Pharmacological Reagents Research Articles

Facile production of native-like κ-bungarotoxin in yeast: An enhanced system for the production of a neuronal nicotinic acetylcholine receptor probe

Research on the mammalian central nervous system has been hindered by the limited number and meager supply of naturally occurring toxins that can be used as pharmacological reagents. The κ-neurotoxins in particular are not found abundantly in nature and are difficult to obtain and isolate in quantities sufficient for research purposes. Here we report the expression and isolation of relatively large quantities of the κ-neurotoxin, κ-bungarotoxin, in an active form using a yeast, Pichia pastoris, expression system. The resultant product of the expression system has a short amino-terminal amino acid extension relative to venom-derived κ-bungarotoxin, but is equivalent to the native toxin in physical and biological properties, as judged by the CD spectra, the ability to form dimers in solution, and the activity on chick ciliary ganglia. The yeast system produces approximately 0.2 mg from a 2 liter culture and the purification takes approximately 2 days. In contrast, E. coli, the only other available expression system for this toxin, produces one-fifth to one-half as much active material from a 5 liter high-density fermentation and the resulting protein takes over a week to purify. No high mol. wt disulfide-bonded aggregates were found in the yeast expression system product, indicating that the product is that of a biologically assisted folding process. This has significant implications not only for the efficient production of native toxin but also for the production of mutant proteins to study the structure-function relationship in these proteins.

Nuclear magnetic resonance (NMR) analysis of ligand receptor interactions: the cholinergic system--a model.

Elucidation of the molecular mechanisms that govern ligand-receptor recognition is essential to the rational design of specific pharmacological reagents. Whereas often the receptor and its binding site are the target of investigation, study of the ligand in its free and bound state can also reveal important information regarding this recognition process. Nuclear magnetic resonance (NMR) spectroscopy can be extremely useful for such studies. In this review, we discuss the attributes of NMR in the study of ligand receptor interactions. The cholinergic receptor and its binding to the neurotransmitter, acetylcholine, and cholinergic antagonists serve as a model system, illustrating the power of ligand analysis by NMR. The results discussed prove that the region of residues alpha 180-205 of the nicotinic acetylcholine receptor are an essential component of the cholinergic binding site and that ligand binding involves a positively charged hydrophobic motif.

A quantitative assay to measure homotypic vacuole fusion in vitro

Homotypic (self) vacuole fusion is the last discernible step in vacuole inheritance ofSaccharomyces cerevisiae and was recently successfully reconstituted in vitro using purified vacuoles, cytosol, ATP, moderate salt concentrations, and a physiological temperature. The following protocol describes a rapid, reliable, and easy technique to quantitate homotypic vacuole fusion in vitro. Frequency of fusion is determined by quantifying alkaline phosphatase activity after fusion has taken place. This method has been successfully used to analyze pharmacological reagents which interfere with certain subreactions and to identify distinct proteins which play key roles in the fusion events.

Induction of opioid receptor-mediated macrophage chemotactic activity after neonatal brain injury.

Macrophages have a prominent role in the injury response of the brain, yet the molecular mechanisms that control their invasion to the site of neuronal degeneration is unknown. After removal of the posterior cortex at birth, there is massive and specific targeting of nonresident macrophages to axotomized neurons in the lateral thalamus. The present study has identified an injury-induced, brain-derived chemotactic factor (BDCF) capable of eliciting chemotactic responses from resident peritoneal macrophages and brain macrophages. Conditioned media collected from tissue slices containing the axotomized central nervous system neurons exhibit BDCF activity. Initial experiments indicated that BDCF is a small peptide and, thus, we used specific pharmacologic reagents to characterize further BDCF activity. Naloxone, a pan opioid receptor antagonist, completely blocks BDCF activity. Although both kappa and mu opioid receptor antagonists failed to modify BDCF-induced macrophage chemotaxis, two specific delta receptor antagonists blocked BDCF. Analysis of BDCF by reverse phase HPLC and RIA revealed peak chemotactic activity in fractions consistent with the presence of an opioid peptide. The results suggest that cells in the brain respond to neuronal injury by producing and releasing opioids that can initiate a specific macrophage response.

