Inhibitory Effect of Calmodulin Antagonists and Calcium Antagonists on Fertilization-Induced Cyanide-Insensitive Respiration in Sea Urchin Eggs

During initial several minutes after fertilization, sea urchin eggs exhibited high rate of respiration which was only slightly inhibited by cyanide. This cyanide-insensitive respiration was inhibited by calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and chlorpromazine, which were added within 1 min after insemination. The inhibitory effect of W-7 on cyanide-insensitive respiration was higher than that of W-5. Cyanide-sensitive respiration of fertilized eggs observed after this initial period was not inhibited by these compounds. Ca²⁺ influx in eggs just after fertilization was inhibited by calcium antagonists but was rather enhanced by calmodulin antagonists. Fertilization-induced stimulation of cyanide-insensitive respiration probably results from calmodulin-dependent reactions which are activated by Ca²⁺ influx.     

Aptamer Antagonists of Myelin-Derived Inhibitors Promote Axon Growth

Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators.     

Mechanisms of Inhibition of Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase by Dihydropyridine Calcium Antagonists

Abstract: Calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interaction between the cyclic nucleotide and Ca²⁺ second-messenger systems. CaMPDE exists as tissue-specific isozymes, and initially these isozymes were designated according to their respective subunit molecular mass. A variety of pharmacological agents have been used to inhibit CaMPDE, and this inhibition occurs mostly via Ca²⁺-dependent association with the proteins. We have examined the effect of dihydropyridine Ca²⁺-channel blockers felodipine and nicardipine on CaMPDE. The results suggest that the 63-kDa (PDE 1B1) and 60-kDa (PDE 1A2) CaMPDE isozymes are inhibited by felodipine and nicardipine by partial competitive inhibition and that these two Ca²⁺ antagonists appear to counteract each other. This study further demonstrates the existence of a specific site, distinct from the active site on CaMPDE, that exhibits high-affinity binding of these drugs. Felodipine and nicardipine have similar affinities for 60-kDa CaMPDE isozymes but bring about different levels of enzyme inhibition, suggesting the possibility of designing specific drugs that can protect the enzyme from inhibition by dihydropyridine Ca²⁺-channel blockers.     

Effects of Protein Phosphatase Inhibitors and Calcium Antagonists on Self-Incompatible Reaction in Buckwheat

Isolated pistils of dimorphic buckwheat (Fagopyrum esculentum Moench.) flowers were treated with phosphatase inhibitors (ocadaic acid and cantharidin) and with calcium antagonists (verapamil, La³⁺, and A23187). They were subsequently cross- or self-pollinated, and the growth of pollen tubes was observed under the fluorescence microscope. All treatments suppressed inhibition of pollen tubes growth suggesting that protein phosphatases and calcium signaling may be involved in self-incompatibility signal transduction in buckwheat. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of Cytokinin-Induced Adventitious Bud Initiation in Torenia Stem Segments by Inhibitors of Serine Proteases

Application of di-isopropyl fluorophosphate (DFP), a highlysensitive inhibitor for serine enzymes, strongly inhibited cytokinin-inducedadventitious bud initiation in Torenia stem segments culturedin vitro. The inhibitory effect was not evident when DFP wasapplied after 3 days of culture. Amount of DFP-binding proteinsremarkably increased in superficial tissues of explants culturedfor 3 and 4 days on a medium containing benzyladenine. At least14 kinds of DFP-binding polypeptides were detected by SDS-polyacrylamidegel electrophoresis and fluorography. DFP-binding to some ofthese polypeptides was inhibited by a prior treatment with phenylmethylsulfonylfluoride and N-p-tosyl-L-lysine chloromethyl ketone. From theseresults, it was suggested that some serine proteases might berelated with biochemical events occurring during the initialstage of adventitious bud differentiation in Torenia stem segments. (Received May 8, 1984; Accepted July 5, 1984)     

Inhibition of Plant Root Growth by Enzymes and Histones

It has been shown previously that root growth can be inhibited by basic, animal proteins. In an effort to see if a plant histone was more efficacious than the animal protein, roots were grown in the presence of wheat histone. Otber basic polymers were also tested. Polycations, including salmine, lysozyme, ribonuclease, wheat germ histone, thymus histone and polylysine inhibit root elongation of barley and wheat. Polyglutamate and lysylglycine at comparable weight concentrations are not inhibitory. No difference in the efficacy of tbe plant and the animal histones could be found with either plant, which suggests that the action is non-specific. Growth of roots inhibited by histone, trypsin, or lysozyme can resume after removal of the polycation. The mechanism whereby polycations influence root growth is not known, but it is clear that the polymeric state of ionic functional groups is of paramount importance in the binding of the polycations to cell surfaces.     

