Degradation of Chitotetraose to Chitobiose in the Axenic Rape Rhizosphere

In the presence of intact roots of 2-week-old axenic rape seedlings,added N, N', N', N'-tetraacetylchitotetraose was degradedto N, N'-diacetylchitobiose during 48 h of incubation. Onlyminute amounts of monomer (N-acetyglucosamine) were formed fromadded chitotetraose or chitobiose. The addition of a purifiedchitin suspension caused no measurable formation of degradationproducts. The formation of chitobiose from added chitotetraose was linear,suggesting the action of a constitutive enzyme during the incubationperiod. Key words: Brassica napus (L.) cv. Brink, rhizosphere, N, N'-diacetylchitobiose, N,N',N',N', tetraacetylchitotetraose     

Metabolism of kaurenoids by Gibberella fujikuroi in the presence of the plant growth retardant,N,N,N-trimethyl-1-methyl-(2′,6′,6′-trimethylcyclohex-2′-en-1′-yl)prop-2-enylammonium iodide

The plant growth retardant, N,N,N-trimethyl-1-methyl-(2′,6′,6′-trimethylcyclohex-2′-en-1′-yl)prop-2-enylammonium iodide, is shown to block gibberellin biosynthesis in Gibberella fujikuroi between mevalonate and ent-kaur-16-ene, probably by inhibiting ent-kaur-16-ene synthetase A-activity. In the presence of the plant growth retardant, cultures of the fungus incorporate (26.5%) added ent-[¹⁴C]-kaur-16-ene into gibberellin A₃. Under the same conditions kaur-16-ene, 13β-kaur-16-ene, and ent-kaur-15-ene are not metabolised to gibberellin analogues.     

Induction of apoptosis using sphingolipids activates a chloride current in Xenopus laevis oocytes

The purpose of this studywas to investigate whether the cell shrinkage that occurs duringapoptosis could be explained by a change of the activity in iontransport pathways. We tested whether sphingolipids, which are potentpro-apoptotic compounds, can activate ionic currents in Xenopuslaevis oocytes. Apoptosis was characterized in our model by adecrease in cell volume, a loss of cell viability, and DNAcleavage. Oocytes were studied using voltage-clamp afterinjection withN,N-dimethyl-D-erythrosphingosine (DMS) or D-sphingosine (DS). DMS and DS activated afast-activating, slowly inactivating, outwardly rectifying current,similar to I_Cl-swell, a swelling-inducedchloride current. Lowering the extracellular chloride dramaticallyreduced the current, and the channel was more selective for thiocyanateand iodide (thiocyanate > iodide) than for chloride. The currentwas blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) andlanthanum but not by niflumic acid. Oocytes injected with apseudosubstrate inhibitor of protein kinase C (PKC),PKC-(19-31), exhibited the same current.DMS-activated current was abolished by preexposure with phorbolmyristate acetate. Our results suggest that induction of apoptosis inX. laevis oocytes, using sphingolipids or PKC inhibitors,activates a current similar to swelling-induced chloride currentpreviously described in oocytes.

     

Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification

The mechanism involved inN-methyl-D-glucamine(NMDA)-induced Ca²⁺-dependentintracellular acidosis is not clear. In this study, we investigated indetail several possible mechanisms using cultured rat cerebellargranule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].When 100 µM NMDA or 40 mM KCl was added, a marked increase in theintracellular Ca²⁺ concentration([Ca²⁺]_i)and a decrease in the intracellular pH were seen. Acidosis wascompletely prevented by the use ofCa²⁺-free medium or1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca²⁺. The following fourmechanisms that could conceivably have been involved were excluded:1)Ca²⁺ displacement of intracellularH⁺ from common binding sites;2) activation of an acid loader or inhibition of acid extruders; 3)overproduction of CO₂ or lactate; and 4) collapse of the mitochondrialmembrane potential due to Ca²⁺uptake, resulting in inhibition of cytosolicH⁺ uptake. However,NMDA/KCl-induced acidosis was largely prevented by glycolyticinhibitors (iodoacetate or deoxyglucose in glucose-free medium) or byinhibitors of the Ca²⁺-ATPase(i.e.,Ca²⁺/H⁺exchanger), including La³⁺,orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca²⁺-ATPaseis involved in NMDA-induced intracellular acidosis in granule cells. Wealso provide new evidence that NMDA-evoked intracellular acidosisprobably serves as a negative feedback signal, probably with theacidification itself inhibiting the NMDA-induced[Ca²⁺]_i increase.

