Polyamines and platelet aggregation

High blood concentrations of the naturally occurring polyamines have been reported in leukemia, psoriasis, cystic fibrosis and polycythemia rubra vera. Spermidine and spermine inhibit $\text{in}$$\text{vitro}$ plate-let aggregation of platelet rich plasma preparations in which ADP and Ristocetin are the agglutinating agents. The proposal is made that these organic cations may modulate $\text{in}$$\text{vivo}$ platelet agglutinability.     

III. Possible mechanisms involved in the presynaptic cholinotoxicity due to ethylcholine aziridinium (AF64A) invivo

AF64A is a toxin which can diminish irreversibly cholinergic transmission $\text{in}$$\text{vivo}$ (1, 2). Disruption of neurotransmitter function $\text{in}$$\text{vivo}$ is specific to the cholinergic system when AF64A is administered in nanomolar quantities (3, 4). The mechanisms involved appear to be mediated presynaptically (2). The neurochemical and behavioral consequences of AF64A administration are reminiscent of similar measures in patients with Alzheimer's disease (5,6). Consequently, we have suggested tentatively that the AF64A treated animal may be explored as a potential animal model of this debilitating disease state (7). In this report we provide a brief overview of our recent findings using this compound $\text{in}$$\text{vivo}$, attempt to correlate these findings with those of others with similar aziridinium agents $\text{in}$$\text{vitro}$, and propose a possible mechanism of action of AF64A $\text{in}$$\text{vivo}$, based on recent observations made in our laboratories.     

Kyotorphin (tyrosine-arginine): Further evidence for indirect opiate receptor activation

Analgesia, opiate receptor binding, and neurochemical effects of kyotorphin (tyrosine-arginine) were studied in the rat. It was found that while kyotorphin, $\text{in vivo}$, causes naloxone reversible analgesia, and affects dopamine metabolism and acetylcholine turnover in the same manner as do morphine and other opiate agents, the dipeptide does not bind to mu, delta or kappa opiate receptors $\text{in vitro}$. Taken together, these data support the concept that there is an indirect action of kyotorphin on opiate receptors.     

Nasal vasoconstrictor activity of a novel PGE2 analog

A novel PGE₂ analog (CL 116,069) was shown to be effective in dogs as a nasal decongestant. Threshold doses were approximately 0.1 μg/kg with intravenous administration and between 0.08 and 4 μg with topical administration. CL 116,069 was compared to 17-phenyl-trinor PGE₂ (CL 116,147), a compound recently studied in humans, and xylometazoline, a well-known nasal decongestant. When given i.v., efficacious doses of xylometazoline tended to raise blood pressure and be shorter acting than the PGs, which did not affect blood pressure. When given topically, all three were long-acting. CL 116,069 usually had the lowest threshold and CL 116,147 usually induced the smallest response. All three agents were more effective than PGE₁ or PGE₂. A 30-day (b.i.d., topical) toxicity test with CL 116,069 produced no inflammation or nasal pathology and no loss in tissue sensitivity. $\text{In}$$\text{vitro}$ examination of xylometazoline and CL 116,069 for vascoconstrictor activity on dog isolated mucosa revealed a response profile similar to that observed with these agents $\text{in}$$\text{vivo}$; i.e., the magnitude of response was comparable for both agents but the t $\text{1}\text{2}$ was only 74 minutes for xylometazoline and greater that 6.5 hours for CL 116,069. The data suggest that CL 116,069 may provide a therapeutic alternative in which constriction of the nasal blood vessels need not be associated with a generalized vasoconstrictor liability.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

Inhibition of invitro sickling by liposome-mediated transport of amino acids into intact human red blood cells

To investigate the role of phenylalanine and tryptophane as potential antisickling agents in intact human SS-red blood cells a liposomal transport system was employed to transfer phenyl-alanine or tryptophane into intact SS-red blood cells. Aromatic amino acids and short peptides containing phenylalanine have been demonstrated to increase the minimum gelling concentration and solubility of deoxy-hemoglobin S in aqueous solution. However, these compounds do not cross the red blood cell membrane under usual incubation conditions. Incorporation of phenylalanine or tryptophane into intact SS-red blood cells $\text{via}$ liposomal transport system markedly inhibited the $\text{in}\text{vitro}$ sickling of deoxy-hemoglobin S. These findings raise the possibility that a nontoxic liposomal transport system which facilitates incorporation of antisickling agents into intact SS-RBC may have significant therapeutic implications in the treatment of sickle cell disease.     

