Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide: A potent and selective fluorescent inhibitor of butyrylcholinesterase

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?7}}\text{M}\text{)}$ but not of acetylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 4 × 10}{}^{\mathrm{?4}}\text{M}\text{)}$. For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate K_i unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a K_d of 4.5 × 10^?7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.     

The effects of cadmium, manganese and aluminium on sodium-potassium-activated and magnesium-activated adenosine triphosphatase activity and choline uptake in rat brain synaptosomes

The effects of Cd²⁺, Mn²⁺ and Al³⁺ on rat brain synaptosomal sodium-potassium-activated and magnesium-activated adenosine triphosphatase (Na-K-ATPase and Mg-ATPase) activity and choline uptake were studied. All three types of metal ions inhibited Na-K-ATPase activity more markedly than Mg-ATPase activity. The rank order of inhibition of Na-K-ATPase was: $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.4 μ}\text{M}\text{) >}\text{Mn}{}_{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 955 μ}\text{m}\text{) >}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 8.3}\text{mM}$). The rank order of inhibition of Mg- was:$\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 316 μ}\text{M}\text{>}\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.5}\text{mM}\text{>}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 21.9}\text{mM}\text{).}\text{Al}{}^{\mathrm{3+}}$ was most potent in inhibiting synaptosomal choline uptake ($\text{ic}{}_{50}\text{= 24μ}\text{M}$ in the absence of Ca²⁺ and 123 μ.M in the presence of 1 mM Ca²⁺). $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 363 μ}\text{M}\text{)}$ was a more effective inhibitor of choline uptake than $\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 1.2?1.5}\text{mM}\text{)}$ . The presence of 1 mM Ca²⁺ did not alter choline uptake, nor did it antagonize the inhibitory actions of the three metals. Our observations that Cd²⁺ and Al³⁺ inhibited synaptosomal choline uptake, but did not show parallel inhibitory effects on Na-K-ATPase activity directly contradicts the ionic gradient hypothesis. These results are also discussed in relation to the in vivo neurotoxicity of cadmium, manganese and aluminium.     

Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: Transmembrane signalling relative to insulin-mimicking cellular effects

Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977); Biochem. Pharmac.27, 2589 (1978); Fedn Proc.37, 1689 (1978); and Biochem. Pharmac.29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The β-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [¹⁴C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140–160% of the basal rate; insulin increased the rate to 190–210% of the basal rate. The relative potencies of the hormones toward H₂O₂ formation and stimulation of the pentose phosphate pathway activity were: insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?11}}\text{M}\text{)}$, desalanine-insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?10}}\text{M}\text{)}$ , proinsulin $\text{(}\text{EC}{}_{50}\text{: 8 × 10}{}^{\mathrm{?9}}\text{M}\text{)}$, and NGF $\text{(}\text{EC}{}_{50}\text{: 10}{}^{\mathrm{?9}}\text{M}\text{)}$. The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an $\text{EC}{}_{50}\text{of}\text{5 × 10}{}^{\mathrm{?7}}\text{M}$, indicating a suboptimal level of H₂O₂ formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4,-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H₂O₂ complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H₂O₂ formation correlated with their abilities to promote lipogenesis from [U-¹⁴C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.     

Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom

J. M. Gutiérrez, J.A. Gpené, G. Rojas and L. Cerdas. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon23, 887–893, 1985. — The polyvalent antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was tested for its capacity to neutralize proteolytic and hemorrhagic activities of ten Costa Rican crotaline venoms. In experiments with preincubation of venom and antivenom, the latter efficiently neutralized proteolytic activities of nine venoms, with ed₅₀ ranging from 50 to 300 μl antivenom/mg venom. The venom of Bothrops nummifer was neutralized less efficiently ($\text{ed}{}_{50}\text{= 760}\text{μl/mg}$). Antivenom was also very effective in neutralizing hemorrhagic activity, having its lowest neutralizing ability against the venom of B. picadoi ($\text{ed}{}_{50}\text{= 430}\text{μl/mg}$ and its highest towards the venom of B. asper (Pacific region) ($\text{ed}{}_{50}\text{= 47 μl/mg}$). There was a significant correlation between the ability of antivenom to neutralize proteolytic and hemorrhagic effects. In spite of the ability of antivenom to neutralize hemorrhage when incubated with venom prior to injection, hemorrhage was only partially neutralized when antivenom was administered i.v. at different time periods after envenomation. This suggests that the rapid development of local hemorrhage, instead of the absence of antivenom antibodies, is the explanation for the poor neutralization observed in these types of experiments.     

