Free Con Research Articles

Preadsorption Effect of Carbon Monoxide on Reactivity of Cobalt Cluster Cations toward Hydrogen.

We report gas-phase reactions of free Con(CO)m+ (n = 3-11, m = 0-2) with H2, expecting a catalytic reaction of coadsorbed CO and H2 on Con+. Preadsorption of CO molecules is found to promote H2 adsorption, in particular, on Con(CO)+ (n = 5, 8-10). Density functional theory (DFT) calculations reveal that the reactivity is governed by the molecular-orbital energy of Con+, which is tuned by preadsorbed CO molecules. Collision-induced-dissociation experiments performed on ConCOH2+ (n = 8-10) imply that at least some of the CO and H2 molecules are bound together on Con+.

Acid phosphatases in seeds and developing of squash (Cucurbita ficifolia)

Changes in protein content and acid phosphatase activity were followed during germination (imbition through seedlings development) in extracts from cotyledons of squash (<i>Cucurbita ficifolia</i>). It has been shown that the activity of acid phosphatase was initially low and than increased to a maximum after 6 days of imbition. Acid phosphates were isolated from cotyledons of seeds and from 6-, 10- and 22-days old seedlings by extraction the proteins with 0.1 M acetate buffer pH 5.1, precipitation with ethanol and by affinity chromatography on con A-Sepharose. Two glycoprotein enzymes AcPase Ba and AcPase Bb which differ in their affinity to immobilized con A were obtained. Both acid phosphatates retained the enzyme activity after binding to free con A. Rocket affinity electrophoresis of AcPase Ba and AcPase Bb, isolated from cotyledons of seeds and seedlings, revealed differences in their ability to bind to con A during seeds germination and seedling develop-ment indicating changes in their sugar component. Con A was found to activate both enzymes. The enzymes cross-reacted with monospecific antibodies raised against grass seed acid phosphatate Ba indicating an antigenic relationship between squash and grass acid phosphatases.

염화제이철 수용액에 의한 스테인레스 강판의 식각에 관한 연구

염화제이철 수용액에 의한 스테인레스 강판의 식각반응에 관한 연구를 수행하였다. 식각액 교반속도, 식각온도, Fe3+ 이온 농도, 식각액의 유리산도 및 비중 등 여러 공정변수가 식각속도에 미치는 영향에 대해 조사하였고, 그 결과, AK(aluminum-killed) 철, 크롬(chlomium) 강, 그리고 스테인레스 강(STS430J1L 합금)의 식각반응이 1차 반응식을 잘 따름을 알 수 있었다. 또한 식각액의 피로도(fatigue ratio)가 16％ 이하로 유지되면 식각액 내에 슬러지가 발생하지 않으며, 식각된 강판의 표면거칠기 또한 좋아짐을 알 수 있었다. 본 연구에서 조사한 범위 내에서는, 식각액의 보메(Baume) 가 증가하면 식각속도는 감소하나 유리산도가 식각속도에 미치는 영향은 미비함을 알 수 있었다. 한편 본 연구의 실험 결과는 공정모델링을 통해 유도된 식각속도식의 계산결과와 잘 일치함을 알 수 있었다.

On the Role of Water Models in Quantifying the Binding Free Energy of Highly Conserved Water Molecules in Proteins: The Case of Concanavalin A.

The ability of ligands to displace conserved water molecules in protein binding sites is of significant interest in drug design and is particularly pertinent in the case of glycomimetic drugs. This concept was explored in previous work [ Clarke et al. J. Am. Chem. Soc. 2001 , 123 , 12238 - 12247 and Kadirvelraj et al. J. Am. Chem. Soc. 2008 , 130 , 16933 - 16942 ] for a highly conserved water molecule located in the binding site of the prototypic carbohydrate-binding protein Concanavalin A (Con A). A synthetic ligand was designed with the aim of displacing such water. While the synthetic ligand bound to Con A in an analogous manner to that of the natural ligand, crystallographic analysis demonstrated that it did not displace the conserved water. In order to quantify the affinity of this particular water for the Con A surface, we report here the calculated standard binding free energy for this water in both ligand-bound and free Con A, employing three popular water models: TIP3P, TIP4P, and TIP5P. Although each model was developed to perform well in simulations of bulk-phase water, the computed binding energies for the isolated water molecule displayed a high sensitivity to the model. Both molecular dynamics simulation and free energy results indicate that the choice of water model may greatly influence the characterization of surface water molecules as conserved (TIP5P) or not (TIP3P) in protein binding sites, an observation of considerable significance to rational drug design. Structural and theoretical aspects at the basis of the different behaviors are identified and discussed.