Root Growth Inhibition by Siduron and Its Relief by Kinetin

The growth of crabgrass (Digitaria ssp.) and barley (Hordeum vulgare) seedlings was susceptible to injury by siduron (1-2(methylcyclohexyl)-3-phenylurea). In both species, root growth was inhibited more than shoot growth and if a functional root system was developed before coming into contact with siduron the plant survived. Kinetin partially overcame the effect of siduron on root growth and increased root length and the number of cells undergoing mitosis per root tip. The effect of siduron seems specific to cell division.     

Antagonists of Growth Inhibition of Crithidia by Allopurinol, a Guanine Analog

SYNOPSIS. Allopurinol, 4-hydroxypyrazolo[3,4- d ] pyrimidine, a relatively non-toxic xanthine-oxidase inhibitor used to treat gout, inhibited growth (ID_0–90 1.0 μg/ml) of the trypanosomatid flagellate of insects Crithidia fasciculata in minimal media in respect to biopterin, folic acid and purine. Allopurinol inhibition was antagonized by thymidine (4.0 μg/ml) when a ) biopterin (≥ 1.0 ng) was present alone; traces of folate entering from the inoculum or contamination of chemicals probably sufficed here to satisfy the folate requirement; b) biopterin (5% pure) 1.0 ng was added together with either pteroylglutamic acid (PGA) 4.0 ng or hypoxanthine 0.01 mg. With biopterin absent, full growth was permitted by PGA 10 ng + hypoxanthine 0.03 mg, and completely inhibited by the standard concentration of allopurinol, but in this instance thymidine did not release the inhibition.
Allopurinol is therefore useful for investigating pteridine function via inhibition analysis with the singularity that folate acts uniquely in trypanosomatids as precursor of biopterin, and the complication that both pteridines catalyze multiple biosyntheses. The biosynthetic step between folate and biopterin is postulated to be a site of inhibition of allopurinol in Crithidia.     

Root Growth Inhibitors from Root Cap and Root Meristem of Zea mays L.

A micro-assay based on the growth inhibition of root segmentsof the seminal roots of Zea mays has been used to investigatethe root-growth-inhibiting substances in root caps and meristemsrespectively of the roots of Zea mays. This micro-assay is sensitiveto 50 pg of IAA or less. Paper chromatography of the acid fractionof methanolic extracts shows the presence of one main inhibitorin root caps and a different main inhibitor in root meristems.Neither is IAA, whose presence in meristems is sometimes indicatedby small inhibitions (or stimulations) at the characteristicRf of IAA. A Commelina leaf-epidermis assay shows the presenceof one stomata-closing ABA-like substance in root caps and onein meristems, one corresponding in Rf to the main root-growthinhibitor from the root cap. The implications of these findingsfor the geotropic responses of roots is briefly discussed.     

ffects of Some Inhibitors on Potassium-and IAA-Induced Adventitious Root Formation of Excised Cucumber Cotyledon

The effects of some inhibitors on potassium- and IAA-induced rooting were studied adopting the root-formation bioassay in the excised cucumber ( Cucumis sativus L. ) cotyledon. 5-fluomuracil at 7 Ï 10-4 – 7 Ï10-1 mmol/L and cycloheximide at 3.5 Ï 10-4 – 1.05 Ï10-2 mmol/L significantly inhibited potassium- and IAA-induced adventitious root formation of the excised cucumber cotyledons, respectively. Na3VO4 at 0.1 – 1.0 mmol/L obviously inhibited potassium and IAA-induced adventitious rooting of the excised cucumber cotyledons, and similar inhibitory effect was found with 2,3,5-triiodobenzoic acid (TIBA) at 2 Ï 10-4 – 2 Ï 10-l mmol/L.There is a close relationship between potassium and IAA-induced adventitious rooting and the promotive effect of potassium on rooting is possibly brought about via affecting the endogenous level of IAA.     

