Sensitivity of starfish oocytes and whole, half and enucleated embryos to cytotoxic neuropharmacological drugs

In early embryos of eight starfish species both usual sensitivity and supersensitivity to cytotoxic neurochemicals have been found. This sensitivity is unaffected by the removal of the cell nucleus. In the starfish Aphelasterias japonica the supersensitivity in the embryos' red halves is higher and in embryos' white halves lower than in the whole embryos.     

The Embryos Obtained from the Karyoplasts of Starfish Oocytes: Their Development and Sensitivity to Cytostatic Neurotransmitter Antagonists

Karyoplasts obtained from full-grown oocytes of the starfish Aphelasterias japonica have practically no cytoplams and are incapable of maturation. Karyoplasts of oocytes of starfishes Marthasterias glacialis and Acanthaster planci have the cytoplasm (10%–15% of the total karyoplast volume) and are often capable of maturation, fertilization and one or several cleavage divisions. The embryoskaryoplasts completely lose supersensitivity and retain usual sensitivity to cytostatic antagonists of neurotransmitters. The assumption is made that the incapability or limited capability of this embryos for development might be due to a deficiency of certain components of the "prenervous" neurotransmitter systems.     

Sensitivity of fragmented and stratified sea urchin embryos to cytotoxic neuropharmacological preparations

The red half embryos and related quarter embryos (yolk and pigment) of Arbacia lixula, obtained by means of centrifugation of the eggs in sucrose gradient, retain the normal level of sensitivity and supersensitivity to cytotoxic neuropharmaca, antagonists of biogenic monoamines. The white half embryos and clear quarter embryos practically lack supersensitivity whereas the granular quarter embryos restore it to the initial level. The non-pigmented blastomers of stratified embryos are characterized by somewhat weakened supersensitivity. A suggestion is put forward that the supersensitive embryos of A. lixula possess a sensibilizing factor which couples the supersensitivity receptors with the processes of cell division and moves together with the yolk granules upon centrifugation. This factor is not observed in the Strongylocentrotus granularis embryos lacking evident supersensitivity.     

The effect of cyclic nucleotides on the sensitivity of early mouse embryos to biogenic monoamine antagonists]

The effect of lipophilic cAMP analogs on the sensitivity of preimplantation mouse embryos of two strains to cytotoxic serotonin and adrenalin antagonists was studied. Dioctanoyl-cAMP significantly decreased the sensitivity of embryos to inmecarb and cyproheptadine: experimental embryos developed to the stage of morula or blastocyst, in contrast to control embryos incubated without this protector. A somewhat weaker effect was observed in experiments with propranolol: embryos incubated in the propranolol-containing medium after the addition of dioctanoyl-cAMP were capable of one to two cleavage divisions. 8-bromomonobutyryl-cAMP partially suppressed the inhibitory effect of cyproheptadine and did not affect the sensitivity of embryos to propranolol. These data suggest cAMP involvement in the regulatory activity of neurotransmitters in the early mouse embryos.     

Antizyme and antizyme inhibitor activities influence cellular responses to polyamine analogs

Summary. Close structural analogs of spermidine and spermine, polyamine mimetics, are potential chemotheraputic agents as they depress cellular polyamines required for tumor growth. Specific mimetic analogs stimulate synthesis of the regulatory protein antizyme (AZ), which not only inactivates the initial enzyme in polyamine biosynthesis but also inhibits cellular uptake of polyamines. The role of AZ induction in influencing cellular uptake of representative analogs was investigated using three analogs produced by Cellgate Inc., CGC-11047, CGC-11102, and CGC-11144, which exhibit markedly distinct AZ-inducing potential. An inverse correlation was noted between the AZ-inducing activity of a compound and the steady-state levels accumulated in cells. As some tumor cells over express AZI as a means of enhancing the polyamines required for aggressive growth, analog sensitivity was examined in transgenic CHO cells expressing exogenous antizyme inhibitor protein (AZI). Although AZI over expression increased cell sensitivity to analogs, the degree of this affect varied with the analog used.     

