Introduction of oxygen into the alkyl chain of N-decyl-dNM decreases lipophilicity and results in increased retention of glucose residues on N-linked oligosaccharides

N-Alkylation of the -glucosidase inhibitor 1-deoxynojirimycin(dNM) dramatically increases its inhibitory potency (Tan etal., J. Biol. Chem., 266, 14504–14510, 1991). However,the possibility of extending the alkyl chain to N-decyl-dNMis limited by an increase of detergent-like (amphiphilic) propertiesof long-chain alkylated dNM derivatives. Substitution of methylenegroups in the N-decyl chain by oxygen reduced the amphiphilicityof N-decyl-dNM derivatives, while retaining their superior inhibitoryproperties. In intact HepG2 cells, the compound N-7-oxadecyl-dNMwas found to result in the most pronounced retention of glucoseresidues on N-linked glycans. Permeabilization of the plasmamembrane with the bacterial toxin Streptolysin O improves theinhibitory properties of the derivatives N-3,6,9-trioxadecyl-,N-7,10,13-trioxatetradecyl-, N-3-oxadecyl- and N-7-oxadecyl-dNM,but not those of dNM. These observations suggest differencesin the mode of entry of the oxygen-substituted dNM derivativesin comparison with dNM. We observed that the dNM derivativeN-3,6,9-trioxadecyl-dNM, devoid of inhibitory activity in intactcells, was inhibitory in Streptolysh O-permeabilized cells.Thus, the permeability barriers posed by plasma membrane andendoplasmic reticulum membrane are not equivalent. The use ofa permeabilized cell system thus allows the elaboration of inhibitoryprinciples for novel bioactive compounds where study of theisolated enzymes may not be possible, and where intact cellsare not a suitable target due to permeability barriers. -glucosidase inhibition N-linked glycosylation oxygen-substituted N-decyl-dNM derivatives permeabilized cells     

Effect of Thiolactomycin on Fatty Acid Synthesis in Higher Plants

Thiolactomycin (TLM), an antibiotic from Nocardia sp. No. 2-200,inhibited fatty acid synthesis in Avena leaves, with the concentrationcausing 50% inhibition being 0.38µg/ml. This antibioticis more inhibitory to the elongation of palmitic to oleic acidthan to the de novo synthesis of palmitic acid in both spinachchloroplasts and Avena leaves, in contrast to the effect ofcerulenin which inhibits de novo synthesis but not fatty acidelongation. On the other hand, TLM is less inhibitory to furtherelongation of stearic acid to very long chain fatty acids inpea seeds. The inhibition rate decreased in the order of synthesisof arachidic, behenic and lignoceric acid. (Received December 26, 1986; Accepted April 24, 1987)     

Identification of a Water-soluble Fungal Inhibitor in the Leaves of Acer platanoides L.

It was confirmed that the leaves of Acer platanoides containan antifungal inhibitory substance. Low concentrations of sterilecold water extracts inhibited the germination of the sporesof Cladosporium herbarum (three isolates), Cladosporium sphaerospermumand Cylindrocarbon radiclcola. In the concentration range 0·06–0·125per cent (w/v) of leaf material the inhibitory response wasdemonstrated to increase linearly as the concentration of leafmaterial increased logarithmically. Inhibitory activity wasfound in leaf samples collected during a period from July toOctober but activity had disappeared from leaves collected inthe following January. The inhibitory activity was located intwo components of the water extract by bioassay tests followingether extraction and separation by chromatography. One of theactive components has been identified as gallic acid by gaschromatography. Gallic acid has also been detected in dew collectedfrom leaf surfaces where it is suggested that it may play animportant part in the colonization of the leaves by fungi.     

