Subnuclear Distribution of the S-100 Protein Specific Binding Sites in Rat Brain

Abstract: Fractionation of isolated brain nuclei previously reacted with ¹²⁵I-labelled S-100 showed that most of the specifically bound radioactivity associated with the nuclear membranes and the nucleoli. Labelling of nucleoli, which indicates the entrance of ¹²⁵I-labelled S-100 into the nucleus, was observed at 37°C, but not at 0–4°C. When tested separately for ¹²⁵I-labelled S-100 specific binding, both the nuclear membranes and the nucleoli were found to bind ¹²⁵I-labelled S-100 in a biphasic manner, the binding displaying a high affinity and a low affinity component, as observed with intact nuclei. However, the binding to nuclear membranes was largely irreversible, while that to nucleoli was fully reversible after any association time.     

GANGLIOSIDES IN NERVOUS TISSUE CULTURES AND BINDING OF 125I-LABELLED TETANUS TOXIN, A NEURONAL MARKER

Abstract— —Continuous cell lines, primary cell cultures derived from embryonic CNS, and homogenates made from adult and embryonic CNS were compared with respect to their lipid pattern and their ability to bind ¹²⁵I-labelled tetanus toxin. In parallel experiments de novo synthesis of gangliosides in the cell lines was studied, using [¹⁴C]glucosamine as precursor. Of the total lipid only gangliosides were specifically labelled by [¹⁴C]glucosamine. The patterns of the de novo synthesized gangliosides corresponded to those present in the respective cells.
Pronounced binding of ¹²⁵I-labelled toxin was only detectable in tissues containing long-chain gangliosides (ganglioside C which represents GDIb and GTI).
Accordingly, hybrid (neuroblastoma x glioma) cells, due to their lack of long-chain gangliosides, bound just-discernible amounts of labelled toxin. When previously exposed to gangliosides, their binding of tetanus toxin tremendously increased.
It was concluded that only the long-chain gangliosides in the neuronal cells are functionally involved in the binding of the tetanus toxin and that these acceptors of tetanus toxin can be transplanted.     

The Specific Interaction of S-100 Protein with Synaptosomal Particulate Fractions. Evidence for the Formation of a Tight Complex Between S-100 and Its Binding Sites

Abstract: The dissociation of the ¹²⁵I-labelled S-100 specifically bound to synaptosomal particulate fractions (SYN) has been studied under a variety of conditions after different association times. The results indicate that after a critical association time of about 20 min at 37°C, the bound protein becomes progressively less accessible to the dissociating agents or conditions employed. These findings support the view that the partial irreversibility of the ¹²⁵I-labelled S-100 binding to SYN could be due to the formation of a tight complex between the protein and its synaptosomal sites. These data are discussed mainly in relation to the particulate-bound fraction of native S-100.     

Characterization of fibronectin binding to Salmonella enteritidis strain 27655R

Abstract The optimal conditions for the binding of fibronectin to Salmonella enteritidis strain 27655R, and the cell-surface components involved in the binding, were identified. Cultivation on colonisation factor antigen (CFA) agar or in CFA broth at 33°C for 24 h were found to be optimal for the expression of fibronectin binding. Such cultures exhibited 88% and 70% binding of ¹²⁵I-labelled fibronectin and its 29-kDa N-terminal domain, respectively. The fibronectin binding was reversed by the addition of unlabelled fibronectin or its 29-kDa fragment. Scatchard plot analysis of the binding showed that the strain possessed one high-affinity ( K _d= 5.8 × 10⁻¹⁰ M) and one low-affinity ( K _d= 2 × 10⁻⁸ M) binding site. The fibronectin-binding could be inhibited by cell surface components of S. enteritidis 27655R released by 30 min treatment at 65°C or 95°C. Inhibition could also be achieved using purified fimbriae. A non-fimbriated mutant of strain 27655R showed a much reduced binding of fibronectin (15%). Electron microscopic analysis showed association of the gold-labelled 29-kDa N-terminal fragment with S. enteritidis 27655R fimbriae. In conclusion, the findings suggest that S. enteritidis (strain 27655R) possesses fibronectin-binding fimbriae.     