Histamine release by fire ant (Solenopsis) venom.

Venoms from the fire ants Solenopsis invicta and S. geminata were free of detectable histamine but caused histamine release from rat peritoneal mast cells in vitro. On a per ant basis, venom from S. invicta ($\text{ed}{}_{50}\text{= 0·12}\text{venom reservoirs/ml}$) was four times as potent as venom from S. geminata ($\text{ed}{}_{50}\text{= 0·54}\text{venom reservoirs/ml}$). Hexane extracts of venom and a synthetic piperidine were as effective as the venom itself in producing histamine release, indicating that the piperidines in the venom are responsible for most of the activity. Intradermal injection of venom from S. geminata into human subjects produced dose-dependent wheals and subjective responses (itch and/or pain). Ten nanograms of histamine produced effects approximately equivalent to the venom of a single ant and the antihistamine diphenhydramine significantly reduced the wheal and subjective responses to the venom. It was concluded that histamine release plays a major role in the action of fire ant venoms.     

The effect of 4-acetamidophenol on prostaglandin synthetase activity in bovine and ram seminal vesicle microsomes.

The effect of paracetamol (4-acetamidophenol) on PG synthetase activity was tested in bull (BSVM) and ram (RSVM) seminal vesicle microsomes. In the presence of glutathione (165 μM) and hydroquinone (45 μM) in BSVM paracetamol (up to 67 μM) had no effect or inhibited ($\text{IC}{}_{50}\text{= 1500 μ}\text{M}$) the enzymic activity. In the absence of cofactors paracetamol (67–667 μM) stimulated the generation of PGs in BSVM. Also in RSVM paracetamol (33–333 μM) stimulated PG and malondialdehyde generation as well as oxygen consumption, provided that the exogenous cofactors were omitted from the incubation mixture. Hydroquinone (150 μM) was able to abolish the stimulatory effect of paracetamol on the enzymic activity. We conclude that paracetamol can replace hydroquinone and other cofactors for cyclo-oxygenation of arachidonic acid in BSVM and RSVM. The inhibitory effect of paracetamol on PG synthetase activity occurs only in the abundance of exogenous cofactors in the microsomal preparation.     

Antiviral, antitumor, and thymidylate synthetase inhibition studies of 5-substituted styryl derivatives of 2'-deoxyuridine and their 5'-phosphates

2′-Deoxyuridine derivatives containing styryl, 3-nitrostyryl, 4-nitrostyryl, and phenylethyl groups substituted at the 5-position of the pyrimidine ring have been evaluated for their effects on vaccinia and herpes simplex virus replication (in primary rabbit kidney cell cultures) and mouse leukemia L-1210 cell culture growth. 5-Phenylethyl-2′-deoxyuridine inhibited herpes simplex (type 1 and 2) virus-induced cytopathogenicity by 50 per cent at a dose (id₅₀) of 10–30 μg/ml. It was inactive against tumor cell growth. The corresponding styryl derivative showed an id₅₀ of 30–70 μg/ml for herpes simplex virus, 20 μg/ml for vaccinia virus, and 280 μg/ml for L-1210 cell growth. 5(E)-(3-Azidostyryl)-2′-deoxyuridine 5′-phosphate inhibited vaccinia replication with an IC₅₀ of 20 μg/ml and L-1210 cell culture growth with an id₅₀ of 80 μg/ml. The nucleotides of these compounds were all potent reversible inhibitors of thymidylate synthetase (Lactobacillus casei) with the following $\text{K}{}_{\mathrm{i}}\text{K}{}_{\mathrm{m}}$ ratios: 3-nitrostyryl, 0.035; 4-nitrostyryl, 0.05; 3-azidostyryl, 0.06; styryl, 0.08; and phenylethyl, 0.31. The photodecomposition of the azidostyryl derivative, a photoaffinity labeling reagent for thymidylate synthetase, was examined at two wavelengths.     