Comparative Effects of Alicyclic Compounds and Quinones on Inhibition of Lettuce Fruit Germination

Germination of lettuce (Lactuca sativa L. cv. Great Lakes) fruitswas inhibited by many alicyclic compounds including monoterpenes.Quinones were particularly active. The hydrocarbons showed littleactivity correlated with their insolubility in water while compoundscontaining a keto group conjugated with a double bond showedhighest activity. As in previous studies the lipophilicity ofthe molecule was one of the main contributing factors to inhibition,provided that the value was not extreme, as for the hydrocarbons.In natural compounds noted for high inhibitory activity, severaldifferent substituents were present in the molecule and it seemslikely that each contributes different factors to the overalleffect. Some of these compounds are well known plant constituentsimplicated in allelopathy. Lettuce germination, Lactuca saliva, monoterpenes, quinones, abscisic acid, lipophilicity, inhibitory activity     

Stimulation by Monovalent Cations of Adenosine Triphosphatase Activity in Extracts of Petiole Tissues3

Adenosine triphosphatase activity has been studied in fractionatedextracts of isolated phloem and xylem tissues of Heracleum mantegazzianumSomm. et Lev. and of petioles of Helianthus annuus L. Enzymeactivity in the microsomal fraction is maximal with ATP as substrate.Monovalent cations stimulate activity, but only below pH 7·0.Divalent cations are inhibitory. Stimulation of ATPase activityby monovalent cations is increased in preparations which haveeither been derived from acetone powders or pre-treated withdithiothreitol or cysteine.     

The Site of Action of General Anaesthetics in Insect Olfactory Receptor Neurons

The effect of volatile anaesthetics such as N₂O, Xe, short-chainalkanes and cyclopropane, at pharmacologically relevant concentrations,on olfactory receptor neurons of insects was tested in electrophysiologicalrecordings. CO₂-receptor neurons in moths and files respondwith increased action potential activity, whereas in adherenceto the Meyer-Overton rule; alkanes of a chain length of 5 andabove are less effective or evoke suppression of action potentials.In olfactiory receptor neurons sensitive to benzoic acid infemale moths of Bombyx mori and in pheromone receptor neuronsof male moths of Antheraea polyphemus, anaesthetics are ineffectiveif applied alone; if superimposed on an excitatory olfactorystimulus, an inhibitory effect occurs, Local stimulation ofonly part of a sensory dendrite reveals that the anaestheticsreversibly block the reception of pheromone or its effect onthe conductance of the receptor cell memebrane. The observedinteractions are consistent with the hypothesis that the anaestheticsdo not interact with the primary transduction process, but ratheraffect a later stage such as the activation of ion channels. ^*Dedicated to H-J. Bestman, on the occasion of his 70th birthday.     

Studies on factor(s) in sweet potato which specifically inhibits germ tube growth of incompatible isolates of Ceratocystis fimbriata

Sweet potato root contained a factor or factors which differentiallyinhibited the growth of various isolates of Ceratocystis fimbriata.The factor scarcely inhibited germ tube growth of sweet potatoisolate, compatible to sweet potato. On the other hand, thegrowth of prune, oak, taro and almond isolates, all incompatibleto sweet potato, was strongly inhibited. The germ tube growthof coffee and cacao isolates, incompatible to sweet potato,were less inhibited. Inhibitory factor was distributed throughvarious fractions when centrifuged on a sucrose density gradient.The germ tube growth of pre-germinated oak isolate became lesssensitive to the inhibitory factor after being treated withpronase, suggesting the interaction of the factor with someprotein-containing surface structure of the fungal cell. Treatmentof the factor by phospholipase c, lipase and pronase causedno changes in its inhibitory activity, whereas periodate treatmentpartially inactivated the factor. These results suggest thatthis inhibitory factor constitutes one of the factors determiningthe specificity in sweet potato-C. fimbriata interactions. (Received July 18, 1977; )     

Comparative Effects of Aromatic Compounds on Inhibition of Lettuce Fruit Germination

Germination of lettuce (Lactuca sativa L. cv. Great Lakes) wasinhibited by aromatic alcohols, aldehydes and carboxylic acidsin a way similar to that by aliphatic members. Increased lipophilicityusually leads to increased inhibitory activity. Thus methylationincreased activity while hydroxylation decreased it. Exceptionswere seen with ortho-hydroxylated compounds and other exceptionsindicated the presence of unrecognized factors. Many of thephenolic compounds often quoted as inhibitors were shown tobe not particularly activeagainst lettuce germination when comparedwith, for instance, abscisic acid or coumarin.     