Binding of vitronectin and plasminogen to Helicobacter pylori

Abstract We have studied how some extracellular matrix proteins, fibronectin, fibrinogen, collagen type I and type IV, plasminogen and vitronectin bind to Helicobacter pylori . Radiolabelled vitronectin and plasminogen bound to the haemagglutinating H. pylori strain 17874 at a high level (53% and 32%, respectively), type IV collagen showed an intermediate level of binding (16%), while binding by ¹²⁵I-labelled fibrinogen, fibronectin and collagen type I remained at a low level (5–7%). Both ¹²⁵I-vitronectin and plasminogen showed a dose-dependent binding to cells of H. pylori 17874. Plasminogen binding by this strain was specific since the binding was inhibited by nonlabelled plasminogen, but not by highly glycosylated glycoproteins such as fetuin and orosomucoid or by a variety of monosaccharides. We have previously shown that ¹²⁵I-vitronectin shows a specific and saturable binding to H. pylori 17874, and that sialic acid-rich glycoproteins such as fetuin and orosomucoid drastically reduced binding. We now report that a simultaneous incubation of ¹²⁵I-vitronectin and ¹²⁵I-plasminogen with cells of H. pylori 17874 showed a total binding approximately similar to the level of binding when either ¹²⁵I-plasminogen, or ¹²⁵I-vitronectin only were incubated with the bacterial cells. Nonlabelled vitronectin inhibited the binding of ¹²⁵I-plasminogen by H. pylori , but nonlabelled plasminogen had no effect on the binding of ¹²⁵I-vitronectin. Our findings suggest that there are different but probably closely localized binding sites for vitronectin and plasminogen on H. pylori 17874.     

Methyl 3β-(4-[125I]Iodophenyl)Tropane-2β-Carboxylate In Vitro Binding to Dopamine and Serotonin Transporters Under "Physiological" Conditions

Abstract: Methyl 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylate ([¹²³I]β-CIT) is a single photon emission computed tomographic radiotracer for in vivo labeling of dopamine (DA) and serotonin (5-HT) transporters. Single photon emission computed tomographic experiments in nonhuman primates showed that [¹²³I]β-CIT in vivo binding to DA transporters had a much slower washout than binding to 5-HT transporters. This observation was not predicted from previously published in vitro studies. These studies, performed at 22°C in nonphysiological buffer, reported similar affinity of [¹²⁵I]β-CIT for DA and 5-HT transporters. We now report [¹²⁵I]β-CIT binding parameters to fresh rat membranes at 22°C and 37°C, in a buffer mimicking the composition of cerebrospinal fluid. At both temperatures, binding to DA transporters was best fit by a twosite model, whereas binding to 5-HT transporters was compatible with one population of sites. At 22°C, [¹²⁵I]β-CIT showed similar affinity to high-affinity DA (0.39 n M ) and 5-HT transporter sites (0.47 n M ). Increasing the incubation temperature from 22°C to 37°C reduced binding to DA transporters by 60%, whereas binding to 5-HT transporters was only marginally affected. In vitro kinetic experiments failed to detect significant differences in on or off rates that could explain the observed in vivo kinetics. These experiments thus failed to explain [¹²³ I]β-CIT in vivo uptake kinetics, suggesting the existence of specific factors affecting the in vivo situation.     

Antigenic heterogeneity of strains of Saccharomyces cerevisiae and Candida albicans recognised by serum antibodies from patients with Crohn''s disease

Abstract Vitronectin, a serum and extracellular matrix protein involved in immunological reactions, interacts with Helicobacter pylori strains. Of the 20 H. pylori strains tested three strains bound more than 50% of the vitronectin added, five strains bound 25–40%, nine strains bound 10–20% and three strains bound 5–8% vitronectin. Two strains, one with high- and one with low-binding properties, were selected for further characterization of ¹²⁵I-vitronectin binding. Binding to the urea-activated ¹²⁵I-labelled vitronectin was fast, saturable and reversible when an excess of unlabelled vitronectin was added to the bacteria with bound ¹²⁵I-vitronectin. The binding was heat- and protease-sensitive, suggesting that the binding was mediated by bacterial cell-surface proteins. Since components such as fetuin and orosomucoid but not asialofetuin inhibited the binding, sialic-acid specific proteins, related to H. pylori sialic-acid specific haemagglutinins, were probably involved.     