Phospholipid membrane stabilization by dimethylsulfoxide and other inducers of friend leukemic cell differentiation

A large number of low molecular weight polar cryoprotective agents have recently been found to induce erythroid differentiation of Friend leukemic cells in vitro. The effect of these agents on membrane fluidity in phospholipid vesicles was studied by determining the solid-to-liquid crystalline phase transition using differential scanning calorimetry. Some of the inducing agents studies were found to raise the normal transition temperature ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ by a few degrees. All of these agents were found to produce a separate transition at a much higher temperature. Changes in the head group of the phospholipid, the pH, the presence of divalent cations, and the addition of other membrane-active compounds were found to significantly influence the inducing agent's effects on the $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ of phospholipid membranes.The ability of the different agents to produce a new transition at a high temperature was found to correlate well with their ability to incude Friend leukemic cell differentiation. The possible mechanisms of action of the chemical inducers, and the significance of the observed membrane effects on differentiation and malignancy are discussed. It is concluded that inducing agents decrease the fluidity and stabilize phospholipid membranes, and that their effects in cell differentiation might be initiated by a similar change in the properties of cell membranes.     

Estrogen-bridged purines: A new series of anti-tumor agents which alter cell membrane properties

An estrogen-bridged adenine derivative was equitoxic to both the P388 murine leukemia and an adriamycin-resistant subline, $\text{P388}\text{ADR}$. The drug rapidly altered several P388 and $\text{P388}\text{ADR}$ membrane properties resulting in impaired nucleoside transport and increased membrane hydrophobicity. Resistance to anthracyclines in $\text{P388}\text{ADR}$ is associated with an operational barrier to drug retention which was reversed by exposure to the estrogen-bridged adenine derivative. These results suggest further exploration of the estrogen-bridged purines as chemotherapeutic agents.     

Inhibitory beta-adrenoceptors in the urinary bladder of the rat

The β-adrenoceptors of the urinary bladder were investigated in the rat $\text{in}$$\text{vivo}$. Isoprenaline, and the β₂-stimulating agents terbutaline and salbutamol elicited relaxation of the detrusor muscle decreasing the intravesical pressure. The responses were not affected by the β₁-blocking agents practolol or H 93/26 but were totally abolished by propranolol and the β₂-blocking agent H $\text{35}\text{25}$. Noradrenaline given after dihydro-ergotamine caused relaxation of the detrusor muscle and this response was completely blocked by propranolol and H $\text{35}\text{25}$. It is concluded that the β-adrenoceptors of the rat urinary bladder belong to the type of inhibitory receptors classified as β₂-receptors in other organs.     

Enzymatic release of nitric oxide from L-alanosine,an antineoplastic antibiotic

L-Alanosine is an antineoplastic drug which is the 3-isonitramino analog of $\text{L}$-aspartic acid. The drug is known to be metabolized to the corresponding 2-oxo acid. Unlike the parent amino acid, the 2-oxo acid is unstable under mild conditions. When the 2-oxo acid is generated $\text{in}\text{vitro}$ by the aerobic action of $\text{L}$-amino acid oxidase on $\text{L}$-alanosine, the reaction mixture contains products capable of diazotizing sulfanilamide and of reducing ferricytochrome $\text{c}$ to ferrocytochrome $\text{c}$. It is thus likely that, as expected from model reactions, the unstable 2-oxo acid derived from $\text{L}$-alanosine decomposes into nitric oxide and other reactive free-radical species. Enzymatically promoted production of highly cytotoxic nitric oxide may pertain to the biological activity of the antibiotic. The reaction should prove extrapolable to the design of other enzyme-activated cytotoxic agents.     