Changes in Phenolic Inhibitors in Seeds of Acer saccharum during Stratification

Phenolics in sugar maple seeds were identified by colour reactions,u.v. spectroscopy and gas-liquid chromatography. Changes intheir inhibitory activity during stratification were also followedusing several bioassays. p-Coumaric, o-coumaric, and ferulicacids were found in all tissues. The highest concentrationswere noted in the embryonic axis and testa, but total amountsper fruit were highest in cotyledons and pericarp. p-Coumaricacid was the principal phenolic in dry seeds, and it declinedsubstantially in all tissues during the first half of stratificationwith little subsequent change thereafter. Decreases in o-coumaricand ferulic acids per seed were small by comparison, neverthelesslosses ranged between 36 and 68% of the original concentrations.Bioassays confirmed that the three endogenous phenolics possessedmarked inhibitory properties that diminished with the progressof stratilication, but only the compound identified as p-coumaricacid showed major inhibition in seed germination tests.     

Structure/activity studies of anti-inflammatory peptides based on a conserved peptide region of the lectin domain of E-, L- and P-selectin

Previously, it was established that the peptide YYWIGIRK-NH₂inhibits both myeloid cell adhesion to selectins in vitro andneutrophil influx into inflammatory sites in vivo (Briggs etal., 1995). Initial structure/activity studies revealed thatat least one Y residue at the N-terminus of the peptide wasessential for these bioactivities but that the C-terminal Kresidue was unnecessary for inhibitory activity. We have nowsynthesized a new series of peptides which contain single residuesubstitutions at each position of the reference peptide, YYWIGIR-NH₂and have tested these peptides for inhibitory activity in aselectin cell binding assay. In addition, peptides containingsingle D-amino acids at selected positions, or an all D-configuredreference peptide sequence, or the retro-inverso version (rigiwyy-NH₂of the reference peptide sequence have also been analyzed forinhibitory activity in the same assays. Finally, the abilityof the reference peptide and a specifically designed controlsequence (YY(AIB)IGIR-NH₂ to discriminate between potentialsynthetic saccharide ligands, including sialyl-Lewis x, Lewisx, and sialyl-N-acetyl-lactosamine, was investigated using isothermaltitration calorimetry. The results of these studies demonstratethat whereas many single amino acid substitutions are toleratedin the peptide without complete loss of inhibitory activity,substitution at some positions (e.g., the W residue) resultsin relatively inactive compounds, clearly pointing to the importanceof these residues in making critical contacts with the appropriatesaccharide ligand. Titration calorimetry revealed that the referencepeptide does not discriminate between Lewis x or sialyl-Lewisx in vitro, but binds these saccharides with nearly 40-foldhigher affinity (K_D 25 µM) than the nonfucosylated trisaccharide,sialyl-N-acetyl-lactosamine. We can infer from these studiesthat the presence of a sialyl group, per Se, is not a requisitefor complex formation between the reference peptide and itssaccharide ligand. Substitution of single D-amino acid residuesat various po sitions in the reference peptide sequence reducesor elimi nates all inhibitory properties. However, the all Dconfigured peptide or the retro-inverso peptide sequence havegreater activity than the all L-configured reference peptidein the in vitro biological assays, and each was an effectiveinhibitor of neutrophil infiltration in a thioglycol late-inducedmouse peritonitis model. These results, combined with the resultsof titration, allow us to conclude that binding between thereference peptide and its saccharide ligand, which affords itsinhibitory properties, is mediated by the presence of a contiguous,nonpolar surface, or face, presented at the N-terminus of thereference peptide, likely encompassing the sequence YYWI. Furthermore,the W plays a critical role in binding, probably through formationof an essential hydrogen bond with a suitably juxtaposed groupcarried on the saccharide ligand. selectin peptides inhibition saccharide ligand     

毛鱼藤酮与鱼藤酮杀虫活性的比较

毛鱼藤酮是从杀虫植物毛鱼藤根中分离的杀虫活性物质。以菜粉蝶、甘蓝蚜、柑桔全爪螨、小菜蛾和黄曲条跳甲为试虫比较测定了毛鱼藤酮与鱼藤酮的毒杀、拒食、生长发育干扰和忌避产卵活性。结果表明：毛鱼藤酮的杀虫活性依昆虫种类而异,对柑桔全爪螨、小菜蛾、黄曲条跳甲的活性与鱼藤酮相当,其LC₅₀没有显著差异,而对甘蓝蚜和菜粉蝶的活性显著低于鱼藤酮。毛鱼藤酮和鱼藤酮对昆虫均具拒食活性,但前者比后者弱。毛鱼藤酮还与鱼藤酮一样对昆虫有一定的生长发育干扰和忌避产卵作用。NADH 泛醌氧化还原酶抑制活性测定结果显示,毛鱼藤酮的抑制活性低于鱼藤酮,二者抑制中浓度（IC₅₀）分别为5.27 nmol·mL^-１和2.58 nmol·mL^-１。根据拒食、受药途径和酶抑制的试验结果分析认为,毛鱼藤酮在浸叶处理时对某些昆虫有较高杀虫活性是由于其比鱼藤酮有较低的拒食活性,导致虫体摄入药剂量增加所致。     

