Philip Rosenberg

Scope of the AIDS Epidemic in the United States

Two-dimensional deconvolution techniques are used here to reconstruct age-specific human immunodeficiency virus (HIV) infection rates in the United States from surveillance data on acquired immunodeficiency syndrome (AIDS). This approach suggests that 630,000 to 897,000 adults and adolescents in the United States were living with HIV infection as of January 1993, including 107,000 to 150,000 women. The estimated incidence of HIV infection declined markedly over time among white males, especially those older than 30 years. In contrast, HIV incidence appears to have remained relatively constant among women and minorities. As of January 1993, prevalence was highest among young adults in their late twenties and thirties and among minorities. An estimated 3 percent of black men and 1 percent of black women in their thirties were living with HIV infection as of that date. If infection rates remain at these levels, HIV must be considered as endemic in the United States.

Dissociation of enzymatic activity from lethality and pharmacological properties by carbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipases A2

Abstract Carbamylation of 9 out of 10 lysine residues in the toxic phospholipase A2 from N. nigricollis venom decreased its lethality at least 8-fold and abolished its direct hemolytic and anticoagulant activities, while the enzymatic activity, as measured on purified substrates, decreased only about 50%. Likewise, carbamylation of 3 out of 5 lysines in the relatively less toxic N. naja atra phospholipase induced detoxification and caused a loss of its blocking activity on the phrenic nerve-diaphragm preparation, while its enzymatic activity on purified substrates was unaltered. Results obtained when 7.4 out of 10 lysines in N. nigricollis phospholipase were carbamylated indicate that basicity is not an absolute requirement for high lethal potency, hemolytic activity or cardiotoxicity. The extent of phospholipid hydrolysis induced in erythrocytes, rabbit plasma, phrenic nerve-diaphragm preparation, brain minces and brain synaptic plasma membranes by incubation with the carbamylated enzymes was in agreement with their enzymatic activities as measured on purified substrates. Levels of phospholipid hydrolysis in heart, lung and kidney of mice given phospholipase intravenously, and in brain synaptic plasma membranes from rats given phospholipase intraventricularly, showed that carbamylated derivatives of N. nigricollis phospholipase A2 lost their ability to reach and/or hydrolyze substrates in vivo. However, the decrease in in vivo phospholipid hydrolysis did not correlate with the decrease in toxicity since, at comparably low levels of phospholipid hydrolysis, some phospholipases were lethal and others were not. Moreover, when intraventricularly administered, both lethal amounts of the native N. naja atra enzyme and its nonlethal carbamylated derivatives produced equally low hydrolysis of synaptic membrane phospholipids. By means of lysine carbamylation, a dissociation between hydrolytic activity and pharmacological properties of phospholipases A2 has been achieved. We suggest, therefore, that the toxicity of pure phospholipases is primarily due to a direct effect which does not correlate with levels of phospholipid hydrolysis and that this direct effect is prominent in the relatively toxic phospholipases while it is less manifest in the relatively non-toxic enzymes.

Relationship between catalysis and toxicological properties of three phospholipases A2 from elapid snake venoms

Abstract Phospholipases A 2 from three elapid snake venoms were studied in order to determine if differences in toxicity correlate with differences in pattern or level of phospholipid hydrolysis. The comparatively toxic basic phospholipase A 2 isolated from Naja nigricollis venom exhibits a cardiotoxic action following iv administration in mice that is not exhibited by the less toxic neutral phospholipase A 2 from Hemachatus haemachatus venom or the acidic phospholipase A 2 from Naja naja atra venom. This cardiotoxic action correlates with high levels of phosphatidylserine hydrolysis in heart. Levels and patterns of phospholipid hydrolysis in heart, lung and kidney following iv administration suggest that only the N. nigricollis enzyme has the ability to penetrate permeability barriers in the heart. No cardiotoxic effects are seen following intraventricular injection of a lethal dose of the phospholipases A 2 . All three phospholipases A 2 (12 μg/ml) abolish the directly and indirectly elicited muscle twitches of the rat phrenic nerve-diaphragm preparation. This block, in normal or altered bathing media, appears to correlate with the level of phospholipid hydrolysis for the N. naja atra enzyme, but not for the N. nigricollis enzyme. These results suggest that N. nigricollis phospholipase A 2 acts by another mechanism in addition to phospholipid hydrolysis.

Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom—II: Pharmacological properties in relationship to enzymatic activity☆

Abstract Despite a remarkable degree of homology in amino acid sequence, the neutral phospholipase A 2 from Hemachatus haemachatus venom is much less toxic than the basic phospholipase A 2 from Naja nigricollis venom, the i.v. ld 50 in mice for the two being, respectively, 8.6 and 0.63 mg/kg. Similarly following intraventricular injection into rats, the neutral phospholipase showed convulsant and lethal dose 50 values of about 7.5 and 15 μg per rat, respectively, whereas corresponding values for the basic phospholipase were 0.5 and 0.5 μg per rat. Death appears to be due to congestion, hemorrhage and edema in the lungs. Consideration of dosages required and times until onset of action suggests that, dependent upon the route of administration, the effect is either mediated via a central action or is due to a direct effect on the cardiac and/or respiratory system in the periphery. The pattern and extent of phospholipid hydrolysis in various brain regions was similar following intraventricular injection of the two phospholipases so that no relationship between phospholipid hydrolysis and lethal potency could be established. Concentrations of 5 and 10 μmg/ml of the N. nigricollis and H. haemachatus phospholipases, respectively, were required to block electrical activity of the isolated single electroplax. The ultrastructural changes produced by both phospholipases were also similar. Parallel to the somewhat greater potency on the electroplax, N. nigricollis phospholipase produced slightly greater overall hydrolysis in the innervated and non-innervated membranes of the electroplax than did H. haemachatus phospholipase. The results suggest that these two phospholipases do not have a specific junctional effect and that the small difference in potency on the junction cannot be responsible for the large difference in lethality observed in mammalian species.

Reversible block of axonal conduction by curare after treatment with cobra venom

Abstract After treatment of squid giant axon with cobra venom (CV), cetyltrimethyl-ammonium chloride (CTA) or a combination of the two, d -tubocurarine (curare) caused reversible block of conduction at concentrations as low as 5 × 10 −4 M. On control axons, 1.4 × 10 −2 M curare had little effect on electrical activity. Other compounds rendered active by the treatment included chlorisondamine and protamine; the actions of physostigmine and di iso propylfluorophosphate were potentiated. Several quaternary ammonium compounds, among them prostigmine and acetylcholine, were not rendered active after pretreatment. Most tertiary amines affected the untreated squid axon action potential in almost the same concentrations that affect the synapses of the single electroplax. In contrast to lipid-insoluble quaternary ammonium compounds, which did not significantly alter the action potential of the untreated axon, three lipid-soluble quaternary compounds were as effective on the axon as on other conducting membranes. The results indicate that a strong lipid barrier surrounds the squid axon. A mechanism for the breakdown of this barrier by CV and CTA is discussed. Possible explanations are presented for the inability of this treatment to permit effects of some lipid insoluble quaternary compounds.

ABILITY OF VENOMS TO RENDER SQUID AXONS SENSITIVE TO CURARE AND ACETYLCHOLINE.

Abstract 1. 1. Cottonmouth-moccasin venom is more potent than copperhead moccasin, fer-de-lance or Western diamondback rattlesnake venom in rendering (+)-tubocurarine (curare) capable of affecting conduction of the squid giant axon. 2. 2. Bee venom is more potent than the snake venoms. It irreversibly blocks axonal conduction in a concentration of 10 μg/ml. However, in lower concentrations (1–2 μg/ml) it does not render curare effective. 3. 3. Following exposure to cottonmouth venom, 8.8·10−4 M acetylcholine markedly decreased axonal electrical activity. 4. 4. Physostigmine was antagonistic to the action of acetylcholine. An explanation for this apparently surprising effect is offered. 5. 5. Cottonmouth venom also rendered the following compounds active: decamethonium, 2-pyridine aldoxime methiodide and 2-benzoylpyridine oxime. The potency of atropine and ethanol was increased by cottonmouth venom. 6. 6. Carbamylcholine in 2.8·10−2 M did not significantly affect conduction after pretreatment with cottonmouth venom while 1·10−2 M nicotine had a weak effect.