Effects of n-Alkanols on the membrane fluidity of chick embryo heart microsomes

The $\text{n-}\text{alkanols}$ from butanol through octanol are membrane perturbing agents that fluidize the microsomal membranes of 20-day-old chick embryo hearts as measured by the fluorescence depolarization of 1,6-diphenylhexatriene. In terms of the aqueous concentrations of $\text{n-}\text{alkanols}$ the fluidizing effect increases with increasing number of carbons per $\text{n-}\text{alkanol}$. In terms of the membrane concentrations of $\text{n-}\text{alkanols}$ the fluidizing effect is roughly equivalent for all the $\text{n-}\text{alkanols}$ studied.     

Comparative kinetic-ionic strength study of two differently charged cytochrome C: effects are limited to overall charge.

The reduction kinetics of two differently charged cytochromes $\text{c}$, horse cytochrome $\text{c}$ and $\text{Rhodosprillum rubrum}$ cytochrome $\text{c}{}_2$, by ferrous EDTA^2? were studied as a function of ionic strength. Since both proteins have nearly the same heme edge region, but have very different overall surface charge, this comparative study served as a direct test of the utility of small nonbinding non-physiological redox agents in the study of the charge of electron transfer sites of redox proteins. Calculations based on the ionic strength-kinetic data yielded protein charges of +10 and +2.3 for cytochrome $\text{c}$ and cytochrome $\text{c}{}_2$ respectively and compared well with values of +9 and +3 for the overall charge of the proteins based on acidic and basic amino acid residues. It is concluded that ionic strength effects upon the redox kinetics with such nonbinding nonphysiological redox agents reflect the influence of the overall protein charge and not the localized charge of the presumed site of electron transfer.     

Selective toxicity of the polyene antibiotics and their methyl ester derivatives.

The methyl ester hydrochlorides of amphotericin B and nystatin are less effective than the parent compounds in causing K⁺ release from human erythrocytes. The parent compounds and the derivatives are of comparable activity toward $\text{Candida albicans}$. The enhanced selective toxicity of polyene methyl ester salts for $\text{C. albicans}$ may mean that these antibiotics will be more effective therapeutic agents for systemic fungal infections.     

Metabolic activation of procarbazine

Procarbazine chemical degradation and rat $\text{in vitro}$ and $\text{in vivo}$ metabolism have been investigated. Procarbazine was rapidly oxidized to the azo derivative in aqueous solution in the presence of oxygen. $\text{In vitro}$ rat liver supernatany and microsomal preparations oxidize the azo function to azoxy isomers and further hydroxylate these metabolites in a manner that may by analogous to 1,2-dimethylhydrazine metabolism. The hydroxylated metabolites are activated species that chemically react to give methylating and alkylating agents. An additional metabolic pathway was observed $\text{in vivo}$. This suggests that procarbazine may be converted to free radical intermediates that decompose to give methane and N-isopropyltoluamide. Procarbazine metabolites have been separated and identified using high performance liquid chromatography and direct probe chemical ionization mass spectrometry.     

The effect of folate and folate analogs upon dihydrofolate reductase and DNA synthesis in kidneys of normal mice

A single subcutaneous injection of folate, homofolate or MTX resulted in the inhibition of the activity of dihydrofolate reductase in homogenates prepared from the kidneys of normal mice. Stimulation of ³H-thymidine uptake occurred in the kidneys of treated animals approximately 30 hr after administration of either folate or homofolate, and reached a peak 72 hr after administration. The effects of folate and MTX on dihydrofolate reductase activity $\text{in}$$\text{vivo}$ were also determined. One hr after administration of 15 mg/kg methotrexate (MTX) or 300 mg/kg folate, enzyme activity $\text{in}$$\text{vivo}$ was inhibited by 90%.³H-deoxyuridine uptake was neither stimulated nor depressed after treatment with MTX. After administration of folate, uptake of ³H-deoxyuridine was stimulated at approximately 30 hr after drug-treatment and reached a peak at 72 hr after folate administration. Treatment with xanthopterin had no effect on the activity of dihydrofolate reductase $\text{in}$$\text{vitro}$. Xanthopterin stimulated uptake of both deoxyuridine and thymidine in an identical manner.The increased DNA synthesis that occurs in animals after treatment with agents that cause renal damage is distinct from the effect these agents have upon dihydrofolate reductase. Nucleoside incorporation after treatment with folate, homofolate, MTX or xanthopterin cannot be predicted on the basis of enzyme inhibition. Treatment with MTX, folate or homofolate results in enzyme inhibition which is not correlated with the uptake of deoxyuridine into DNA.     