Developmental Role of Juvenile Hormone Metabolism in Lepidoptera

SYNOPSIS. Lepidopteran juvenile hormone (JH) esterase appearsto have a functional role in the regulation of embryogenesis,larval growth and development, and adult reproduction. In preovipositionaland newly laid eggs of the tobacco hornworm, Manduca sexta,JH esterase activity was elevated presumably to metabolize maternalJHs, and then declined after blastoderm formation. Also, a singlepeak in hemolymph JH esterase activity was found prior to ecdysisin the second through the fourth instar of M. sexta, the functionof which is unclear. However, in the last instar, elevated hemolymphJH esterase activity was noted prior to wandering and againprior to ecdysis to scavenge the last traces of JH necessaryfor normal development. The hemolymph JH esterase is likelyof multiple tissue origin for the prewandering peak with thefat body excluded as a source for the prepupal peak; an inhibitoryfactor from the brain and JH regulate JH esterase biosynthesis.In adult cabbage loopers, Trichoplusia ni, elevated hemolymphJH esterase activity appeared to be important in reducing theJH titer and preventing egg maturation. Structure/activity datawith trifluoromethylketones were incorporated into the designof a novel, JH esterase inhibitor, the sulfone and hydrate ofoctylthio-1,1,1- trifluoropropan-2-one, with selective and persistent,in vivo inhibitory activity. The topical application of thiscompound to last instar larvae and virgin adults of T. ni producedjuvenizing effects (delayed pupation and induced egg maturation/oviposition,respectively) providing direct evidence of a functional rolefor JH esterase in lepidopteran development.     

Amitriptyline reduces olfactory neuron adenylyl cyclase activity

Adenylyl cyclase plays an important role in olfactory signaltransduction. Recently, a novel type III adenylyl cyclase hasbeen localized in olfactory neurons (Pfeuffer et al., 1989;Bakalyar and Reed, 1990). Because amitriptyline (AMI), a tricyclicantidepressant, appears to have an inhibitory effect on adenylylcyclase activity in other in other neuronal tissue (Yamaokaet al., 1988; Wong et al., 1991), we measured the effect ofAMI on forskolin-stimulated adenylyl cyclase activity in membranepreparations of olfactory mucosa from adult rats. In the presenceof 5'-guanylyl-imidodiphosphate, AMI (0.5–8.0 µM)inhibited forskolin-stimulated adenylyl cyclase activity ina dose-dependent manner. To determine whether this effect wasspecific for olfactory neurons, as opposed to other cells inthe olfactory epithelium, rats were unilaterally bulbectomizedin order to reduce selectively the number of olfactory neuronson the side ipsilateral to the bulbectomy. In membrane preparationsfrom unilaterally bulbectomized animals we saw significantlylower adenylyl cyclase activity in ipsilateral olfactory mucosa,compared with adenylyl cyclase activity from non-bulbectomizedmucosa. These results indicate that AMI inhibition of adenylylcyclase activity is primariy localized in olfactory neurons.     