Local Delivery of Vascular Endothelial Growth Factor Accelerates Reendothelialization and Attenuates Intimal Hyperplasia in Balloon-Injured Rat Carotid Artery

Most strategies designed to reduce restenosis by the use of pharmacological or biological reagents involve direct inhibition of vascular smooth muscle cell (SMC) proliferation. Alternatively, SMC proliferation might be indirectly inhibited if reendothelialization could be specifically facilitated at sites of balloon-induced arterial injury. Accordingly, we investigated the hypothesis that application of an endothelial cell (EC)-specific mitogen to a freshly denuded intimal surface could accelerate reendothelialization and thereby attenuate intimal hyperplasia. The left carotid artery of 31 Sprague-Dawley rats was subjected to balloon injury, after which 16 rats were treated with a 30-minute incubation with 100 micrograms of vascular endothelial growth factor (VEGF), an EC-specific mitogen. Control animals (n = 15) received a 30-minute incubation with 0.9% saline. At 2 weeks after balloon injury, carotid artery reendothelialization was markedly superior in the VEGF-treated group compared with the control group (14.59 +/- 1.12 versus 7.96 +/- 0.51 mm2, P < 0.005). The extent of reendothelialization measured at 4 weeks after balloon injury remained superior for arteries treated with VEGF (18.04 +/- 0.90 mm2) versus saline (13.42 +/- 0.84 mm2, P < .005). Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries treated with VEGF versus the control group at both the 2-week and 4-week time points. Immunostaining for proliferating cell nuclear antigen (PCNA) disclosed a threefold increase in PCNA-positive cells in the neointima of control arteries versus VEGF-treated arteries at 2 weeks after injury. Application of VEGF, an EC-specific growth regulatory molecule, may be effectively used in vivo to promote reendothelialization and thereby indirectly attenuate neointimal thickening due to SMC proliferation.

A soluble chimeric form of the L1 glycoprotein stimulates neurite outgrowth.

Cerebellar neurons, cultured on monolayers of 3T3 fibroblasts or on a polylysine/extracellular matrix-coated substratum, responded to a soluble recombinant L1-Fc chimera by extending longer neurites than controls. The response was inhibited by pretreating neurons with antibodies to L1 or antibodies to the fibroblast growth factor (FGF) receptor. The response could also be inhibited by a range of pharmacological reagents that inhibit various steps in the signal transduction cascade which underlie a neurite outgrowth response to basic FGF. The response was of a similar magnitude and not additive with that induced by L1 expressed in a cellular substrate. These data show that L1 in neurons is capable of directing a neurite outgrowth response to a soluble L1-Fc chimera, and that neuronal FGF receptor function is required for this response. The data also show that the ability of cell adhesion molecules (CAMs) to stimulate neurite outgrowth can be dissociated from their ability to function as substrate-associated adhesion molecules and point to the potential of using CAM-Fc chimeras to promote nerve regeneration.

Involvement of tumor necrosis factor-alpha in glomerular injury.

Many in vivo and in vitro studies provide strong support for the concept that TNF-α released from either infiltrating or intrinsic glomerular cells may significantly contribute to glomerular injury in experimental models of toxic or immunologically mediated renal diseases. The sequence of the possible events involved in this process is depicted in Fig. 1. The real challenge now is first to investigate further the changes induced by TNF-α in its target cells of the glomerulus, especially by determining the post-receptor signaling. This may suggest novel approaches for intervention. Secondly, it is necessary to expand TNF-α studies by analyzing the interactions of TNF-α with other inflammatory mediators including cytokines. Finally, pharmacological reagents that can interfere with the generation or the action of TNF-α have to be developed. They include drugs like thalidomide which selectively inhibits TNF-α production by enhancing the degradation of TNF-α mRNA [41], as well as specific TNF-α receptor blockers or antagonists directed against receptors known to be involved in the recruitment and activation of blood-borne cells. There also might be implications for in vivo gene transfer. The availability of synthetic genes should allow TNF-α gene expression in glomerular cells to be blunted by either using antisense constructs or introducing the gene of inhibitory cytokines including IL-6 or IL-10.