Inhibition of enkephalin degradation in the guinea pig ileum

Guinea pig ileum tissue preparations contain enzymes which degrade both leucine and methionine enkephalin by cleavage of the N-terminal tyrosine residue. Similar enkephalin degrading activity is also found in the fluid bath surrounding ileum tissue preparations and appears to arise from serum and broken cell enzymes. Chelating agents such as 1,10-phenanthroline and 8-OH quinoline are effective inhibitors of enkephalin destruction by these enzymes but in the concentrations necessary to inhibit all enzyme activity, they disturb the contractility of the ileum during $\text{in}$$\text{vitro}$ bioassays. The presence of enkephalin degrading enzymes and the lack of appropriate peptidase inhibitors may hinder the determination and quantification of enkephalin release in this tissue.     

Response of the mixed function oxidase system of rat hepatic microsomes to parathion and malathion and their oxygenated analogs

$\text{In}$$\text{vitro}$ inhibition of ethylmorphine metabolism in rat hepatic microsomes by parathion, malathion, malaoxon and paraoxon was not well correlated with their effects on NADPH oxidation, cytochrome C reduction or the reduction of cytochrome P-450. A parallel relationship was observed between inhibition of ethylmorphine metabolism by parathion, malathion and malaoxon and the binding affinity of these agents to microsomal cytochrome P-450 obtained from rats pretreated with an anticholinesterase agent, Bis-[?-nitrophenol] phosphate.     

Selective killing of Leishmania infected mouse macrophages by 6-methylpurine 2′-deoxyriboside

6-methylpurine 2′-deoxyriboside killed mouse macrophages infected with amastigotes of $\text{Leishmania donovani}$ and $\text{Leishmania mexicana}$, but did not affect the growth of non-parasitized cells. $\text{Leishmania}$ extracts cleaved the non-toxic 6-methylpurine 2′-deoxyriboside to 6-methylpurine, a potent adenine antimetabolite for mammalian cells. By eliminating macrophages latently infected with $\text{Leishmania donovani}$ amastigotes, 6-methylpurine 2′-deoxyriboside could augment the effects of leishmanicidal agents $\text{in vivo}$.     

Chloroquine susceptibility in malaria: dependence on exposure of parasites to the drug.

Chloroquine-resistant $\text{P.}$$\text{berghei}$ is as susceptible to chloroquine as chloroquine-susceptible $\text{P.}$$\text{berghei}$ when adequately exposed for short periods of time (1 hour) $\text{in}$$\text{vitro}$. In both cases 3.1 mM chloroquine causes a significant decrease in infectivity of the parasites whereas 0.31 mM chloroquine is without effect. Since there is no evidence that chloroquine has a peculiar mechanism of action $\text{in}$$\text{vitro}$, these results support the hypothesis of inadequate exposure of intracellular parasites as the cause of chloroquine resistance.     

A functional cyclooxygenase enzyme is not required for mediation of the pleiotropic effects of human alpha or beta interferon

Aspirin, indomethacin, and phenbutazone at 50 μM concentration inhibit cyclooxygenase in cultured human foreskin fibroblasts as evidence by the suppression of the major prostaglandin species which aaccumulate in the culture medium. In contrast to data reported for mouse interferon on target mouse cells, these agents have no effect on the introduction of antiviral activity by human α and β interferons. Similarly, these agents have no effect on interferon induced inhibition of cell growth $\text{in vitro}$ or on interferon induced natural killer cell activity.