Regularization by Free Additive Convolution, Square and Rectangular Cases

The free convolution is the binary operation on the set of prob- ability measures on the real line which allows to deduce, from the individual spectral distributions, the spectral distribution of a sum of independent uni- tarily invariant square random matrices or of a sum of free operators in a non commutative probability space. In the same way, the rectangular free con- volution λ allows to deduce, from the individual singular distributions, the singular distribution of a sum of independent unitarily invariant rectangular random matrices. In this paper, we consider the regularization properties of these free convolutions on the whole real line. More specifically, we try to find continuous semigroups (μt) of probability measures such that μ0 = δ0 and such that for all t> 0 and all probability measure ν, μtν (or, in the rectan- gular context, μtλν) is absolutely continuous with respect to the Lebesgue measure, with a positive analytic density on the whole real line. In the square case, for , we prove that in semigroups satisfying this property, no measure can have a finite second moment, and we give a sufficient condition on semi- groups to satisfy this property, with examples. In the rectangular case, we prove that in most cases, for μ in a λ-continuous semigroup, μλν either has an atom at the origin or doesn't put any mass in a neighborhood of the origin, and thus the expected property does not hold. However, we give suffi- cient conditions for analyticity of the density of μλν except on a negligible set of points, as well as existence and continuity of a density everywhere.

Most effective saccharide inhibitors of immobilized concanavalin A‐cell binding

This laboratory has been using a derivatized bead assay to study cell surface properties in many experimental systems. Here we test its efficacy in the yeast/concanavalin A (Con A) binding model to determine if immobilized Con A is inhibited by the same saccharides that inhibit free Con A binding. This system offers an excellent test case for the efficacy and validity of the derivatized bead assay. Con A binds to yeast because yeast are rich in cell surface mannose residues, a Con A preferential binding sugar. Here we examine the effects of 30 different sugars (0.05 M) on the binding of yeast to agarose beads derivatized with Con A, in a total of 3998 trials, with an average of over 133 replicates for each sugar. The most inhibitory sugars included D(+) melezitose, D(+) trehalose and maltotriose, the same sugars that effectively inhibit binding of free Con A. The results suggest that the bead assay is an effective approach to study the binding properties of cells, and most important, it is so rapid that hundreds of trials can be done in the time it would take to do one trial using conventional assays (supported by NIH NIGMS SCORE, RISE, MARC, the ITQ program, and the Joseph Drown Foundation).

Electronic Properties of Small Free Co Clusters

We present a systematic study of the average magnetic moments and the non-metal–metal transition of free CoN clusters having different geometries: hexahedral, octahedral and decahedral. The electronic structure is calculated with a parameterized Hubbard Hamiltonian with spd electrons within the unrestricted Hartree-Fock approximation, and spill-over effects are considered. We compare our calculations with other theoretical calculations in the literature. We also comment on the experimental results.