Biological activity of optically pure C-1 altered abscisic acid analogs in Brassica napus microspore embryos

We have examined the effects of stereochemically pure analogs of abscisic acid (ABA) on three responses in Brassica napus microspore embryos. The analogs used include modifications to natural (S-) (+)-ABA (= N-ABA) at the C-1 and C-1 positions. At the C-1 position, the carboxylic acid function was replaced with an alcohol, aldehyde, or methyl ester functional group, and at the chiral C-1 position both enantiomers were prepared. The rationale for choosing these particular analogs was that they had previously shown some potential as slow release forms of ABA (Gusta LV, Ewan B, Reaney MJT, Abrams SR (1992) Can J Bot. 70:1550–1555). The responsiveness of microspore-derived embryos of B. napus to these analogs was investigated. Three types of responses were evaluated: (1) the inhibition of precocious germination; (2) induction of oleosin gene expression; and (3) induction of napin gene expression. All of the structural analogs of ABA tested were effective in the three assays, regardless of functional group substitution or stereochemistry. However, the three assays showed differential sensitivity to the various analogs. The U-forms of abscisyl alcohol and abscisyl aldehyde were very effective in inhibiting precocious germination (greater than natural ABA). Oleosin mRNA accumulation responded most effectively to U-abscisyl alcohol, while the N-abscisyl aldehyde and ABA methyl ester were the most effective at inducing napin mRNA accumulation. This work highlights the distinct differences in activity which result from using stereochemically pure analogs. In addition, surprisingly potent responses are reported in one or more of the assays for abscisyl aldehyde and abscisyl alcohol.Abbreviations ABA abscisic acid - LEA late embryogenic abundant - HPLC high performance liquid chromatography - MOPS 4-morpholinepropanesulfonic acid - SDS sodium dodecyl sulfate     

Structure-activity relationships of ABA analogs based on their effects on the gas exchange of clonal white spruce (Picea glauca) emblings

ABA analog structure-function relationships were determined by testing an array of 19 different ABA analogs on 1-year-old clonal white spruce ( Picea glauca [Moench.] Voss) raised from somatic embryos. The contribution of specific structural features to analog activity was determined from the relative effect of aeroponically applied analog solutions (10⁻³ M ) on seedling gas exchange. Seedling transpiration rate (E) and carbon assimilation rate (A) were measured continuously during treatment by means of a whole plant cuvette system. The analogs were racemic about the C-1' chiral center and were derived from changes imposed on six regions of the ABA molecule. The activity of optically pure (+)-S-ABA and (−)-R-ABA were also determined. Analog activity was reduced by changing the oxidation level at C-1 from the carboxylic acid. The ring C-2', C-3' double bond was important but not essential to activity. The activity lost through changes in ring structure and C-1 oxidation level was, in many cases, almost fully restored by replacing the C-4, C-5 double bond with a triple bond. Therefore, analogs with a triple bond at C-4 were more active than their equivalents with a dienoic side chain. Fluorination of the C-7' methyl caused a relatively moderate reduction in analog activity. Truncation of C-1 and C-2 from the side chain reduced activity to near zero. The unnatural (−)-ABA enantiomer was inactive.     

Binding specificity of the mouse cerebral cortex receptor for small cholecystokinin peptides

Prior studies have shown that the cerebral cortex cholecystokinin (CCK) receptor can bind CCK and gastrin analogs with high affinity. In the present work the brain CCK receptor had approximately a three times greater affinity for CCK₈ than its C-terminal tetrapeptide (CCK₄) while the C-terminal tripeptide (CCK₃) was 1000-fold less potent than CCK₄. Thus the C-terminal tetrapeptide appears to be the minimal C-terminal CCK sequence required for high affinity binding. Since brain membranes degrade various peptides including CCK, we also evaluated the stability of CCK analogs under the conditions used to measure receptor binding by the following three methods: (1) Studies of degradation-resistant analogs in binding assays; (2) analysis of analog degradation by high performance liquid chromatography (HPLC); and (3) determination of the change in potency of CCK analogs in competitive binding studies subsequent to preincubation with brain membranes. These studies indicated that degradation of analogs by the brain membranes although significant did not account for the differences in potency of analogs in competitive binding studies. Therefore, the observed differences in potencies of the analogs tested are due to the receptor affinity and not sensitivity of the analog to degradation.     