Inhibition of Cottonseed Choline- and Ethanolaminephosphotransferases by Calcium during Postgerminative Growth

Activities of choline- and ethanolaminephosphotransferase (CPT and EPT) were reproducibly high in microsomes from imbibed seeds of cotton (Gossypium hirsutum, L.). Initial studies showed that both activities dramatically declined during postgerminative growth when demand for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesis was high. Addition of CaCl₂ (0.1 millimolar) or aliquots of supernatant fractions (150,000g, 60 minutes) from cotyledons of 48-hour-old seedlings to imbibed-seed microsomes reduced the CPT and EPT activities to levels approximating those found in 48-hour microsomes. Inhibition by supernatants was completely reversed by adding EGTA (1.0 millimolar), but not by boiling the supernatants. EGTA (1.0 or 5.0 millimolar) relieved inhibition in cellular fractions whether it was added to the homogenization media or the assay reaction mixtures. A time course of CPT and EPT activities in cellular fractions prepared with 1.0 millimolar EGTA showed that activities were well developed in imbibed seeds, doubled coincidentally to a peak at 36 hours, then declined during the next 12 hours to levels approximating those in imbibed seeds. Greater than 90% of the CPT and EPT activities were pelletable (150,000g, 60 minutes) at all ages examined. Calcium apparently was artificially released upon homogenization, to a progressively greater extent in older cotyledons, and severely inhibited CPT and EPT activities. This is the only time course of CPT and EPT activities reported for cotyledons of any oilseed; it is substantially different from that in oil-storing endosperm.     

Inhibition of Hydrolytic Enzyme Activities and Plant Pathogen Growth by Invertase Inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, α - l -arabinofuranosidase and β -glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.     

Enkephalins and Opiate Antagonists Control Calmodulin Distribution in Neuroblastoma-Glioma Cells

The calcium binding protein calmodulin and the opiate receptor binding sites are unevenly distributed in various subcellular fractions of neuroblastoma-glioma NG108-15 cells. The crude mitochondrial-membrane fraction of these cells contains two membrane fractions that are separable by sucrose gradient centrifugation. These two differ in the content of both calmodulin and opiate receptors. Leucine enkephalin and D-Ala2-methionine enkephalinamide decrease the amount of membrane-bound calmodulin in the NC108-15 cells in a time- and dose-dependent manner, whereas the opiate antagonists naloxone and levallorphan have an opposite effect. Naloxone blocks the effect of leucine enkephalin and dextrallorphan has no significant effect. The opiate alkaloids entorphine and phenazocine induce changes similar to that of the enkephalins whereas morphine is inactive even at high concentrations. The alteration in the amount of membrane-bound calmodulin after a short incubation (15 min) with the enkephalins or with naloxone is reflected as an opposite change in the amount of calmodulin in the cell cytosol. Naloxone and levallorphan also increase the number of opiate receptors in NG108-15 cells but dextrallorphan has no such effect. Modulation of the intracellular distribution of calmodulin by opioid peptides and alkaloids may control the activity of various membrane-bound and cytosolic systems that are calmodulin- and/or calcium-dependent.     

Glycine and L-Alanine as Antagonists of Growth Inhibition by the Branched-Chain Amino Acids in Spirodela polyrhiza

Growth inhibition of the duckweed Spirodela polyrhiza (L.) Schleidenby exogenously supplied L-leucine, L-valine, or L-isoleucinewas antagonized by simultaneously supplied glycine or L-alanine.Calculations, using the kinetic parameters for the uptake ofthese amino acids, indicated that the antagonisms to a considerabledegree resulted from competitive inhibition of the uptake ofthe growth-inhibiting amino acids. However, the antagonismsresulted partly from an interaction between growth inhibitorand antagonist inside the cells.     

Bacterial Inhibitors Formed During the Adventitious Growth of Micro-organisms in Chicken Liver and Pig Kidney

Inhibitory activity (detected by Bacillus cereus var. mycoides) , identical with that encountered in survey samples, was induced in homogenized fresh chicken liver or pig kidney samples by incubating at 30°C. relative potency of each of three active components as detected by t.l.c./bio-autography of extracts of freeze-dried material was ascertained. Three strains of Streptococcus faecalis and a Lactobacillus sp. were responsible for production of the inhibitory activity, which was not produced by any of these organisms in synthetic liquid media.