Glycan microheterogeneity of α1-antitrypsin in serum and meconium from normal and cystic fibrosis patients by crossed immunoaffinoelectrophoresis with different lectins (Con A, LCA, WGA)

In order to test whether abnormalities of glycosylation occur in cystic fibrosis (CF), the glycan microheterogeneity of α 1-antitrypsin ( α 1-AT) was studied in serum and meconium from normal individuals and patients with cystic fibrosis, by crossed immunoaffinoelectrophoresis (CIAE) using free Concanavalin A (Con A), Lens culinaris lectin (LCA) and wheat germ agglutinin (WGA). Thre3 main results emerged from this study: (1) modification of glycosylation in serum α 1-AT from patients with cystic fibrosis were only significant with free Con A and WGA; this probably results from a reduced synthesis of the bi-antennary side-chains or by their increased catabolism. (2) Differences in isoforms found in α 1-AT from normal individuals and patients with CF using free Con A, LCA, were more pronounced in the meconium than in the serum; this may provide a useful test in diagnosis of cystic fibrosis. (3) There was parallelism between the behaviour of α 1-AT in serum and meconium from patients with CF using LCA, Con A; this may be explained by different types of levels of disfunction affecting a glycosylation mechanism.

Concanavalin A induces neural tissue and cartilage in amphibian early gastrula ectoderm

We have studied in vitro differentiation of explants of the amphibian ( Rana temporaria) early gastrula ectoderm after treatment with various concentrations (50–300 μg/ml) of ‘free’ and Sepharose-bound concanavalin A (Con A). The explants were incubated with Con A for 3 h at 20°C; the rolling up of the explants was prevented by using special weights. We have demonstrated that: (1) free Con A has an inducing action on the explants in the concentration range 100–300 μg/ml medium; (2) when treated with Con A the explants produce neural tissue (50–70%), cartilage (20–40%) and, rarely, lentoids (5–10%); (3) the frequency of neural and cartilage inductions was similar at various Con A concentrations; (4) α-methyl- d-mannoside pyranoside inhibited the Con A effects; (5) Sepharose-bound Con A had no effect on the explants, although it was bound to the cell surface of the ectoderm inner layer. Possible mechanisms of the neuralizing and chondrogenic effects of Con A on ectodermal explants are discussed.

Allergens in Pollen from Mugwort (<i>Artemisia vulgaris </i>L.)

A crude and a partly purified extract of mugwort pollen were characterized with particular emphasis on the glycoprotein allergen Ag7. Crossed immunoaffinoelectrophoresis with immobilized Con A in the intermediate gel demonstrated binding of Ag7 to this lectin, indicating that Ag7 contains terminal <i>α</i>-<i>D</i>-mannopyranosyl and/or <i>α-D</i>-glucopyranosyl residues. Crossed immunoaffinoelectrophoresis with free Con A in the first-dimension gel demonstrated microheterogeneity in the carbohydrate moiety of the allergen. Crossed radio-immunoelectrophoresis analysis using sera from 26 mugwort-allergic patients indicated that Ag7 is an important allergen in mugwort pollen. Heat stability experiments demonstrated the presence of at least three heat-resistant allergens, one of these being Ag7. Crossed immunoelectrofocusing indicated a pi value for Ag7 of 4.3.

Antitumor Lectin-Trypsin Inhibitor Conjugate<xref ref-type="fn" rid="FN2">2</xref>

Concanavalin A (Con A) and trypsin inhibitor isolated from Acacia confusa were covalently linked with N-succinimidyl-3-(2-pyridyldithio)propionate. Con A-A. confusa trypsin inhibitor (ACTI) conjugate covalently bound (Con A-ACTI) retained about 42% of the trypsin inhibitory activity present in the native ACTI and had a higher hemagglutinating activity than did the native Con A. Con A-ACTI had a greater resistance to tryptic digestion than did the mixture of Con A and ACTI. The conjugate entered sarcoma 180 tumor cells, whereas the free ACTI did not. A single dose of the conjugate injected ip into noninbred N:NIH(S) white mice bearing sarcoma 180 had a remarkable effect of increasing the survival of tumor-bearing mice, while the mixture of an equivalent dose of free Con A and ACTI was not effective.