Cytotoxic ribonuclease chimeras. Targeted tumoricidal activity in vitro and in vivo.

Monoclonal antibodies to the transferrin receptor or to the T cell antigen, CD5, were chemically linked to mammalian RNase A and found to specifically inhibit protein synthesis in antigen-positive cells. Antibody-mediated specificity of these cytotoxic ribonuclease chimeras (CRCs) was demonstrated in three ways. 1) Toxicity was due to the chemical linkage of RNase to antibody, as the individual components added separately or in combination did not inhibit protein synthesis; 2) the anti-transferrin receptor CRCs inhibited protein synthesis in those cells expressing the human transferrin receptor (K562, U251, Jurkat cells) but had no detectable toxicity to cells lacking the human transferrin receptor (Vero or NIH 3T3 cells); 3) free antibody to either the human transferrin receptor (454A12 or 5E-9) or to the T cell antigen, CD5 (T101), blocked the cytotoxicity of the respective CRC. Two CRC species, designated P1 and P2, that differed in size and stoichiometry of RNase A to antibody, were purified by size-exclusion high performance liquid chromatography. The higher molecular weight P1 conjugate had an IC50 of 20-30 nM, whereas the P2 conjugate had a higher IC50 of 300-500 nM. Bioactivity could be reversibly increased more than 10-fold by freezing. The cytotoxicity of the CRCs was examined in vivo in a solid tumor animal model. Intratumoral injections of an anti-transferrin receptor CRC into established U251 human glioblastoma tumors grown in the flanks of nude mice prevented tumor growth, whereas RNase A mixed with antibody was ineffective. CRCs, therefore, express cytotoxicity in vitro and in vivo. Mammalian nucleases coupled to antibodies may be utilized as cell type-selective cytotoxins and have potential as pharmacologic reagents. The systemic toxicity and immunogenicity observed with mammalian derived cytotoxins may be significantly less than that of the currently employed plant- and bacterial-derived immunotoxins.

Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures.

We have isolated a set of ligand-binding DNA sequences from a large pool of random sequence DNAs by selection and amplification in vitro, using similar methods to those described for the isolation of ligand-binding RNAs. The ligand-DNA interactions are both sequence- and ligand-specific, and are dependent on proper folding of the single-stranded DNA. Some ligands led to the isolation of more DNA sequences than RNA sequences, and vice versa. Analysis of individual sequences reveals that ligand binding is DNA-specific; RNAs of identical sequence could not interact with the same ligands. Ligand-binding DNAs might be more suitable than RNAs as potential pharmacological reagents because of the greater stability of DNA. The apparent primacy of RNA in the early evolution of life may have been due to its availability rather than to its functional superiority.

Induction of an activation antigen on human endothelial cells in vitro

This study describes the expression characteristics of a cell membrane protein recognized by a monoclonal antibody ENA1, which was obtained by immunizing mice with human umbilical vein endothelial (HUVE) cells cultured with a mixture of interleukin 1 and tumor necrosis factor-alpha. The expression of this ENA1 antigen could also be induced by lipopolysaccharide and phorbol esters. Expression was only demonstrated on HUVE cells and human umbilical arterial endothelial cells, pretreated with one or with a mixture of these reagents. No expression was detected on human fibroblasts, renal epithelial cells or on mesothelial cells derived from omental tissue, either pretreated or not with the aforementioned inducers of the antigen. Furthermore, no reactivity was observed with either polymorphonuclear cells, peripheral blood lymphocytes or the monocytic cell line U937. Time course experiments revealed that the expression of the ENA1 antigen was time dependent. Maximal expression on HUVE cells was observed after 5 h of incubation with activator, after which a decline in expression occurred. Induction of expression could be completely blocked by the mRNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide, indicating that de novo synthesis occurs. Other pharmacological reagents tested had no effect on the induction of ENA1 expression. The putative role of the newly described antigen is discussed in relation to the current knowledge of molecules involved in adhesion of immune cells in inflammatory processes.