     

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA

The involvement of cAMP- andCa²⁺-mediated pathways in theactivation of tyrosine hydroxylase (TH) gene expression by nicotine wasexamined in PC-12 cells. ExtracellularCa²⁺ and elevations inintracellular Ca²⁺ concentration([Ca²⁺]_i)were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in[Ca²⁺]_iwas inhibited by blockers of either L-type or N-type, and to a lesserextent P/Q-, but not T-type, voltage-gatedCa²⁺ channels. With continualnicotine treatment,[Ca²⁺]_ireturned to basal levels within 3-4 min. After a lag of~5-10 min, there was a smaller elevation in[Ca²⁺]_ithat persisted for 6 h and displayed different responsiveness toCa²⁺ channel blockers. This secondphase of elevated[Ca²⁺]_iwas blocked by an inhibitor of store-operatedCa²⁺ channels, consistent with theobserved generation of inositol trisphosphate.1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor2',5'-dideoxyadenosine (DDA) prevented thenicotine-elicited phosphorylation of cAMP response element bindingprotein. DDA also blocked the elevation of TH mRNA only when addedafter the initial transient rise in [Ca²⁺]_iand not after 1 h. This study reveals that several temporal phases areinvolved in the induction of TH gene expression by nicotine, each ofthem with differing requirements forCa²⁺ and cAMP.

     

Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe

We examined the regulation of theNa⁺/H⁺exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinalC2/bbe cells, which spontaneously differentiate and have little basalapical NHE activity. Unidirectional apical membrane²²Na⁺influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of5-(N,N-dimethyl)amiloride-inhibitableflux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHEactivity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulatedby serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na⁺ influx), NHE2 and NHE3exhibited similar patterns of regulation, suggesting that the secondmessenger effects observed were not secondary to effects on cell pH.Thus, in contrast to their expression in nonepithelial cells, NHE2 andNHE3 expressed in an epithelial cell line behave similarly toendogenously expressed intestinal apical membrane NHEs. We concludethat physiological regulation and function of epithelium-specific NHEsare dependent on tissue-specific factors and/or conditionalrequirements.

     

Flowering and Endogenous Levels of Plant Hormones in Lemna Species

The occurrence and endogenous level of various plant hormoneswere measured for the short-day plants Lemna paucicostata 151and 381 and the long-day plant Lemna gibba G3 to determine whetherany of them are involved in the photoperiodic control of flowering.ABA, IAA, GA₁, GA₂₉, GA₃₄, GA₅₃, trans- and cis-zeatin, trans-and cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenine and N⁶-(²-isopentenyl)adenosine were definitely detected in each species, while GA₄was only detected in L. gibba G3 and GA₂₀ was only detectedin L. paucicostata 151. The endogenous levels of ABA and IAAwere in the range of 1–7 ng/g fr wt and were not significantlydifferent in vegetative and flowering plants. The endogenousgibberellin levels were generally higher in Lemna grown underlong-day rather than short-day conditions. The endogenous cytokininlevels were almost the same in both flowering and vegetativeplants of L. paucicostata 151 and 381. In L. gibba G3, however,the level of cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenineand N⁶-(²-sopentenyl) adenosine were higher in vegetative thanin flowering plants. These results indicate that there is not necessarily a directrelation between endogenous plant hormone levels and flowering,and that the chemical basis for the photoperiodic control offlowering cannot be explained solely by changes in hormone levels.The possibility remains, however, that one or more of the planthormones has some influence of secondary importance on the floweringprocess in Lemna. (Received January 29, 1986; Accepted July 12, 1986)     