Factors in venoms leading to block of axonal conduction by curare

Abstract Several enzyme and other fractions of venoms were studied to determine the component (or components) responsible for rendering axonal conduction sensitive to the action of acetylcholine and curare. A phospholipase A (EC 3.1.1.4)-rich fraction from cobra venom and phospholipase D (EC 3.1.4.4) rendered the squid axon sensitive to the action of curare. Venom phosphodiesterase (EC 3.1.4.1), apparently also active, was found to have considerable phospholipase A activity. A phospholipase A-poor fraction of cobra venom, l -amino acid oxidase (EC 1.4.3.2), Cobroxin, lysolecithin and phospholipase C (EC 3.1.4.3) were all ineffective in rendering curare active on the squid axon. All the enzymes and venom products tested were relatively weak in their direct effects on conduction except for the phospholipase A-rich fraction. Heating a solution of cottonmouth venom at an alkaline pH destroyed almost all of its phospholipase A activity whereas heating at an acid pH had little effect on this enzymic activity. Eastern diamondback rattlesnake venom hydrolyzes egg lecithin at a rate only slightly less than that of cottonmouth venom. The ability of several venoms to hydrolyze beef lecithin was also compared. The results support the assumption that the phospholipase A of venoms is responsible for their ability to increase the permeability of the squid axon allowing thereby acetylcholine and curare to penetrate into the axon and to affect conduction.

Maintenance of axonal conduction and membrane permeability in presence of extensive phospholipid splitting.

The importance of phospholipids for the maintenance of axonal conduction and membrane permeability has been investigated by treating squid giant axons with phospholipases, lysosphospholipids and fatty acids. It was found that phospholipase C, from Cloistridium welchii, induces an extensive splitting of axonal phospholipids, without however blocking conduction or increasing the penetration of a lipid-insoluble compound into the axoplasm of squid giant axons. These findings are in sharp contrast with the results obtained with phospholipase A, electrophoretically separated from Ringhals venom, where a close relationship between phospholipid splitting, block of axonal conduction, and increased penetration has been reported. Phospholipase B from bovine pancreas, and D from cabbage, did not markedly hydrolyze the axonal phospholipids nor did they affect conduction or penetration. Both purified lysolecithin and a mixture of lysophosphatides, prepared by the action of venom phospholipase A on the phospholipids of squid axons, blocked conduction and increased penetration into giant axons. In contrast to phospholipase A, which affects giant axons only if surrounded by adhering small nerve fibers, lysolecithin and the lysophosphatide mixture acted equally well on giant axons with or without adhering small nerve fibers. The other product of phospholipase A action, free fatty acids, was inert on the giant axon. On the basis of these and earlier findings, it is concluded that extensive splitting of axonal phospholipids by phospholipases is compatible with maintenance of normal function unless the hydrolytic products are themselves active. This conclusion would explain the marked differences in action found between phospholipases A and C.

In vivo reactivation by pam of brain cholinesterase inhibited by paraoxon

Abstract Pyridine-2-aldoxime methiodide (PAM) injected in amounts of from 25 to 150 mg/kg from 0.5 to 1.5 hr after 0.15 mg/kg paraoxon caused a significant increase in the cholinesterase levels of cerebral cortex, pons, cerebellum, region of area postrema in medulla, and the remainder of the medulla of rabbits killed 3 hr after the injection of paraoxon. Reactivation seemed most marked in the pons, region of area postrema and the remainder of the medulla. Similar results were obtained when rabbits were killed 24 hr after paraoxon, and there appeared to be no difference whether the first dose of PAM was given 1 or 5 hr after the inhibitor. A chloroform extraction procedure was used which appears capable of removing at least some of the extracellular paraoxon present in the brain tissue and uncombined with the enzyme at the time of sacrifice of the rabbits. It is concluded that sufficient amounts of PAM are able to penetrate into the brain to cause marked reactivation of inhibited cholinesterase. Possible reasons are discussed why other workers have not observed in vivo reactivation of brain cholinesterase by PAM.

Rapid and Reversible Block of Electrical Activity by Powerful Marine Biotoxins

Puffer-fish poison and clam poison reversibly inhibit conduction in single nerve fiber preparation of frog in a concentration of 3 x 10-10M. In the isolated electroplax of Electrophorus electricus higher concentrations block both transmission and conduction. Neither toxin is a potent acetylcholinesterase inhibitor. The mechanism of action of these toxins in blocking transmission and conduction has not yet been established.