Cordycepin rapidly collapses the intermediate filament networks into juxtanuclear caps in fibroblasts and epidermal cells

The nucleoside analog 3′-deoxyadenosine (cordycepin) rapidly collapses the intermediate filaments into juxtanuclear caps in interphase fibroblasts and keratinocytes. A minimum of 80 μg/ml cordycepin or 20 μg/ml cordycepin in combination with 2 μ/ml of the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenosine (EHNA) to inhibit its degradation is required to see these effects. This is the same concentration required for cordycepin to arrest cells at the onset of mitosis and depolymerize the microtubules to small asters. Cordycepin enters the cells rapidly and is phosphorylated to 3′-dATP with a concomitant drop in ATP levels. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on either the intermediate filaments or microtubules. In addition, similar effects are not produced by a variety of other adenosine analogs with alterations in the 2′- and 3′-ribose positions. Although other pharmacological reagents result in alterations of the fibroblastic intermediate filaments, cordycepin is unusual because of the rapiditiy with which the fibroblastic intermediate filaments collapse into the juxtanuclear caps. The juxtanuclear caps have a morphology different from that of the perinuclear bundles of intermediate filaments that arise after long-term depolymerization of the microtubules. The keratin fibers in the epidermal cells retract to a perinuclear ring when treated with cordycepin

Cordycepin disrupts the microtubule networks and arrests Nil 8 hamster fibroblasts at the onset of mitosis.

The nucleoside analogue 3'-deoxyadenosine (cordycepin) arrests dividing cells at the onset of mitosis in prometaphase. The microtubules in the arrested prometaphase cells depolymerize to two small asters. A minimum of 80 micrograms/ml cordycepin or 20 micrograms/ml cordycepin in combination with 2 micrograms/ml of the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA) to inhibit its degradation is required to see these effects. Analysis of cell extracts by high-pressure liquid chromatography indicates that cordycepin enters the cells rapidly and is phosphorylated to 3'-dATP. The intracellular concentration rises almost linearly from 0.7 mM after 15 min to 7 mM by 210 min. Concomitantly the ATP concentration shows a rapid drop from the 4 mM present in controls. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on the microtubules. In addition, similar effects are not produced by a variety of other adenosine analogues with alterations in the 2' and 3' ribose positions. Although other pharmacological reagents arrest cells at the onset of mitosis, cordycepin is unusual because of the collapse of the microtubule networks to two small asters that radiate from the microtubule-organizing center. 3'-dATP can replace the requirement for ATP or GTP in the vitro polymerization of microtubules from microtubule protein: however, at limiting concentrations of nucleotide it requires approximately two times the concentration of 3'-dATP as ATP to support an equivalent level of microtubule polymerization. This suggests that the effects of cordycepin in vivo may be the result of the depletion of cellular ATP pools and the altered ability of 3'dATP to substitute for ATP-dependent reactions. Current experiments are testing this hypothesis.

The stability of soman and its stereoisomers in aqueous solution: toxicological considerations.