Transferrin Receptor on Chick Fibroblast Cell Surface and the Binding Affinity in Relevance to the Growth Promoting Activity of Transferrin

We examined the transferrin (Tf) receptor of chick skin fibroblasts using chick ¹²⁵I-Tf. When the cells were incubated with ¹²⁵I-Tf on ice, most of the cell-associated ¹²⁵I-Tf was found on the cell surface; on the other hand, a large part of it was located inside the cells when incubated at 37°C. By equilibrium binding assay, the number of Tf receptors per cell was determined as 6.7 × 10³. Dissociation constant was estimated to be 2.6 × 10⁻⁸ M.
The binding of ¹²⁵I-Tf was competitively inhibited by the addition of chick unlabeled Tf. Weaker inhibition was observed when bovine Tf was used as a competitor. Horse Tf had no effect on the binding of chick Tf. This agrees well qualitatively with chick cell growth-promoting activites of these Tfs.
Removal of Fe from Tf affected the affinity for its receptors. About 5- to 10-fold higher concentrations of chick apo–Tf was needed to achieve the same degree of inhibition of ¹²⁵I-Tf binding as that made by chick Fe-Tf.     

Vitronectin and type-I collagen binding by Staphylococcus aureus and coagulase-negative staphylococci

Abstract Binding of ¹²⁵I-labelled type-I collagen and ¹²⁵I-labelled vitronectin (human serum spreading factor or S-protein) was studied using Staphylococcus aureus and coagulase-negative staphylococci of different species. Binding of collagen and vitronectin was time dependent for S. aureus ISP 546, and S. haemolyticus E 2498/86. Co-operative binding of vitronectin and collagen by staphylococcal cells was demonstrated. Binding to S. haemolyticus E 2498/86 was more rapid and was enhanced in vitronectin/collagen mixtures than for either protein separately. Furthermore, pre-incubation of staphylococcal cells with unlabelled collagen enhanced vitronectin binding. When cells of S. haemolyticus E 2498/86 were treated with pronase E, proteinase K, subtilopeptidase A or trypsin, vitronectin-binding was decreased by 50% or more, whereas collagen-binding was protease resistant. For the strains of S. aureus tested, both vitronectin and collagen binding were found to be protease sensitive. Type-I collagen peptides inhibited collagen-binding to S. haemolyticus E 2498/86, whereas vitronectin-binding was not affected perhaps indicating different receptors for these proteins. The binding of both collagen and vitronectin was shown to be reversible, since bound ¹²⁵I-collagen and ¹²⁵I-vitronectin were displaced after adding excess of the homologous protein.     

The protective effect of some food ingredients on Staphylococcus aureus MF31

The upper limiting temperature of growth of Staphylococcus aureus MF31 in heart infusion broth (HI) was about 44°C but addition of monosodium glutamate (MSG) and soy sauce permitted the organism to grow above this temperature. This effect is similar to that of NaCl. Tomato ketchup, Worcestershire and HP sauces added to HI did not allow growth at the non-permissive temperature of 46°C but death was delayed. Staphylococcus aureus died in unsupplemented chicken meat slurry at 46°C but grew at 48°C in slurry supplemented with 5.8% NaCl and survived incubation for 18 h at 50°C in slurry supplemented with 5.8% NaCl and 5% MSG. Cultures grown at 37°C had a D ₆₀ value of 2 min in 50 mmol/l Tris (pH 7.2) buffer. Cultures grown at 46°C in HI containing 5.8% NaCl had a D ₆₀ value of 8 min in Tris buffer. Addition of 5.8% NaCl plus 5% MSG to the buffer increased the D ₆₀ by a factor of about 7 for both cultures. In storage experiments at room temperature, the culture grown at 37°C and at 46°C plus 5.8% NaCl died at about the same rate in salami. In milk powder, however, the count of 37°C culture decreased from 10⁹/g to 10⁶/g in 5 weeks while the count of 46°C culture remained unchanged. In cottage cheese, freeze-dried rice and macaroni, the 37°C cultures also died more rapidly. It is suggested that cultures grown at 46°C plus 5.8% NaCl may be suitable for experiments with artificially contaminated foods.     