Rat uterine contractility and the activities of uterine adenyl cyclase and phosphodiesterase during the estrus cycle

A study has been made of the activities of rat uterus adenyl cyclase and phosphodiesterase, and the inhibitory effect of theophylline on uterine contractions at various stages of the estrus cycle. The activities of adenyl cyclase and phosphodiesterase at proestrus were found to be 2.52 ± 0.28 pmoles/min/mg of protein and 1.90 ± 0.30 nmoles/min/mg of protein, respectively. Activities of both the enzymes increased from proestrus to peak values at metestrus (early metestrus for adenyl cyclase and late metestrus for phosphodiesterase) and then fell until the following proestrus. Theophylline inhibition of oxytocin-induced maximum uterine contractions was found to be greatest at early metestrus. Similarly, the oxytocin concentration producing 40 per cent maximum contraction in the presence and absence of the theophylline

$\text{Oxytocin}\text{(E}{}_{40}\text{)+}\text{theophylline}\text{Oxytocin}\text{(E}{}_{40}\text{)}$

was also highest at early metestrus. These findings indicate that, at early metestrus, cellular turnover of cyclic AMP may be high and that the exaggerated inhibition of oxytocin- induced maximum contraction and the high value of

$\text{Oxytocin}\text{(E}{}_{40}\text{)+}\text{theophylline}\text{Oxytocin}\text{(E}{}_{40}\text{)}$

could be due to extensive accumulation of cyclic AMP produced from theophylline inhibition of phosphodiesterase.     

Dopaminergic effects of buspirone,a novel anxiolytic agent

The novel anxiolytic drug buspirone raised striatal levels of the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) 1 hr after oral administration. This effect was dose-dependent with a peak at 60 min. No changes were observed in the levels of 3-methyxytyramine (3MT), the extraneuronal metabolite of dopamine. Noradrenaline, serotonin and its metabolite 5-hydroxyindoleacetic acid (5HIAA) were not affected. Buspirone displaced [³H]spiroperidol from striatal binding sites, with an ic₅₀ (1.8 × 10^?7 M), comparable to that of clozapine ($\text{ic}{}_{50}\text{= 1.4 × 10}{}^{\mathrm{?7}}\text{M}$) but considerably lower than that of haloperidol (4.7 × 10^?9 M). Buspirone was only a weak inhibitor of dopamine-stimulated adenyl cyclase. Buspirone was not active on the binding of trifluoperazine to calmodulin and did not modify calmodulin-induced activation of phosphodiesterase (PDE). Repeated administration of buspirone did not increase the number of DA receptors. These data show that, although buspirone has antidopaminergic activity, it can hardly be classified as a classic neuroleptic agent.     

Characteristics of [3H]fentanyl binding to the opiate receptor

The present study characterises the binding of the highly lipophilic opiate agonist [³H]fentanyl to homogenates of the rat central nervous system. At 25°C, association of [³H]fentanyl with its binding site was rapid ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.5}\text{min}$). Dissociation from the binding site was biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{'}\text{s}\text{= 4.0}\text{and}\text{100}\text{min}$) suggesting the existence of high and low affinity binding sites. Scatchard plots of saturation isotherms were curvilinear, confirming the presence of high (K_D = 0.46 nM) and low K_D = 4.26 nM) affinity binding sites. Increasing temperature and the concentration of sodium ion decreased the [³H]fentanyl binding. Opiate agonists, antagonists and mixed agonist-antagonists were all potent (IC₅₀'s < 20 nM) in displacing [³H]fentanyl and displacement by levorphanol and dextrorphan indicated that [³H]fentanyl binding was stereospecific. The μ and δ selective peptides, morphiceptin and [D-Ala²,D-Leu⁵]enkephalin, had IC₅₀ values of 87 and 9.2 nM respectively. The regional distribution of [³H]fentanyl binding was in the rank order striatum ? midbrain > hypothalamus > cortex > hippocampus > brainstem > spinal cord > cerebellum. Comparison of [³H]fentanyl, [³H]naloxone and [³H-d-Ala², d-Leu⁵]enkephalin binding in the hypothalamus-thalamus (μ-enriched) compared with the frontal cortex-striatum (δ-enriched) indicated that the pattern of [³H]fentanyl labelling was similar to that obtained with [³H]naloxone, but differed from that obtained with [³H-d-Ala²,d-Leu⁵]enkephalin. These characteristics suggest that [³H]fentanyl binds to the μ-opiate receptor. These findings are discussed in relation to the high lipid solubility of fentanyl as compared with morphine.     

