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R S Jacobs

Publications and source records attributed to R S Jacobs.

At least 37 records · Page 2Linked to original sources
PubMed1991

Two-step inactivation of bee venom phospholipase A2 by scalaradial.

Scalaradial (SLD), a marine natural product isolated from the sponge (Cacospongia sp., possesses anti-inflammatory properties in vivo and in vitro (Pharmacologist 32: 168, 1990). In this study we characterize its effects against bee venom phospholipase A2 (PLA2; EC 3.1.1.4). SLD is a potent inactivator of bee venom PLA2 with an IC50 value of 0.07 microM. Inactivation of bee venom PLA2 occurred in a time-dependent, irreversible manner. The rate of inactivation followed first-order reaction kinetics and was dependent on the concentration of SLD. Kinetic analysis suggested a two-step mechanism of inactivation: an initial apparent noncovalent binding (Ki = 4.5 x 10(-5) M) followed by covalent modification. The rate of inactivation was reduced markedly in the presence of excess phosphatidylcholine, suggesting that modification of the enzyme occurs at or near the substrate binding site.

Anti-Inflammatory Agents↗
PubMed1990

Inactivation of human synovial fluid phospholipase A2 by the marine natural product, manoalide.

The marine natural product, manoalide (MLD), was investigated to determine if this drug inhibited purified human synovial fluid phospholipase A2 (HSF-PLA2). Utilizing classical Michaelis-Menten kinetics, apparent Km and Vmax values for HSF-PLA2 of 1.34 mM and 0.47 mumol [3H]palmitic acid released/min/mg protein were obtained using dipalmitoylphosphatidylcholine (DPPC) as the substrate, and 38.0 microM and 18.8 mumol [3H]arachidonic acid released/min/mg protein with Escherichia coli as a natural substrate. These kinetic parameters were utilized subsequently to evaluate the inhibitory effects of manoalide on HSF-PLA2. Inhibition of HSF-PLA2 by MLD was concentration and time dependent with IC50 values of 0.2 and 0.02 microM for DPPC and E. coli respectively. Dialysis studies and examination of DPPC or E. coli hydrolysis versus enzyme concentration indicate that MLD is an irreversible inhibitor of HSF-PLA2. Substrate specificity was also examined in the absence and presence of MLD using dipalmitoylphosphatidylethanolamine (DPPE) as a substrate. MLD inhibited the hydrolysis of DPPE (greater than 90% inhibition at 2 microM), and preliminary results indicate that DPPC was more readily hydrolyzed than DPPE under the substrate conditions of the assay. While the cellular source of secreted HSF-PLA2 is unknown, these studies indicate that MLD can inactivate secreted phospholipase A2 isolated from patients with inflammatory joint disease.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

Luffolide, a novel anti-inflammatory terpene from the sponge Luffariella sp..

Luffolide (4) is a minor metabolite of the sponge Luffariella sp. from Palau. The structure of luffolide was determined by single crystal X-ray analysis. Luffolide is relatively unstable and undergoes a complex cyclization reaction to give the hexacyclic products 5 and 6. Luffolide (4) has some of the anti-inflammatory properties of manoalide (1): this may help to define the chemical reaction between manoalide (1) and phospholipase A2.

Animals↗
PubMed1989

Synthesis, in vitro acetylcholine-storage-blocking activities, and biological properties of derivatives and analogues of trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol).

Eighty-four analogues and derivatives of the acetylcholine-storage-blocking drug trans-2-(4-phenylpiperidino)-cyclohexanol (vesamicol) were synthesized, and their potencies were evaluated with the acetylcholine active-transport assay utilizing purified synaptic vesicles from Torpedo electric organ. The parent drug exhibits enantioselectivity, with (-)-vesamicol being 25-fold more potent than (+)-vesamicol. The atomic structure and absolute configuration of (+)-vesamicol were determined by X-ray crystallography. The absolute configuration of (-)-vesamicol is 1R,2R. Structure-activity evidence indicates that (-)-vesamicol does not act as an acetylcholine analogue. Alterations to all three rings can have large effects on potency. Unexpectedly, analogues locking the alcohol and ammonium groups trans-diequatorial or trans-diaxial both exhibit good potency. A potent benzovesamicol family has been discovered that is suitable for facile elaboration of the sort useful in affinity labeling and affinity chromatography applications. A good correlation was found between potencies as assessed by the acetylcholine transport assay and LD50 values in mouse.

Acetylcholine↗
PubMed1989

Manoalide: structure-activity studies and definition of the pharmacophore for phospholipase A2 inactivation.