Supersensitivity to β-Lactam Antibiotics in Escherichia coli Caused by D-Alanine Carboxypeptidase IA Mutation

Escherichia coli cells acquired supersensitivity to various β-lactam antibiotics by dacA mutation, a defect in D-alanine carboxypeptidase IA activity. The mutant cells were rather less sensitive to mecillinam than the dacA⁺ cells. This mutation did not result in either thermosensitivity of cell growth or appreciable increase of the generation times in usual rich media, but the resulting appearance of supersensitivity to β-lactam antibiotics suggests that the cell wall or envelope of this mutant is somewhat abnormal and thus that D-alanine carboxypeptidase IA is involved in cell wall or envelope synthesis.     

Synthesis and biological activity of cyclic ADP-carbocyclic-ribose analogs: structure-activity relationship and conformational analysis of N-1-carbocyclic-ribose moiety

Several cyclic ADP-carbocyclic-ribose analogs 3-10 modified in the N-1-carbocyclic-ribose moiety were synthesized. Their Ca2+-releasing activity was estimated in sea urchin eggs to show that the 3"-deoxy analog 6 shows 5 times more potent activity than cADPcR, but the 2",3"-didieoxy-2",3"-unsunsaturated analog 3 has very weak activity. We also calculated their stable conformation and found that 3 and 6 were significantly different in their stable conformation.     

Novel bis-, tris-, and tetrakis-tertiary amino analogs as antagonists at neuronal nicotinic receptors that mediate nicotine-evoked dopamine release

A series of tertiary amine analogs derived from lead azaaromatic quaternary ammonium salts has been designed and synthesized. The preliminary structure-activity relationships of these new analogs suggest that such tertiary amine analogs, which potently inhibit nicotine-evoked dopamine release from rat striatum, represent drug-like inhibitors of α6-containing nicotinic acetylcholine receptors. The bis-tertiary amine analog 7 exhibited an IC₅₀ of 0.95 nM, while the tris-tertiary amine analog 19 had an IC₅₀ of 0.35 nM at nAChRs mediating nicotine-evoked dopamine release.     

Inhibition of rat prostate tumor growth by an octapeptide analog of somatostatin

Analogs of a potent octapeptide analog of somatostatin (SRIF)

were synthesized. Aromatic substitutions for Tyr resulted in little change in inhibitory potency on growth hormone (GH) secretion in the rat. Substitutions for Val or (D)Trp resulted in analogs with diminished activity. Substitution of (D)Nal for (d)Phe increased duration of GH inhibition. Final weights of subcutaneously implanted prostate tumors (R3327) were 41% lower in rats treated with an N-terminal 4-chloro-(D)phenylalanyl analog as compared to vehicle treated controls. The analog had no effect on testicular weight or final plasma testosterone levels. SRIF analogs may represent an alternative treatment for prostate cancer that would be free of the untoward reproductive effects of other treatments (e.g. LH-RH or castration).     

The role of tyrosine 67 in the cytochrome c heme crevice structure studied by semisynthesis.

Tyr-67 of mitochondrial cytochrome c is thought to be involved in important hydrogen bonding interactions in the hydrophobic heme pocket of the protein (Takano, T., Dickerson, R. E. (1981) J. Mol. Biol. 153:95-115). The role of this highly conserved residue in heme pocket stability was studied by comparing properties of semisynthetic (Phe-67) and (p-F-Phe-67) analogs with those of native cytochrome c and a "control" analog, (Hse-65)cytochrome c. The (Phe-67) and (p-F-Phe-67) analogs have well-developed 695-nm visible absorption bands and are active in a cytochrome c oxidase assay. The reduction potentials of both analogs are lower than the native protein by approximately 50 mV. Although both analogs bind imidazole with higher affinity than the native protein, only the (p-F-Phe-67) analog has a 3- to 5-fold lower binding constant for cyanide. Only the (Phe-67) analog was significantly more stable toward alkaline isomerization. These results are not consistent with stabilization of the native protein heme pocket via hydrogen bonding of Tyr-67 to Met-80. An alternative steric role for Tyr-67 is proposed in which the residue controls the heme reduction potential by limiting the number of internal H2O molecules in the heme pocket.     

Synthesis of a new 8-spin-labeled analog of adenosine 5'-phosphate and its interaction with AMP nucleosidase.