Kinetic studies on the metabolism of ethylmorphine by isolated hepatocytes from adult rats

Hepatocytes of adult rats were isolated by infusion of a hyaluronidase collagenase mixture. High yields of cells excluding trypan blue were obtained. These cells, in Hank's buffer containing rat serum and 0.1% glucose, $\text{N-}\text{demethylate}\text{[}{}^3\text{H-CH}{}_3\text{-N]}$ethylmorphine. The formaldehyde initially formed is further metabolized to tritiated water. Fifteen per cent of the original metabolic activity was observed after 21 hr at 37° in 5% CO₂-air, and cumulative metabolism is linear for up to 90 min under these conditions. The K_m for the N-demethylation of [³H-CH₃-N]ethylmorphine is 50 μM, 20 per cent of the value observed for this reaction by musomal preparations. An active transport of the substrate into the cell is postulated to account for this difference.     

Effect of diphenylhydantoin on the uptake and catabolism of l-[3H]norepinephrine in vitro in rat cerebral cortex tissue

The effect of diphenylhydantoin on the accumulation of [³H]norepinephrine in vitro was examined in brain slices prepared from rat cerebral cortex. High concentrations of diphenylhydantoin (10^?3 M) caused a significant reduction in the 5-min accumulation of [³H]norepinephrine. On the other hand, 10^?5–10^?4 M diphenylhydantoin facilitated the 20-min accumulation of [³H]norepinephrine. This facilitative action of diphenylhydantoin was (1) associated with a reduction in oxidative catabolism of [³H]norepinephrine and (2) abolished by the 2-hr pretreatment of rats with 100 mg/kg of nialamide (i.p.). The inhibitory action of diphenylhydantoin on the oxidative catabolism of [³H]norepinephrine was observed in both whole and lyzed crude synaptosomal preparations. When diphenylhydantoin and pargyline were compared, it was found that pargyline $\text{(}\text{id}{}_{50}\text{= 1.5 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ was 37 times more effective than diphenylhydantoin $\text{(}\text{id}{}_{50}\text{= 5.5 × 10}{}^{\mathrm{?5}}\text{M}\text{)}$ in inhibiting the oxidative deamination of [³H]norepinephrine. These results suggest that diphenylhydantoin alters norepinephrine metabolism in cerebral cortex slices by an inhibitory action on (1) monoamine oxidase activity and (2) the neuronal uptake system.     

Interaction of aliphatic N-hydroxylamines with microsomal cytochrome P450: nature of the different derived complexes and inhibitory effects on monoxygenases activities.

Primary N-hydroxylamines, RR′R″CNHOH, produce difference spectra of liver microsomes from variously pretreated rats with peaks around 420 nm and troughs around 392 nm which are interpreted as the result of the hydroxylamines binding to cytochrome P450-Fe (III) through their oxygen atom. The hydroxylamines interact with dithionite-or NADPH-anaerobically reduced micro-somes giving peaks around 423 nm. In the presence of NADPH, oxygen and microsomes, all the hydroxylamines of the type RR'CHNHOH lead to 455 nm difference spectra which should correspond to cytochrome P450-Fe (II)-RR′CHNO complexes. These hydroxylamines also act as strong inhibitors of aniline hydroxylase, P-nitroanisole-O-demethylase and 7-ethoxycoumarin-O-dealkylase activities of PB-pretreated rat liver microsomes. In most cases, they are better inhibitors than metyrapone or SKF 525A. These inhibitory effects of hydroxylamines are related to their ability to lead to 455 nm absorbing complexes; For instance, 1-hydroxylamino-2phenyl-ethane exhibits the best apparent Ks for the 455 nm absorbing complex formation and is also the best inhibitor of microsomal $\text{7-}\text{ethoxycoumarin}\text{-O-}\text{dealkylase}\text{(}\text{I}{}_{50}\text{= 0.5 μ}\text{M}\text{)}$.     