5-Methoxytryptamine Inhibits Cyclic AMP Accumulation in Cultured Retinal Neurons Through Activation of a Pertussis Toxin-Sensitive Site Distinct from the 2-[125I]Iodomelatonin Binding Site

Abstract: Melatonin and 5-methoxytryptamine inhibited forskolin-stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC₅₀ values of 0.8 n M for melatonin and 7.2 n M for 5-methoxytryptamine. Inhibition of cyclic AMP formation by 5-methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5-methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5-methoxytryptamine but not that to 2-iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2-[¹²⁵I]iodomelatonin. Melatonin displaced specific 2-[¹²⁵I]iodomelatonin with a K _i value (0.8 n M ) similar to the EC₅₀ for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[¹²⁵I]iodomelatonin binding at very high concentrations ( K _i = 650 n M ). Pretreating cultured cells for 24 h with 2-iodomelatonin or melatonin, but not with 5-methoxytryptamine, reduced subsequent 2-[¹²⁵I]iodomelatonin binding. Thus, 5-methoxytryptamine appears to inhibit forskolin-stimulated cyclic AMP formation at a site distinct from the 2-iodomelatonin binding site.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

METABOLIC STUDIES ON 125I-LABELLED BETA-TRACE PROTEIN, WITH SPECIAL REFERENCE TO SYNTHESIS WITHIN THE CENTRAL NERVOUS SYSTEM

Abstract— By means of single radial immunodiffusion on agar, the concentration of beta-trace protein in serum was estimated to about 0.4 mg per 100 ml, i.e. seven times lower than the concentration in CSF. The turnover time in serum was found to be about 1.2 h and the daily amount of beta-trace protein metabolized was approx. 240 mg. The turnover time in CSF was about 3 h after injection of ¹²⁵I-labelled beta-trace protein into the lumbar spinal space. The data suggest a synthesis of beta-trace protein within the CNS of about 30 mg daily, i.e. one-eighth of the intravascularly metabolized beta-trace protein.     

In Vitro Effect of Tetanus Toxin on GAB A Release from Rat Hippocampal Slices

Abstract Ca²⁺-dependent K⁺-stimulated γ-aminobutyric acid release from rat hippocampal slices was reduced about 30% by pre-incubation of the slices with 10⁴ mouse LD₅₀/ml tetanus toxin for 3 h at 37°C.     

ETA and ETB Specific Ligands Synergistically Antagonize Endothelin-1 Binding to an Atypical Endothelin Receptor in Primary Rat Astrocytes

Abstract: Using a whole-cell binding procedure with long incubations at low temperature and subsequent acid stripping, we have characterized an atypical endothelin (ET) receptor in primary rat cortical astrocyte cultures. We found the following: (a) no competition for ¹²⁵I-ET-1 binding by the ET_A antagonists BQ-123 and LU 135252 or the ET_B agonist IRL 1620; (b) weak competition by the ET_B antagonist BQ-788 and by the predominant ET_B ligand ET-3; (c) potent synergistic competition of ET_A and ET_B ligands in combination for ¹²⁵I-ET-1 binding; (d) potent competition of ET-1 with any of the radioligands used, ¹²⁵I-ET-1, ¹²⁵I-IRL 1620, and [³H]BQ-123; (e) lack of competition of IRL 1620 and BQ-123 with the respective other radioligand; (f) shifting of the amount of acid-strippable ¹²⁵I-ET-1 binding from 20 to 80% by ET_B ligands and to 4% by ET_A ligands; and (g) as a control, typical ET_A and ET_B binding characteristics of the RAT-1 fibroblast and the U373MG astrocytoma cell line, respectively, under our assay conditions. The unusual binding properties of astrocytic ET receptors described in this study appear to be the result of several binding sites in the receptor for different ET ligands or ligand epitopes.     