The synthesis of a new 8-spin-labeled analog of AMP, 8-[[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)carbamoyl]methyl]thio]adenosine 5'-phosphate (8-slAMP), is described. The procedure is facile and results in high yields. 8-slAMP is a competitive inhibitor of AMP nucleosidase with a Ki of 19 microM as compared to a Km of 100 microM for AMP. The analog is not a substrate for the enzyme and does not displace MgATP2- from the allosteric sites under the usual assay conditions. The EPR spectrum of the bound spin probe reveals a highly immobilized nitroxide group. Binding studies with 8-slAMP at 8 degrees C indicate three independent binding sites (Kd = 1.4 microM) per molecule of enzyme (Mr = 320,000). These properties make 8-slAMP a good spin probe for AMP nucleosidase. The analog may also be useful for other proteins known or suspected of binding AMP analogs in a syn conformation.     

Use of Drosophila deoxynucleoside kinase to study mechanism of toxicity and mutagenicity of deoxycytidine analogs in Escherichia coli

Most bacteria, including Escherichia coli, lack an enzyme that can phosphorylate deoxycytidine and its analogs. Consequently, most studies of toxicity and mutagenicity of cytosine analogs use ribonucleosides such as 5-azacytidine (AzaC) and zebularine (Zeb) instead of their deoxynucleoside forms, 5-aza-2′-deoxycytidine (AzadC) and 2′-deoxy-zebularine (dZeb). The former analogs are incorporated into both RNA and DNA creating complex physiological responses in cells. To circumvent this problem, we introduced into E. coli the Drosophila deoxynucleoside kinase (Dm-dNK), which has a relaxed substrate specificity, and tested these cells for sensitivity to AzadC and dZeb. We find that Dm-dNK expression increases substantially sensitivity of cells to these analogs and dZeb is very mutagenic in cells expressing the kinase. Furthermore, toxicity of dZeb in these cells requires DNA mismatch correction system suggesting a mechanism for its toxicity and mutagenicity. The fluorescence properties of dZeb were used to quantify the amount of this analog incorporated into cellular DNA of mismatch repair-deficient cells expressing Dm-dNK and the results showed that in a mismatch correction-defective strain a high percentage of DNA bases may be replaced with the analog without long term toxic effects. This study demonstrates that the mechanism by which Zeb and dZeb cause cell death is fundamentally different than the mechanism of toxicity of AzaC and AzadC. It also opens up a new way to study the mechanism of action of deoxycytidine analogs that are used in anticancer chemotherapy.     

Rat heart organ culture for the study of trophic substances involved in the maintenance of normal sensitivity to norepinephrine: possible involvement of cAMP and search for other factors

In organ culture conditions, in the absence of in vivo factors, the newborn rat right atria acquire a high sensitivity to agonists similar to that seen before sympathetic innervation and after denervation. In the present study, we examined the effects of various extracts and substances on the development of supersensitivity to norepinephrine (NE) to obtain information on the in vivo factors that regulate myocardial sensitivity. Addition of rat serum, right atrial extract, superior cervical ganglionic extract, vas deferens extract, carbachol, insulin, cortisone, thyroxin, and neuropeptide Y in the culture medium did not prevent the development of supersensitivity. Addition of NE completely inhibited the development of supersensitivity. This effect of NE was blocked by sotalol but not by phentolamine. Addition of calcitonin gene related peptide, forskolin, and 8-bromo-cAMP partially inhibited the development of supersensitivity. These results are consistent with the view that NE released from sympathetic nerve terminals in the newborn atria maintains myocardial sensitivity at normal level by acting on beta-adrenergic receptors, and that the effect may be partially mediated by a rise in intracellular cAMP concentration.     

Mathematical models for continuous culture growth dynamics of mixed populations subsisting on a heterogeneous resource Base: I. Simple competition

The classical Monod model for bacterial growth in a chemostat, based on a Michaelis-Menten kinetic analog, is restated in terms of an approximate Lotka-Volterra formulation. The parameters of these two formulations are explicitly related; the new model is easier to work with, but yields the same results as the original. The model is then extended to the case where multiple alternate substrates may be growth limiting, using the corresponding kinetic analogs for multiple-substrate enzymes. Again, one is led to a Lotka-Volterra analog. In the multiple-substrate model, however, coexistence of multiple genotypes is possible, in contrast to the single-substrate model. The usual Lotka-Volterra conditions for existence and stability of pure or mixed equilibria may all be translated into corresponding statements about the parameters of the chemostat system. Possible extensions to deal with metabolic inhibition, cross-feeding, and predation are indicated.