Manoalide is a potent antiinflammatory marine natural product and a direct inactivator of venom phospholipase A2 (PLA2; EC 3.1.1.4). Manoalide has been shown to irreversibly inhibit PLA2, with the corresponding modification of a selective number of lysine residues. The mechanism of inactivation has not yet been elucidated and structure-activity relationship studies were, therefore, performed in order to determine the contributions of the various functional groups incorporated in the gamma-hydroxybutenolide, alpha-hydroxydihydropyran, and trimethylcyclohexenyl ring systems to the efficacy (irreversibility) and potency of this series of inhibitors. These studies indicate that 1) the presence of the hemiacetal in the alpha-hydroxydihydropyran ring is required for irreversible binding of manoalide, 2) the gamma-hydroxybutenolide ring is involved in the initial interaction of manoalide with PLA2, and 3) the hydrophobic nature of the trimethylcyclohexenyl ring system allows nonbonded interactions between manoalide and PLA2 that enhance the potency of these analogs. These structure-activity relationship studies suggest that the closed ring form of manoalide is the predominant molecular species that accounts for the selective and potent inhibition of PLA2 by manoalide. Elucidation of the mechanism awaits further detailed physicochemical studies on the structure of the manoalide (analog)-protein adducts in model systems and using PLA2.

Aldehydes↗
PubMed1989

Selective inhibition of cytokinesis in sea urchin embryos by low concentrations of stypoldione, a marine natural product that reacts with sulfhydryl groups.

Stypoldione is a marine natural product that inhibits cells division in marine embryos and in mammalian cell cultures. The mechanism responsible for the ability of the compound to inhibit cell division is not known. The compound was found in early studies to inhibit polymerization of tubulin into microtubules in vitro, which lead to the suggestion that inhibition of microtubule polymerization in cells might be responsible for the ability of the compound to inhibit cell division. More recently, stypoldione was found to react covalently with the sulfhydryl groups of a number of proteins including tubulin and with sulfhydryl groups of peptides and small molecules. Thus, stypoldione could potentially react with a large number of cellular targets. In the present study, we have examined the effects of stypoldione on the organization of microtubules and chromatin in cells, in relation to the ability of the compound to inhibit cell division. We used indirect immunofluorescence light microscopy of fixed and stained sea urchin embryos during the first and second divisions after fertilization, with stains specific for tubulin and DNA. We found that stypoldione exerted qualitatively different effects on cell division and microtubule organization and function at different concentrations. At the lowest effective concentrations, 5-10 microM, stypoldione selectively inhibited cytokinesis. Mitotic division occurred normally, usually with no discernible perturbation of microtubule organization or function, and cells became multinuclear. At somewhat higher concentrations, 20-40 microM, stypoldione blocked embryos before streak stage of the first division and, although microtubules were present, their organization was perturbed and they often formed unusual "spiral aster" arrays. At 80 microM and above, microtubules in blocked cells were largely absent. Thus, stypoldione uncouples cytokinesis from mitosis at the lowest effective concentrations and, although it can disrupt microtubules at relatively higher concentrations, it inhibits cell division at the lowest effective concentrations by a selective action on cytokinesis through a mechanism that does not appear to involve disassembly of microtubules.

Animals↗
PubMed1988

Inactivation of phospholipase A2 by manoalide. Localization of the manoalide binding site on bee venom phospholipase A2.

The marine natural product manoalide (MLD), a potent inhibitor of phospholipases, completely inactivates bee venom phospholipase A2 (PLA2) by an irreversible mechanism. It has been proposed [K. B. Glaser and R. S. Jacobs, Biochem. Pharmac. 36, 2079 (1987)] that the reaction of MLD with PLA2 may involve the selective reactivity of MLD to a peptide sequence, possibly a Lys-X-X-Lys peptide. Localization of the MLD binding site on bee venom PLA2 demonstrated that upon MLD modification of bee venom PLA2 the only change in amino acid content was an apparent loss of Lys, corresponding to approximately three of the eleven Lys residues present. Selective chemical modification of Lys residues with [14C]maleic anhydride demonstrated that all eleven Lys residues on bee venom PLA2 were accessible to this reagent (11.6 mol maleyl group incorporated/mol of PLA2). Pretreatment of PLA2 with MLD (less than 0.7% residual activity) resulted in a molar ratio of 8.7, also consistent with the loss of three Lys residues upon modification by MLD. Reverse phase high performance liquid chromatography (RP-HPLC) of the cyanogen bromide (CNBr) digestion product of MLD-treated PLA2 produced three peaks (A280). The second peak showed the most intense absorbance at 434 nm. This material corresponded to residues 81-128, as determined by gas-phase microsequence analysis. Sequencing failure was observed at Lys-88 in the MLD-treated fragment. The control carboxymethylated-PLA2 fragment corresponding to residues 81-128 sequenced beyond Lys-88 without significant change in the expected yield. These data suggest that Lys-88 may correspond to one of the three MLD-modified Lys residues. The minor absorbance at 434 nm of the CNBr fragments containing residues 42-80 and 1-36 as compared to the fragment of residues 81-128 suggests that the major MLD binding fragment residues in residues 81-128.