2-羟基-4-甲氧基苯甲醛缩苯胺对菜青虫酚氧化酶的抑制作用

为筛选对十字花科蔬菜害虫菜青虫Pieris rapae（L.）酚氧化酶具有高抑制活性的化合物,为寻找新型害虫控制剂提供线索,采用酶标仪微量法以室内合成、筛选的高活性化合物2-羟基-4-甲氧基苯甲醛缩苯胺为抑制剂,研究了其对菜青虫酚氧化酶的抑制活性及抑制类型。结果表明,供试化合物对菜青虫酚氧化酶的抑制中浓度（IC₅₀）为0.116 mmol/L;该化合物为典型的可逆非竞争型抑制剂,抑制常数（K_i）为1.96 mmol/L。该化合物直接对靶标酚氧化酶产生作用,而不是通过影响酶结构内的铜离子来产生作用的。     

Stimulation of Nitrate Reductase by Light and Ammonium in Spirodela oligorrhiza

Light-enhanced nitrate reductase (NR) activity was 8 times greaterthan the dark control. Exogenous application of sucrose, glucoseand fructose increased the induction of NR in the light as wellas in the dark, whereas glycolate had no effect. DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethyl urea] completely inhibited thedevelopment of NR in light. Sucrose, when added with DCMU, reversedthis inhibitory effect NR in vivo was more stable in light thanin darkness, the half-lives being 9.6 h and 6.4 h, respectively.The addition of sucrose did not change the half-life of NR ineither light or darkness. Ammonium, the end product of the inorganicnitrogen assimilatory pathway, stimulated the NR activity whereasamino acids decreased it. Key words: Spirodela oligorrhiza, nitrate reductase, ammonium, light     

The roles of myosin ATPase activity and myosin light chain relative content in the slowing of type IIB fibers with hindlimb unweighting in rats

We tested the hypothesis that slowing of shortening velocity generated by type IIB fibers from hindlimb-unweighted (HU) rats resulted from a reduced ATPase activity and/or a reduction in the relative content of myosin light chain 3f isoform content (MLC_3f). After 2, 3, and 4 wk of HU, maximal unloaded shortening velocity (V_o) of single permeabilized semimembranosus muscle fibers was determined by the slack test. Subsequently, the myosin heavy chain and the relative content of MLC were determined by SDS-PAGE. The ratio of MLC_3f to MLC_2f was determined by densitometric analysis. In addition, myofibrils were prepared from permeabilized fibers (soleus and semimembranosus muscles) and assayed for resting myosin ATPase and Ca²⁺-activated myosin ATPase. After HU, V_o declined by 28–40% and the MLC_3f/MLC_2f ratio decreased by 32 to 48%. A significant correlation between the relative amount of MLC_3f and V_o was found (r = 0.48, P < 0.05). Resting myosin ATPase rates were not different between myofibrils prepared from corresponding muscles of control and HU rats (P = 0.86). Ca²⁺-activated myosin ATPase activities also were not different between myofibrils prepared from corresponding muscles of control and HU rats (P = 0.13). These data suggest that the slowing of maximal unloaded shortening velocity in type IIB fibers with HU is, at least in part, due to a relative change in the essential light chain composition, a decrease in the relative amount of MLC_3f and most likely a concomitant increase in MLC_1f. However, this reduction in V_o is independent of myosin ATPase activity. unloading shortening velocity; myosin light chain 3f     

Inhibition of zipper-interacting protein kinase function in smooth muscle by a myosin light chain kinase pseudosubstrate peptide

As a regulator of smooth muscle contractility, zipper-interacting protein kinase (ZIPK) appears to phosphorylate the regulatory myosin light chain (RLC20), directly or indirectly, at Ser19 and Thr18 in a Ca²⁺-independent manner. The calmodulin-binding and autoinhibitory domain of myosin light chain kinase (MLCK) shares similarity to a sequence found in ZIPK. This similarity in sequence prompted an investigation of the SM1 peptide, which is derived from the autoinhibitory region of MLCK, as a potential inhibitor of ZIPK. In vitro studies showed that SM1 is a competitive inhibitor of a constitutively active 32-kDa form of ZIPK with an apparent K_i value of 3.4 µM. Experiments confirmed that the SM1 peptide is also active against full-length ZIPK. In addition, ZIPK autophosphorylation was reduced by SM1. ZIPK activity is independent of calmodulin; however, calmodulin suppressed the in vitro inhibitory potential of SM1, likely as a result of nonspecific binding of the peptide to calmodulin. Treatment of ileal smooth muscle with exogenous ZIPK was accompanied by an increase in RLC20 diphosphorylation, distinguishing between ZIPK [and integrin-linked kinase (ILK)] and MLCK actions. Administration of SM1 suppressed steady-state muscle tension developed by the addition of exogenous ZIPK to Triton-skinned rat ileal muscle strips with or without calmodulin depletion by trifluoperazine. The decrease in contractile force was associated with decreases in both RLC20 mono- and diphosphorylation. In summary, we present the SM1 peptide as a novel inhibitor of ZIPK. We also conclude that the SM1 peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues. inhibitory peptide; calcium sensitization     

