lipopolysaccharides

Culture medium content of free lipopolysaccharide (LPS) components spontaneously released from a Citrobacter freundii culture grown in minimum synthetic medium was determined during early (8-hr culture) and late (24-hr culture) phases of growth. As judged by Limulus-lysate test, free LPS occurred in the medium as early as after 8 hrs of incubation, i.e. at the beginning of log growth phase. As the culture continued to grow the LPS amount released into culture medium kept rising, reaching 30% of endotoxin present in 24-hr Citrobacter culture. The released LPS complex was isolated by separation and its physicochemical, immunochemical and biological properties were determined and compared with those of cell-bound endotoxin recovered from cells by phenol extraction. Comparisons revealed distinct differences in the chemical composition and the degree of heterogeneity; free LPS was less heterogeneous. Immunologically, free LPS differed from bound LPS in the structure of macromolecules, but was identical with it in some antigenic determinants. The biological activity of free LPS preparation was greater than that of cell-bound LPS.     

Triiodothyronine bound to red blood cells is not available for transport through the blood-brain barrier

Many steroid and thyroid hormones and some drugs are bound by circulating red cells. Red cell-bound ligand may not be physiologically inert, as recent studies show that red cell-bound drug is available for uptake by brain. To investigate whether triiodothyronine (T₃) is available for uptake by brain in vivo from the circulating red cell pool, the present investigations measure the effects of human erythrocytes on rat brain uptake of [¹²⁵I]T₃ in vivo. The fraction of circulating T₃ available for uptake in vivo in the presence of 0, 2, 5, 10, 22, or 44% red cells was essentially identical to the fraction of [¹²⁵I]T₃ unbound in vitro. Therefore, [¹²⁵I]T₃ bound to red cells obtained from normal volunteers is not available for uptake by brain in vivo.     

Incorporation of fucose in the intact heart and dissociated heart cells of the chick embryo

The incorporation of [³H]fucose into cell-bound and medium-released TCA-precipitable fractions was determined in intact hearts and dissociated heart cells of the 4-day chick embryo. The amount of released label was found to be much greater in the dissociated cells than in intact hearts both in absolute quantities and in proportion to cell-bound label.     

Nuclear condensation and free radical scavenging: a dual mechanism of bisbenzimidazoles to modulate radiation damage to DNA

The complexing of histones with DNA and the resulting condensation of chromatin protects mammalian cell, from radiation-induced strand breakage. In the present study, benzimidazoles DMA and TBZ showed marked radioprotection through drug-induced compaction of chromatin and direct quenching of free radicals generated by radiation. The mammalian cells were incubated with 100 μM concentration of DMA and TBZ and irradiated at 5 Gy; both the ligands showed nuclei condensation suggesting a probable mechanism to protect DNA from radiation damage. The bisubstituted analogs of Hoechst 33342 are found to be better free radical scavengers and protect DNA against radiation-induced damage at a lower concentration than the parent molecule. Both the ligands also quenched free radicals in isolated free radical system suggesting their dual mode of action against radiation-induced damage to DNA. Molecules binding to the chromatin alter gene expression, whereas in this study both the ligands have not shown any profound effect on the nucleosome assembly and gene expression in vitro and in vivo. Both ligands afford a 2-fold protection by altering DNA structure as well as through direct free radical quenching in bulk solution in comparison to the parent ligand, which acts only through quenching of free radicals. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tumor Necrosis Factor Receptor Expression in HIV-1-Infected CD4+ T Cells