The paucity of information regarding the characteristics of soman (pinacolyl methylphosphonofluoridate) in aqueous solution has limited its use as a toxicological or pharmacological reagent. We report here on the stability of soman under conditions in which it may normally be found during use and storage in the laboratory. Solutions of 1 mM soman in normal saline were not hydrolyzed after 5 months of storage at -90 degrees C. Samples that were repeatedly thawed but not allowed to warm to room temperature and then immediately refrozen showed no apparent hydrolysis. Portions of the same solution, stored in the refrigerator just above freezing, exhibited 50% hydrolysis after 150 days. When portions of this solution were stored at 21 degrees C, the time for 50% hydrolysis was in excess of 5 days. This rate of hydrolysis was the same for all four of the soman stereoisomers. In buffered solutions at pH 7.4, 8.0 and 8.6 the half-times were 6.6, 3.2 and 2.2 h at 27 degrees C and 4.8, 1.6 and 1.2 h at 37 degrees C, respectively. Hydrolysis rates were not significantly influenced by the presence of a carbodimide stabilizer in the agent. There is no reason to expect any deviation from a direct correlation between total soman concentration and toxicity.

Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits human tumor cell growth in vitro.

A murine hybridoma has been obtained that produces a monoclonal antibody against the human transferrin receptor. In contrast to previously characterized monoclonal antibodies that recognize the transferrin receptor, this antibody, designated 42/6, blocks the binding of transferrin to its receptor and inhibits the growth of the human T leukemic cell line, CCRF-CEM, in vitro. Inhibition of cell growth was dose dependent, and as little as 2.5 micrograms of purified antibody per ml had a detectable effect, even though transferrin was present in the tissue culture medium in large molar excess. Cells grown in the presence of antibody for 7 days accumulated in S phase of the cell cycle. The addition of iron to antibody-treated cultures in the form of ferric complexes or ferrous sulfate did not overcome the growth inhibitory effects of the anti-transferrin-receptor antibodies. This result suggests that either transferrin is the only means by which CCRF-CEM leukemic cells can be provided with sufficient iron in vitro or that other factors in addition to iron starvation are involved in the antibody-mediated growth inhibition. The inhibition of cell growth by 42/6 monoclonal antibody suggests that monoclonal antibodies against proliferation-associated cell surface antigens, such as the transferrin receptor, may be useful pharmacological reagents to modify cell growth in vitro.

Inhibition of mouse spleen cell function by diphtheria toxin fragment A coupled to anti-mouse Thy-1.2 and by ricin A chain coupled to anti-mouse IgM

In order to study the mechanisms involved in the receptor-mediated uptake of effector proteins, a disulfide-linked conjugate of fragment A of diphtheria toxin and monoclonal anti-mouse Thy-1.2 has been synthesized and used to inhibit T-cell function in the mouse spleen. The hybrid was synthesized by coupling cystaminyl anti-Thy-1.2 with reduced fragment A under conditions maximizing disulfide interchange and was purified by affinity chromatography on NAD-Sepharose and Sephadex G-200 gel filtration. Low concentrations of the purified hybrid were active in inhibiting the PHA stimulation of thymidine incorporation by those mouse spleen cells which exhibit Thy-1.2 on their surface (T cells), while the lipopolysaccharide-stimulated B-cell population was unaffected. In similar experiments, purified, monospecific, goat anti-mouse IgM was coupled to the toxic A subunit of the plant toxin ricin. Incubation of spleen cells with this hybrid protein resulted in the preferential loss of the B-cell population as shown by decreased response to lipopolysaccharide stimulation while the phytohemagglutinin stimulation was unaffected. It is concluded that these disulfide conjugates may act as functional analogs of diphtheria toxin and ricin, but with altered cell specificity, and are useful probes for B-cell and T-cell studies. Conjugates of this type may offer a new class of pharmacologic reagents with specificity toward a unique class of target cells.

The reactions of the sponge Cliona celata to applied stimuli

1. 1. The contractile tissues of Cliona celata were stimulated in various ways. 2. 2. Electrical stimuli were without effect. 3. 3. Pharmacological reagents were believed to have a toxic effect on the choanocytes of Cliona celata. No direct action on the contractile cells was observed. Change in pH had a similarly toxic effect. 4. 4. The response to mechanical stimulation showed a graded response to increase in stimulus strength and a short latent period, but there was no evidence of temporal summation or facilitation. Interpapillar conduction did not occur; intrapapillar conduction was most likely due to stretch.