Amino Acid Sequence↗
PubMed1988

A further study of the neuromuscular effects of vesamicol (AH5183) and of its enantiomer specificity.

1. The effects of vesamicol (2-(4-phenylpiperidino) cyclohexanol), an inhibitor of acetylcholine storage, and its two optical isomers have been studied on neuromuscular transmission in rat and frog muscle, and on nerve conduction in frog nerve. 2. Racemic vesamicol produced a pre-block augmentation of twitch tension that also occurred in directly-stimulated muscle. This effect is thus at least partially due to an increase in muscle contractility. 3. (-)-Vesamicol was approximately 20 times more potent than (+)-vesamicol in blocking twitches elicited at 1 Hz. This degree of stereoselectivity is similar to that measured for inhibition of acetylcholine uptake by isolated synaptic vesicles. Both enantiomers were equally weak in reducing nerve action potential amplitude in frog nerve. 4. Further studies with the active isomer, (-)-vesamicol, showed that, like that produced by racemic vesamicol, the neuromuscular block was highly frequency-dependent. The block was not reversed by choline or neostigmine, but was partially reversed by 4- or 3,4-aminopyridine. 5. Preliminary electrophysiological studies showed that vesamicol reduced miniature endplate potential amplitude in rapidly-stimulated frog nerve-muscle preparations. Addition of lanthanum ions increased the frequency of miniature endplate potentials and led to the appearance of apparently normal-sized potentials amongst those of reduced amplitude. 6. The results show the close agreement between pharmacological and biochemical observations indicating the suitability of the rat diaphragm as a test model for substances of this nature. The degree of reversibility of the vesamicol-induced neuromuscular block by aminopyridines was unexpected, and it is suggested that in the presence of a drug which greatly increases release, a pool of acetylcholine is capable of being released which is not normally releasable after block of storage by vesamicol. It is also considered possible that the results from the intracellular recording studies may be explained in these terms.

Anesthetics, Local↗
PubMed1988

Regulation of eicosanoid biosynthesis in vitro and in vivo by the marine natural product manoalide: a potent inactivator of venom phospholipases.

The marine natural produce manoalide has been reported to inactivate venom phospholipase A2 from several sources and phospholipase A2 from polymorphonuclear leukocytes. In this investigation, the regulation of eicosanoid production was studied both in an in vitro and in an in vivo model. The release of arachidonic acid and prostaglandin E2 was inhibited by manoalide when cultured mouse peritoneal macrophages were stimulated with phorbol myristate acetate (apparent IC50 = 0.23 microM), calcium ionophore A23187 (apparent IC50 = 0.23 microM) and zymosan (apparent IC50 = 0.18 microM). Leukotriene C4 production was inhibited by manoalide when macrophages were stimulated by A23187 (IC50 = 0.35 microM) but was enhanced when the cells were stimulated with zymosan. In an in vivo model, manoalide antagonized zymosan-induced peritoneal writhing in the mouse (ED50 = 0.71 mg/kg) and inhibited the i.p. release of 6-keto-prostaglandin F1 alpha (ED50 = 0.2 mg/kg) and leukotriene C4 (ED50 = 0.24 mg/kg). Our results suggest that: 1) manoalide modifies arachidonic acid release and metabolism to prostaglandins and leukotrienes in mouse cultured peritoneal macrophages stimulated by phorbol myristate acetate, calcium ionophore A23187 and zymosan and 2) the analgesic properties of manoalide seem to be partially correlated with reduced eicosanoid production in zymosan-stimulated mouse peritoneal exudates.

6-Ketoprostaglandin F1 alpha↗
PubMed1987

Inactivation of bee venom phospholipase A2 by manoalide. A model based on the reactivity of manoalide with amino acids and peptide sequences.

The marine natural product manoalide (MLD), a potent irreversible inhibitor of bee venom phospholipase A2 (PLA2), was shown to produce a chromophore (lambda max = 437 nm) during incubation with the enzyme. MLD also developed an identical chromophore when incubated with free lysine (Lys), cysteine (Cys) or tryptophan (Trp) but not with their N-alpha-amino-blocked analogs. These results suggest that the chromophore product was dependent on the presence of two nucleophilic groups which react by an ordered mechanism rather than by simple random collision. Lys polymers prevented MLD from inhibiting PLA2, whereas monomeric Lys did not. The optimal active polymer of Lys appeared to be a tetralysine (L4) peptide, and a degree of selectivity was obtained when the Lys residues were in a 1,4-Lys arrangement. The rate of chromophore development with PLA2 and the rate of inactivation of PLA2 by MLD appear to be independent processes. Based on these data, it is possible that the irreversible inactivation of PLA2 may involve an ordered reaction with a peptide sequence in PLA2 containing a 1,4-Lys arrangement.