Thiophene Content in Normal and Transformed Root Cultures of Tagetes erecta: A Comparison with Thiophene Content in Roots of Intact Plants

Transformed roots of Tagetes erecta were obtained followinginfection of stems of sterile plantlets with Agrobacterium rhizogenesstrain TR105. The thiophenes detected were 5-(4-hydroxy-l-butenyl)-2,2'-bithienyl,5-(4-acetoxy-l-butenyl)-2,2'-bithienyl, 5-(3-buten-l-enyl)-2,2',-bithienyl and 2.2': 5', 2'-terthienyl. The thiophene patternwas the same in normal root cultures and roots of the intactplant. Transformed roots showed a higher growth rate and a higherbiomass yield than normal root cultures on a hormone-free media. Key words: Transformed roots normal roots, Tagetes erecta, thiophenes, Agrobacterium rhizogenes     

Physiology and Growth of Wheat Across a Subambient Carbon Dioxide Gradient

Two cultivars of wheat (Triticum aestivum L.), 'Yaqui 54' and'Seri M82', were grown along a gradient of daytime carbon dioxideconcentrations ([CO₂]) from near 350-200 µmol CO₂ mol^-1air in a 38 m long controlled environment chamber. Carbon dioxidefluxes and evapotranspiration were measured for stands (plantsand soil) in five consecutive 7·6-m lengths of the chamberto determined potential effects of the glacial/interglacialincrease in atmospheric [CO₂] on C₃ plants. Growth rates andleaf areas of individual plants and net assimilation per unitleaf area and daily (24-h) net CO₂ accumulation of wheat standsrose with increasing [CO₂]. Daytime net assimilation (P_D, mmolCO₂ m^-2 soil surface area) and water use efficiency of wheatstands increased and the daily total of photosynthetic photonflux density required by stands for positive CO₂ accumulation(light compensation point) declined at higher [CO₂]. Nighttimerespiration (R_N, mmol CO₂ m^-2 soil surface) of wheat, measuredat 369-397 µmol mol^-1 CO₂, apparently was not alteredby growth at different daytime [CO₂], but R_N /P_D of stands declinedlinearly as daytime [CO₂] and P_D increased. The responses ofwheat to [CO₂], if representative of other C₃ species, suggestthat the 75-100% increase in [CO₂] since glaciation and the30% increase since 1800 reduced the minimum light and waterrequirements for growth and increased the productivity of C₃plants.Copyright 1993, 1999 Academic Press Atmospheric carbon dioxide, carbon accumulation, evapotranspiration, light compensation point, net assimilation, respiration, Triticum aestivum, water use efficiency, wheat     

Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle

The patch-clamptechnique was used to determine the ionic conductances activated by ATPin murine colonic smooth muscle cells. Extracellular ATP, UTP, and2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increasedoutward currents in cells with amphotericin B-perforated patches. ATP(0.5-1 mM) did not affect whole cell currents of cells dialyzedwith solutions containing ethylene glycol-bis(-aminoethylether)-N,N,N',N'-tetraaceticacid. Apamin (3 × 10⁷M) reduced the outward current activated by ATP by 32 ± 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and2-MeS-ATP increased the open probability of small-conductance, Ca²⁺-dependentK⁺ channels with a slopeconductance of 5.3 ± 0.02 pS. Caffeine (500 µM) enhanced the openprobability of the small-conductance K⁺ channels, and ATP had no effectafter caffeine. Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS,10⁴ M), a nonselectiveP₂ receptor antagonist, preventedthe increase in open probability caused by ATP and 2-MeS-ATP. PPADS hadno effect on the response to caffeine. ATP-induced hyperpolarization inthe murine colon may be mediated byP_2y-induced release of Ca²⁺ from intracellular stores andactivation of the 5.3-pSCa²⁺-activatedK⁺ channels.