Growth and Aspartate Kinase Activity in Wheat Cell Suspension Culture: Effects of Lysine Analogs and Aspartate-Derived Amino Acids

The effects of lysine analogs and aspartate-derived amino acidson the growth of wheat cell suspension culture were studied.S-(2-Aminoethyl)-L-cysteine (AEC), -hydroxylysine (DHL) andtrans-lysene caused complete growth inhibition at 1.0 mM. Thegrowth inhibition of lysine analogs were, in the order of decreasingeffectiveness; AECDHL, trans-lysene>oxalysine, homolysineand lysyne. cis-Lysene and methyllysine were not inhibitoryeven at concentrations of 10 mM. Lysine effectively relievedgrowth inhibition induced by the lysine analogs. Lysine plusthreonine showed concerted inhibition, which was relieved bythe addition of methionine. Activity of aspartate kinase extracted from wheat cell suspensionculture was strongly inhibited by L-lysine; 0.75 to 1 mM oflysine was required for half-maximal inhibition. Threonine andmethionine, individually or in combination with lysine, showedno inhibitory effect on the enzyme activity. S-Adenosylmethionine,when added with lysine in equimolar concentrations, enhancedthe feedback inhibition by lysine, lowering the concentrationof lysine for half-maximal inhibition to 0.13 mM. The aspartatekinase isolated from the cells cultured in the presence of 5mM lysine did not differ in regulatory properties from the enzymefrom the cells cultured without lysine. AEC at 5 mM inhibitedthe enzyme activity by 50%. Other lysine analogs were not inhibitoryto the enzyme activity even at 10 mM. Growth inhibition of wheat suspension culture by aspartate-derivedamino acids and lysine analogs were discussed in relation totheir inhibitory effects on aspartate kinase activity. (Received October 25, 1985; Accepted February 26, 1986)     

A volume-sensitive protein kinase regulates the Na-K-2Cl cotransporter in duck red blood cells

When Na-K-2Cl cotransport is activated in duckred blood cells by either osmotic cell shrinkage, norepinephrine,fluoride, or calyculin A, phosphorylation of the transporter occurs ata common set of serine/threonine sites. To examine the kinetics andregulation of the activating kinase, phosphatase activity was inhibitedabruptly with calyculin A and the subsequent changes in transporterphosphorylation and activity were determined. Increases in fractionalincorporation of ³²P into thetransporter and uptake of ⁸⁶Rb bythe cells were closely correlated, suggesting that the phosphorylation event is rate determining in the activation process. Observed in thismanner, the activating kinase was 1)stimulated by cell shrinkage, 2)inhibited by cell swelling, staurosporine, orN-ethylmaleimide, and3) unaffected by norepinephrine orfluoride. The inhibitory effect of swelling on kinase activity wasprogressively relieved by calyculin A, suggesting that the kinaseitself is switched on by phosphorylation. The kinetics of activation bycalyculin A conformed to an autocatalytic model in which thevolume-sensitive kinase is stimulated by a product of its own reaction(e.g., via autophosphorylation).

     

7H-Pyridocarbazole, a Probe to Study the Role of Diphosphatidylglycerol in the Functioning of the Cytochrome Oxidase Complex of Mitochondria

Ditercalinium, the generic name for 7H-pyridocarbazole dimer,is a planar aromatic cation which exhibits strong inhibitoryeffects on the respiratory chain of plant mitochondria. By enzymaticassays and spectroscopic studies, the inhibition is shown tooccur at the level of the cytochrome c oxidase complex. It issuggested that the mechanism of inhibition could be due to thecomplexation of the aromatic cation with the diphosphatidylglycerolenvironment essential for cytochrome c oxidase activity ratherthan to a drug-enzyme direct interaction. 7H-pyridocar-bazolecan be used as an appropriate tool to study the role that negativelycharged acidic phospholipids, as diphosphatidylglycerol, playin the functioning of cytochrome c oxidase. (Received July 10, 1989; Accepted February 19, 1990)