Turnover of cytidine and uridine components of acid-soluble pool and RNA of cytoplasmic ribosomes after repeated phenobarbital administration

The administration of phenobarbital in drinking water (1 g/l) to rats that had received labeled orotic acid three days before does not affect the half-life of uridine components of the liver nucleotide pool $\text{(t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5.9}\text{days}\text{)}$. The half-life of the cytidine components in the control group is 6.2 days. In the experimental group the decrease of specific activity of cytidine components of the pool is biphasic. The decrease is faster during the first six days of phenobarbital administration $\text{(t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.5}\text{days}\text{)}$ whereas a slower decrease $\text{(t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 8.2}\text{days}\text{)}$ is observed during the subsequent administration of the drug. The decrease of specific activity of uridylic acid in RNA from cytoplasmic ribosomes is faster in the control group $\text{(t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.9}\text{days}\text{)}$ than in the experimental group $\text{(t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5.9}\text{days}\text{)}$. Similarly, the decrease of the specific activity of cytidylic acid isolated from the ribosomal RNA is markedly slowed down $\text{(t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of controls = 5.9 days, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{of experimental group}\text{= 7.5}\text{days}\text{)}$.     

Bradykinin receptor-like binding studied with iodinated analogues

The biological potencies of iodinated derivatives of Tyr¹ kallidin, Tyr⁵ bradykinin, and Tyr⁸ bradykinin were determined on isolated rat uteri and bovine uterine strips. Monoiodotyr¹ kallidin was the most potent of the derivatives (0.9 times as active as bradykinin on rat uterus). Monoiodotyr⁸ bradykinin was 0.2 and iodotyr⁵ bradykinin was less than 0.001 times as active as bradykinin. Using mono-[¹²⁵I]-Tyr¹ kallidin as a receptor probe, we demonstrated receptor-like binding in a subcellular fraction from bovine myometrium. Binding of bradykinin showed an apparent K_assoc. of 10¹⁰ M^?1, and was highly specific. The optimum pH for saturable binding of [¹²⁵I]-Tyr¹ kallidin to the homogenized myometrium was 6.0 to 6.5. Several cations inhibited binding, with calcium the most effective ($\text{id}{}_{50}\text{= 20}\text{mM}$), and potassium the least effective ($\text{id}{}_{50}\text{= 220}\text{mM}$). The problems encountered in using radioactive bradykinins to search for and study receptors in bradykinin target tissues are discussed.     

Antitumor effects of bis-thiosemicarbazones— inhibition of deoxyribonucleic acid synthesis in vitro

A group of bis-thiosemicarbazones was evaluated for potential antitumor activity, using the L1210 murine leukemia in cell culture. Drug levels required to inhibit DNA synthesis by 50 per cent, under standard conditions, were determined. The most potent of the agents examined had the structure X[CH₂CR₁=NNHCSNHR₂]₂ where X = C or S and R₁ = R₂ = CH₃. Optimal activity was also obtained with R₁ = H and R₂ = CH₃ only when X = S. The most potent derivatives inhibited DNA synthesis by 50 per cent within 10 min at 10^?6 M levels (id₅₀). Metal chelates of several compounds tested were extremely potent inhibitors of DNA synthesis ($\text{id}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{M or less}$). Insolubility in water and short duration of action in vivo may limit effectiveness of the bis-thiosemicarbazones.     

Isolation and characterization of the main toxic fraction from the venom of the false horned viper (Pseudocerastes fieldi)

R. Batzri-Izraeli and A. Bdolah. Isolation and characterization of the main toxic fraction from the venom of the false horned viper (Pseudocerastes fieldi). Toxicon20, 867–875, 1982—The venom of Pseudocerastes fieldi was subjected to gel filtration on Sephadex G-75. Most of the protein and lethality of the venom were eluted in a major symmetrical peak (C). The lethality of this peak is confined to a basic protein fraction, Cb (pI > 9.5) separable by DEAE-cellulose chromatography. Two proteins with molecular sizes close to 16,000 daltons were isolated from this fraction by preparative acidic gel electrophoresis in the presence of Triton X-100. One of the proteins (CbII) is lethal to mice ($\text{ld}{}_{50}\text{= 1}\text{mg/kg}$) and shows phospholipase A activity as well as direct hemolytic activity. The other protein (CbI) does not reveal any known biological activity. However, upon recombination of the two a synergistic lethal activity is evident ($\text{the}\text{ld}{}_{50}\text{of the mixture = 0.25 mg/kg}$). It is suggested that CbI may be a specifier which potentiates the toxicity of the phospholipase A at the target site.