We investigated whether HIV-1 can regulate tumor necrosis factor receptor (TNFR) expression in SupT-1, a CD4 + T-cell line. The cells were infected with HIV-1 containing 1,000 cpm RT activity, as early as day 3 after infection and all along the culture the supernatant level of core protein p24 was >250 pg/ml, and on days 6 and 9 after infection, p24 was found in 10 % of the cells as determined by indirect immunofluorescence assay. The cells were growing without loss of viability. The study of TNFR expression was based on a microassay for measurement of binding of ¹²⁵I-TNFα to cells, in which free and cell-bound ligand separation was performed by centrifugation through oil. Scatchard analysis of TNFα binding on days 6 and 9 after infection revealed a 90 % increase in the expression of high-affinity membrane receptors in HIV + SupT-1 culture compared with uninfected cells (mean +/-S.D. = 501 +/-148.5 vs. 263 +/-77.8 receptors/cell, n = 9, P< 0.001) with no change in dissociation constants (mean +/? S.D. = 4.36 +/?1.06 vs. 4.00 +/?1.12 × 10^?10 m ).     

High activity extracellular glucose (xylose) isomerase from a Chainia species

Summary A sclerotia-forming actinomycete of the genus Chainia secreted high levels of glucose (xylose) isomerase when grown in submerged culture on a wheat bran - yeast extract medium. Maximum activity (4 units/ml) was obtained after 3–4 days when the cell bound activity was 0.19 units/ml. The two enzymes differed significantly in pH optima (extracellular, 9.5; cell-bound, 7.0) and in their adsorption behaviour on CM and DEAE celluloses. Both Mg⁺⁺ and Co⁺⁺ are required by the cell-bound enzyme for its optimum activity while either Mg⁺⁺ or Co⁺⁺ is necessary for the extracellular enzyme.NCL Communication 3320     

Negative and positive ion chemical ionization mass spectrometry of dioximes and their nickel complexes

The negative ion mass spectra of Ni(LH)₂ (where LH₂ is glyoxime, methylglyoxime, dimethylglyoxime and diphenylglyoxime), in the presence of ammonia or methane at 0.5 torr, are reported and compared with the spectra of the free ligands. In each case, the base peak of the complex is the molecular negative ion and the extent of fragmentation was found to decrease gradually going from the glyoximato to the diphenylglyoximato derivative. In the chemical ionization mass spectra of the free ligands, the [M]⁻ ion is absent in all cases. The base peak is [M  H]⁻ for methylglyoximine, dimethylglyoxime and diphenylglyoxime and [M  H  H₂]⁻ for glyoxime. The fragmentation occurs largely by loss of H, OH, H₂O and NO species. The positive ion chemical ionization mass spectra of the same complexes show very abundant [M + H]⁺ and [M]⁺ ions and weak fragments, whilst a rather high fragmentation is observed for the corresponding free ligands.     

Retention of intact concanavalin A and Lens culinaris lectin in transformed lymphoblasts during mitogenic response.

The fate of cell bound mitogens during mitogenic response of mouse cortisone resistant thymocytes (CRT) was studied using ¹²⁵I-concanavalin A (Con A) and ¹²⁵I-Lens culinaris lectin (LcH). A majority of autoradiographic grains derived from ¹²⁵I-lectins bound to a CRT population were distributed in a single broad peak. Pulse labeling of CRT with ¹²⁵I-Con A or ¹²⁵I-LcH in the initial 60 min of incubation followed by 48 hr of culture with unlabeled mitogens revealed that transformed lymphoblasts carried over half of cell-bound ¹²⁵I-mitogens. ¹²⁵I-Con A and ¹²⁵I-LcH found in the lymphoblasts in the above pulse experiments were electrophoretically identical to the native mitogens. A significant loss of cell-bound ¹²⁵I-mitogens was observed only after extensive cell division.     

Binding and cytochemical detection of cell-bound concanavalin A

We have investigated the relationship between the total amount of cell-bound concanavalin A (con A), as determined in binding experiments with ³H-conA, and the amount of cell-bound conA detected with horseradish peroxidase on normal murine fibroblasts (3T3). By comparing prefixed and non-prefixed cell membranes a discrepancy was found between the amount of cell-bound conA and the amount of cytochemically detected conA. This discrepancy was interpreted to substantiate the theory that conA binding sites can move within the membrane. Incubation of non-prefixed cells with conA induced redistribution of binding sites on the cell membrane. The redistribution resulted in changes in detectability of conA by horseradish peroxidase. The use and limitations of horseradish peroxidase in the study of cell transformation and of changes in agglutinability by conA are discussed.     