Studies on the Myoneural Physiology of Echinodermata

ABSTRACT Spontaneous activity is a well attested characteristic of holothurian muscle (Hill, 1926; du Buy, 1936a, Wells 1942). Some preparations show regular rhythmical contractions and have been used to test pharmacological reagents (Lutz, 1930; Wyman &amp; Lutz, 1930; Iriye &amp; Dille, 1941), while in other preparations the spontaneous activity is so variable that this is not considered possible (Riesser, 1933 ; Bacq, 1939; Ambache &amp; Sawaya, 1953; Welsh, 1954). For certain preparations there has been no record of spontaneous activity (Morin, 1931 ; Fischer, 1938 ; Steinbach, 1940 ; Galambos, 1941 ; Prosser, Curtis &amp; Travis, 1951 ; Prosser, 1954; van Weel, 1955). The variations between different preparations suggest that spontaneous activity is not an inevitable property of holothurian muscle, and in fact Tao (1927) and Buddington (1937) have shown that such activity depends, in part, on the presence of intact nervous tissue. Buddington (1937) studied the cloacal breathing of Thyone, and showed that the characteristic patterns of the spontaneous contractions of isolated muscles of the cloacal complex relate to the inherent functions of each muscle in the behaviour of the animal.

The pigmentary effector system.—II

In a previous paper dealing with the pigmentary response evoked by pituitary (posterior lobe) administration in the common frog, emphasis has been laid on the necessity of discriminating between the alternatives of nervous and endocrine factors in controlling colour changes in Amphibia. In the present communication some account of the pigmentary responses evoked by physiological reagents is discussed, before passing on to more direct methods of experiment. To investigate the action of drugs on melanophores, the ideal method would be to study the effects of the latter on the isolated skin. In common with other investigators, we have, however, found that when frog’s skin is placed in Ringer’s solution a condition of extreme melanophore contraction supervenes, so that this mode of treatment is useless for demonstrating the action of drugs whose application induces expanded melanophores to contract. As Spaeth, who has studied the effect of drugs on the melanophores of fish scales, rightly insists, the administration of drugs to the intact animal is fraught with formidable objections. But the disadvantages are preeminently of the nature of limitations. Of these the most obvious are: first, the difficulty in discriminating between local, peripheral, central, or reflex effects, when the seat of action of the drug is not homogeneous, e . g . in the case of atropine which has peripheral (parasympathetic) as well as central effects, or of veratrine, which acts directly on contractile tissue, and reflexly through its excitatory effect on sensory nerve endings; secondly, the possibility of inducing death, or interfering unduly with the circulation, with quantities subminimal to produce pigmentary response. However, if these limitations are recognised, the study of drug administration can, in the light of existing pharmacological knowledge, be used to explore the possible existence of a nervous mechanism of control in colour change. In this connection both direct experiment and the study of drug administration, have prompted a bewildering series of conflicting statements from a body of investigators so numerous that it is impossible to mention them individually. Fuchs, in his extensive survey of the literature—there are about 150 papers on Amphibian pigmentary changes—comments upon this lack of unanimity: “Wir müssen diese verschiedene Reaktionen auf ein und dasselbe Reagens als physiologische Artverschiedenheiten ansehen.” Foremost among those who have studied pigmentary responses to drugs in Amphibia are Biedermann, Bimmermann, Carnot, and Fuchs (1906). The reagents investigated by previous workers include veratrine, strychnine, santonin, curare, brucine, coniine, morphine, atropine, and eserine; but none have systematically applied modern knowledge of pharmacological reagents, using those whose action is most instructive. Apart from Lieben’s important work (1906) on adrenalin, the only observation which is specially interesting is Fuchs’ discovery that nicotine in amount adequate to produce motor paralysis induces darkening of the frog’s skin.