Amino Acids↗
PubMed1987

Effect of pseudopterosin A on cell division, cell cycle progression, DNA, and protein synthesis in cultured sea urchin embryos.

Pseudopterosin A is a diterpene riboside isolated from Pseudopterogorgia bipinata, a soft coral of the order Gorgonacea. Pseudopterosin A inhibits the first cleavage in fertilized sea urchin eggs (Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, and Lytechinus pictus) with an IC50 of 25 microM. In time of addition studies, Pseudopterosin A (4 X 10(-5) M) progressively blocked the first cleavage when added within the first 50 min post fertilization; when added after 50 min the first division occurred normally. Cell cycle studies show that Pseudopterosin A had to be in continuous contact with the sea urchin eggs during the initial 45 min post fertilization to significantly inhibit the first cleavage. Shorter drug contact time progressively reduced the degree of inhibition, suggesting that inhibition of cytokinesis by Pseudopterosin A was correlated with mitosis. Pseudopterosin A (4 X 10(-5) M) inhibited the uptake and incorporation of [3H]thymidine during the S2 phase. This concentration of Pseudopterosin A also inhibits protein synthesis as measured by the uptake and incorporation of [3H]phenylalanine. In this case the inhibition started before the S1 phase. Cytological examination revealed that sea urchin embryos did not progress beyond early prophase. Notably, the nuclear envelope remained intact and chromatin was condensed into chromosomes in the arrested embryos. These synchronously dividing embryos did not show any abnormalities such as lysis, swelling, or morphological changes different from control embryos.

Animals↗
PubMed1986

Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2.

The marine natural product manoalide (MLD) was shown to directly inactivate bee venom phospholipase A2 (PLA2). Inactivation was pH dependent (maximum inactivation occurred at pH 8.0), time dependent and concentration dependent. The IC50 was estimated at 0.05 microM and virtually complete inactivation of the enzyme occurred at 4.0 microM. The time-dependent loss of PLA2 activity suggested that inactivation does not follow typical Michaelis-Menten kinetics. Reversibility was studied directly by dilution and dialysis; both methods were ineffective in dissociating the MLD-PLA2 complex. A kinetic plot of initial velocity (v) versus [PLA2] supported our hypothesis that MLD apparently inactivates bee venom PLA2 by an irreversible mechanism.

Bee Venoms↗
PubMed1986

Mechanism of action of the marine natural product stypoldione: evidence for reaction with sulfhydryl groups.

Stypoldione, a marine natural product that possesses an o-quinone functional group, has been shown to inhibit a variety of biological processes including cell division. We found that stypoldione binds covalently to sulfhydryl groups of thiol-containing compounds via addition of sulfur to the C-4' position of the quinone ring. We examined the ability of stypoldione to add to sulfhydryl groups of a number of thiol-containing substances, including glutathione, thiophenol, beta-mercaptoethanol, and the protein tubulin. We suggest that the biological actions of stypoldione may be caused by the addition of this compound to thiol groups of biological molecules.

Cell Division↗
PubMed1986

The pseudopterosins: anti-inflammatory and analgesic natural products from the sea whip Pseudopterogorgia elisabethae.

The Caribbean sea whip Pseudopterogorgia elisabethae (Octocorallia, Cnidaria) has been found to contain the pseudopterosins, a newly described class of natural products, which have been characterized as diterpene-pentoseglycosides. The pseudopterosins possess anti-inflammatory and analgesic properties that exceed, in our assays, the potencies of existing drugs such as indomethacin. As anti-inflammatory agents, the pseudopterosins appear to modify the arachidonic acid cascade by an as yet undefined mechanism of pharmacological action.

Animals↗
PubMed1985

Irreversible autonomic actions by lophotoxin suggest utility as a probe for both C6 and C10 nicotinic receptors.

The marine natural product lophotoxin has produced a non-reversible antagonism of parasympathetic and sympathetic functions that are known to be mediated by C6 sub-type nicotinic receptors. Transmission through anuran paravertebral ganglia was eliminated in 20-40 min by 10-30-min treatments with 16-32 microM lophotoxin, in a time course resembling the onset of block of C10 sub-type nicotinic receptors at the neuromuscular junction and in cultured BC3H-1 cells. The action persisted through 16 h of washout. Nerve conduction was unaffected. Somewhat longer treatments (80 min) of in vitro ileal sections resulted in loss of sensitivity to nicotine, but not to acetylcholine, for at least 5 h. These data indicate that lophotoxin can serve as a more universal nicotinic receptor probe than the alpha-neurotoxins, which may bind to both C6 and C10 sub-types, but block only the C10.

Animals↗