Induction of immunity and specific unresponsiveness in vitro by cell-bound antigen: I. Symmetry in enhancement of both responses

Pulse treatment of lymphoid cells from rabbits with solubilized antigens from T2 phage results in the firm binding of small but highly active amounts of antigen. Binding of phage antigens to viable, nonviable, or disrupted cells enhances their ability to evoke antibody formation or specific unresponsiveness in the primary in vitro response of rabbit spleen cells. Transfer of sonicate containing the equivalent of 10₂ to 10₃ antigen-pulsed cells carrying 10^?8 to 10^?7 μg phage protein nitrogen into spleen cell cultures regularly evokes antibody formation, while introduction to such cultures of 10^?3 μg phage protein nitrogen in cell-bound form evokes unresponsiveness. These findings indicate a 10- to 100-fold amplification of tolerogenic and immunogenic activities of cell-bound over soluble T2 antigen.     

The effects of cell-released protease nexin on the measurement of thrombin binding to mouse cells

We previously showed that fibroblast-like cells release protease nexin into their growth medium. Protease nexin links to thrombin and mediates the cellular binding of thrombin via the protease nexin part of the complex to a site different from that for unlinked thrombin (1,2). To determine the effect that cell-released protease nexin had on the measurement of total cell-bound thrombin, we separately measured the cellular binding of both ¹²⁵I-thrombin and ¹²⁵I-thrombin-protease nexin complexes. Scatchard analysis of our binding data indicates that the cellular binding affinity of linked ¹²⁵I-thrombin is about 19-fold higher than that of unlinked ¹²⁵I-thrombin. We show that protease nexin acts to increase the apparent affinity of ¹²⁵I-thrombin for cellular binding sites.     

β-Adrenergic stimulation inhibits calcium efflux and alters the morphology of cultured arterial muscle cells

β-Adrenoreceptor stimulation with isoproterenol (IP) rapidly and reversibly rounded and arborized smooth muscle cells (SMC) cultured from rat aorta. The arborized SMC remained firmly attached to the substratum via a network of long, dendritelike processes. Arborization of the SMC correlated closely with increases in cellular cAMP produced by IP and a variety of other compounds. The intracellular calcium antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) also rounded and arborized the SMC. Antitubulin compounds potently blocked arborization by IP, dibutyryl cAMP, and TMB-8. The release of cell-bound ⁴⁵Ca²⁺ was followed in the presence and absence of IP. IP strikingly increased the amount of ⁴⁵Ca²⁺ that remained cell bound between 20 and 120 min Propranolol and colchicine prevented IP from inhibiting the release of cell-bound ⁴⁵Ca²⁺. These results suggest that the modulation of Ca²⁺ transport is involved in the arborization of cultured SMC by cAMP.     

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T₂RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T₂RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T₂RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T₂RB3, T₂Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.     

Effect of lipids and detergents on the production and solubilization of extracellular lipase inYarrowia lipolytica

Triglycerides, oleic acid but not fatty acid-containing, nonionic detergents (Spans, Tweens) were able to stimulate the synthesis of cell-bound and soluble lipase ofYarrowia lipolytica grown in a complex medium containing citrate and urea. The optimal concentration of olive oil for induction was 0.5% (W/V). The combined effect of a high ionic strength (0.75 mol/L KCl) and of digitonin (2 mmol/L) at pH 7.6 resulted in solubilization of 80% of the cell-bound lipase and a significant activation of the enzyme. Comparison of twoY. lipolytica strains showed the effects of Mg²⁺ and Fe³⁺ concentrations in the medium on the synthesis of the enzyme to be strongly strain-dependent.