Avian lymphokines: an improved method for chicken IL-2 production and assay. A con A-erythrocyte complex induces higher T cell proliferation and IL-2 production than does free mitogen

Optimixed production conditions and a functional assay of avian T cell growth factor (TCGF) or interleukin 2 (IL-2) are described. Treatment of lymphocytes with mitogen (Con A)-coated chicken red blood cells (MRC) resulted in markedly enhanced mitogenic response and IL-2 secretion compared to stimulation with free Con A. A positive correlation ( r = 0.89) was found between mitogenic response and IL-2 activity of conditioned media. Enrichment of target cells, i.e., Con A lymphoblasts, by Percoll R consistently improved the sensitivity of the IL-2 assay. The half-life time of chicken IL-2 at 40°C was 9.7 ± 1.7 h, which was considerably shorter that the value obtained for murine IL-2, i.e., 53.1 ± 8.5 h. High concentrations of conditioned media were found to contain a dialysable factor that suppressed IL-2 promoted blast proliferation. The relevance of the data for in vitro analysis of T cell function as well as for establishing T cell lines in the chicken system are discussed.

The pathway of lectin-mediated lymphocytotoxicity by Con A-coupled Sepharose beads

Soluble concanavalin A (Con A) can effectively mediate nonspecific target cell lysis by cytolytic T lymphocytes (LDCC). Because Con A bound to Sepharose beads (Con A-Seph) is also effective, it has been concluded by Z. K. Ballas, W. R. Green, and C. S. Henney. ( Cell. Immunol. 59, 411, 1981) that Con A-mediated “activation” of the cytolytic cell to kill in LDCC can occur without intracellular penetration of the lectin. No preincubation of either effector or target cells with Con A-Seph has been performed. Exploiting the previous finding of G. Berke ( Immunol. Rev. 72, 5, 1983) that in LDCC Con A exerts its effect(s) strictly by affecting the target rather than by bridging effector and target cells and activating the effector, identical results with Con A-Seph are shown. Preincubation of Con A-Seph with the target but not with the effector cells results in substantial killing. Moreover it is shown that the ability of Con A-Seph to mediate LDCC can be attributed to free Con A dissociating from the beads (about 1%) during the assay. Evidence is presented to indicate that the dissociated Con A, not unlike free Con A, reacts with the target cells, thereby rendering them recognizable by the effector cells. It is concluded that the activity of Con A-Seph may not be taken as evidence for Con A-mediated activation of the cytolytic cell, as suggested by Ballas et al., and that the putative Con A-mediated lymphocyte activation relevant to killing still remains to be demonstrated. Evidence contradicting Con A-mediated activation of the effector and supporting the target cell modification theory has been discussed by G. Berke, V. Hu, E. McVey, and W. R. Clark ( J. Immunol. 127, 776, 1981).

Immunochemical characterization and serum concentrations of pregnancy-associated serum proteins in mice.

The glycoprotein structure of two pregnancy-associated murine proteins, alpha 1-PAMP and alpha 2-PAMP, was analysed by using concanavalin A (Con A) affinity electrophoresis: alpha 1-PAMP was completely precipitated by free Con A, whereas alpha 2-PAMP showed heterogeneity. The isoelectric points of alpha 1-PAMP and alpha 2-PAMP were determined at 4.2 and 4.0 respectively by using crossed immunoelectrofocusing. The alpha 2-PAMP was detectable only in the serum of pregnant mice and fetuses. Maternal serum concentrations increased from Day 8 of pregnancy, remained at high values between Days 14 and 18, and could not be detected by 2 days post partum.

A simple method for the production of migration inhibitory factor by concanavalin A-stimulated lymphocytes

A simple method for preparing culture supernatants containing macrophage migration inhibitory factor (MIF) by pulse exposure of guinea pig lymphocytes to concanavalin A (Con A), is described. The method is based on the property of originally nonadherent lymphocytes to attach to the flask surface in the presence of Con A. The Con A-induced lymphocyte ‘monolayer’ can be easily rinsed, allowing the removal of free Con A without repeated, cell damaging centrifugation. The Con A-pulsed lymphocytes are cultured for 24 h in medium and supernatants containing MIF but free of undesired Con A are regularly obtained.