Radioiodination of Enterotoxin from Clostridium perfringens Type A using Chloramine T

Procedures were examined for labelling enterotoxin isolated from Clostridium perfringens type A. with ¹²⁵I using chloramine T as the oxidizing agent. The iodination method was evaluated critically to establish the optimal conditions for the preparation of iodinated enterotoxin with a high specific radioactivity and without impairing the immunospecificity and biological activity. The use of 250 μg/ml of chloramine T in the reaction mixture. 500–1000 μCi of Na¹²⁵I/10 μg of enterotoxin and a reaction time of 40 s at pH 7–0 produced ¹²⁵I-enterotoxin of both high specific radioactivity and immunospecificity which retained its biological activity. No damage or aggregate formation due to the iodination process was observed. Enterotoxin labelled with high specific activity (135 μCi μg) showed extensive dissociation of ¹²⁵I when stored at 4°C and—20°C. In contrast, toxin labelled with low specific activity (7 μgCi/μg) was stable for as long as two months. The immunoreactivity of all labelled preparations was essentially unchanged after storage for one month.     

Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines

Abstract. Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level. The two types of cultures exhibited significant ( P <0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus. The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture. Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10⁻¹⁰−10⁻⁹ M . Slow cultures bound more [¹²⁵I]-insulin than fast cultures at 24 h in culture. These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.     

The Specific Interaction of S-100 Protein with Synaptosomal Particulate Fractions. Modulation of Binding by Fractional Occupancy of Sites and by the Physical State of Membranes

Abstract: The nonlinearity of single components of the Scatchard plot of S-100 binding to synaptosomal particulate fractions (SYN) and the observation that dilution of the ¹²⁵I-labeled S-100 site complex results in a greater extent of dissociation of the tracer in the presence than in the absence of an excess of unlabeled S-100 suggest that sites change their binding behavior depending on fractional occupancy. To study this aspect of the interaction in more detail, ¹²⁵I-labeled S-100 binding experiments were conducted in the presence of, or after preincubation of SYN with various concentrations of, unlabeled S-100. The results indicate that: (a) S-100 synaptosomal sites do change their binding behavior depending on fractional occupancy; and (b) the nonrapid equilibrium between bound S-100 and the medium, which has been referred to as the formation of a tight complex between S-100 and its binding sites, is related to the activation of high-affinity sites. However, no univocal interpretation of these data in terms of binding model can be offered at present, as the binding models currently employed in the analysis of ligand-site interactions can each account for only part of the results described in this report. In any case, data obtained by studying ¹²⁵I-labeled S-100 binding to untreated SYN at 2°C and to prefixed SYN at 37°C indicate that the physical state of membranes influences both the extent of the interaction and the binding behavior of the sites.     

Tetanus Toxin Inhibition of K+ -Stimulated [3H]GABA Release from Developing Cell Cultures of the Rat Cerebellum

Abstract: The effect of tetanus toxin pretreatment on K⁺ -stimulated [³H]γ-aminobutyric acid release from neuron-enriched cerebellar cell cultures at various stages during their development in vitro was assessed. Tetanus toxin had little inhibitory effect on immature (1-3-day-old) cultures, but markedly reduced K⁺-evoked [³H]γ-aminobutyric acid release from 7- and 14-day-old cultures (∼80% inhibition). It is suggested that cerebellar neurons in culture develop tetanus toxin-sensitive transmitter release mechanisms similar to their in vivo counterparts.     

A Comparison of Toxin Production by Clostridium botulinum Type E in Irradiated and Unirradiated Vacuum Packed Fish

S ummary . The rates of toxin production at 10° by inocula of 10⁵, 10⁴, 10³ and 10² spores of Clostridium botulinum type E in vacuum packed herring, cod and haddock were compared with that of equivalent numbers of spores surviving γ-irradiation at 0.3 Mrad. There was little difference between the rates of toxin production in unirradiated and irradiated fish. More toxin was produced in irradiated herring than in unirradiated, but in haddock the reverse was obtained. In cod about equal amounts of toxin were found without trypsinization; after trypsinization toxin levels were generally higher in irradiated samples.