Mechanism of the Growth-Promoting Effect of Serum Albumin on Concanavalin A-Activated Lymphocytes: Protective Effect of the Plasma Proteins

The concentration of concanavalin A (Con A) required for optimal growth of lymphocytes is 10 to 20 times greater in the presence of plasma than in the presence of serum albumin (BSA). This shift in Con A requirement is mainly caused by Cohn fractions III and IV, which bind and probably remove free Con A. Aymphocytes, once they are activated by Con A, lose all proliferative activity unless protected by certain proteins. Cohn fraction VI and beta-lactoglobulin were found to be most effective in protecting the cells. The protective proteins do not promote growth; only BSA promotes growth of Con A-activated lymphocytes. The mechanism of the growth-promoting effect of BSA on Con A-activated lymphocytes was investigated. The continous presence of BSA in the culture medium durdly reduces, whereas protein-free medium abolishes, growth. The mechanism of the growth-promoting effect of BSA is not concerned with regulating the uptake of Con Aby lymphocytes. Red blood cells, crenated by washing in protein-free medium, revert immediately to their normal globular shape by the addition ofBSA or FAFBSA, whereas the protective proteins fail to do so, indicating a direct effect of BSA on the external surface of the erythrocyte membrane and presumably also of the lymphocyte. The hypothesis is proposed that BSA promotes growth by conveting total structural integrity to the cell surface membrane of Con A-treated lymphocytes.

Modulation of lymphocyte receptor mobility by locally bound concanavalin A.

Binding of concanavalin A (con A) to the lymphocyte surface at room temperature leads to restriction of the mobility of a variety of cell surface receptors including those from immunoglobulin (Ig), O, H-2, beta2-microglobulin, Fc receptors results in "co-capping" of Ig, H-2, theta, Fc receptors, as well as receptors for other lectins. Addition of colchicine to the cell suspensions reverses this effect and allows Con A receptors as well as other receptors to form patches and caps. Capping of Con A receptors, and beta2-microglobulin in the absence of ligands specific for these receptors. Receptors binding Limulus hemagglutinin and wax bean agglutinin, as well as those interacting with carbohydrate-specific antibodies, were partially co-capped with Con A but receptors for wheat germ agglutinin were not co-capped, excluding the possibility that restriction of receptor mobility by Con A resulted simply from cross-linkage of mobile receptors to immobilized glycoproteins (Con A receptors). Latex beads and platelets coupled to Con A were bound to lymphocytes under the same conditions as free Con A. Binding of these particles to local regions of the cell surface resulted in restriction of the mobility of those receptors that could be co-capped with free Con A. In contrast to the findings with free Con A, however, addition of colchicine resulted in capping of the bound particles but did not cause co-capping of either the unbound Con A receptors or other receptors. These findings support the hypothesis that modulation occurs via a submembranous assembly containing microtubules, and they further suggest that the transitions of this assembly induced locally by Con A may be propagated via cooperative processes.

Lymphokines

Incubation of guinea pig lymph node lymphocytes with insoluble concanavalin A (Con A) in serum-free medium yields sizable amounts of migration inhibition (MIF) and mitogenic factors (MF). Various controls confirmed that the activities detected in supernatants from such cultures are not due to free soluble Con A released during culture. Whereas guinea pig MIF does not act upon human and horse monocytes, guinea pig MF stimulated human but not horse lymphocytes. Upon stimulation by insoluble Con A, guinea pig MIF and MF appear to be produced essentially by T lymphocytes. The technique proposed for producing lymphokines appears convenient for further purification and biochemical characterization of culture supernatants.

Activation of B lymphocytes by locally concentrated concanavalin A.

AbstractB lymphocytes are not stimulated by soluble concanavalin A (Con A). However, Con A cross‐linked at the bottom of tissue culture petri dishes could activate DNA synthesis in B cells. Addition of free Con A to such cultures inhibited the proliferative response to insoluble Con A. T lymphocytes were not activated by locally concentrated Con A, although they responded to soluble Con A. In the presence of both soluble and cross‐linked Con A there was a shift in the dose response curve to soluble Con A, the magnitude of which varied with the concentration of cross‐linked Con A.