     

Significance of Cyclic Nucleotides in Amaranthin Synthesis by Amaranthus caudatus Seedlings

Explants of 72–76 h old Amaranthus caudatus seedlingssynthesize the betalain pigment amaranthin in response to light.Light can be replaced with a cytokinin or a cyclic nucleotidewith an N⁶-substituent. Cyclic 3'5'-AMP shows only weak activityand that only at high unphysiological concentrations. Even cyclic2'3'-AMP, which docs not act as a ‘second messenger’,induces amaranthin synthesis to a greater degree than cyclic3',5'-AMP. But N⁶-monobutyryl-cyclic 3',5'-AMP and N⁶-2'-O-dibutyryl-cyclicAMPshow high activity, higher even than kinetin at its optimumconcentration of 10^–5 M. 2'-O-Monobutyryl-cyclicAMP, onthe other hand, is considerably less active, suggesting thatN⁶-substitution of the adenine ring is responsible for the enhancedactivity. N⁶-Propionyl, butyryl and valeryladenines are allhighly active, indicating that the cyclic monophosphate moietyis unnecessary for this response. All the compounds tested,including cyclic 3',5'-AMP, show additive effects, but thereis no amplification of the response, typical of second messengeraction. Inhibition of amaranthin synthesis imposed by hadacidin, isrelieved by kinetin, DBc AMP, N⁶-monobutyryl-cAMP and N⁶-butyryladenine. Cyclic 3',5'-AMP is weakly active in this regard. Asnatural cytokinins are N⁶-substituted adenine compounds, andas only N⁶-substituted cyclic nucleotides are able to mimicthe effect of cytokinin, it is concluded that these cyclic nucleotidesfunction as cytokinin analogues and not as ‘second messengers’'. Amaranthus caudatus, amaranthin, cytokinins, cyclic nucleotides     

N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X(7) receptors

Activation of membrane P2X₇ receptors by extracellular ATP [or its analog 2',3'-O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-D-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-D-glucamine and YO-PRO-1 enter through a common permeation pathway, the "dilating" P2X₇ receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2X₇ receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-D-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2X₇ receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-D-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-D-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway. ATP; cation channel; permeability; quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide     

Effects of a New Plant Growth Regulator Prohexadione Calcium (BX-112) on Shoot Elongation Caused by Exogenously Applied Gibberellins in Rice (Oryza sativa L.) Seedlings

The effects of a novel plant growth regulator (PGR) prohexadionecalcium (BX-112; calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate)on shoot elongation caused by exogenously applied GA₁, GA₃,GA₄₎ GA₁₉ and GA₂₀ were investigated in rice (Oryza sativa L.cv. Nihonbare and cv. Tan-ginbozu) seedling test. Dependingon the dose, BX-112 reduced shoot elongation in both cultivarscaused by GA₁₉ and GA₂₀, but not by GA₁. When a high dose ofBX-112 (e.g. 250 ng/plant and over) was applied with GA₁, orGA₄, shoot elongation was even promoted. This promotive effect,however, was not observed with GA₃. These results suggest thatBX-112 inhibits gibberellin (GA) biosynthesis in the rice plantat the 3ß- and 2ß-hydroxylation of GAs,namely steps of activation and inactivation, respectively. (Received September 6, 1989; Accepted November 27, 1989)     

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration([Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.

     

Genes Encoding the Group I Intron-containing tRNALeu and Subunit L of NADH Dehydrogenase from the Cyanobacterium Synechococcus PCC 6301

A part of the tRNA^Leu (UAA) gene containing a 240-nucleotidegroup I intron was amplified by PCR from cyanobacterium SynechococcusPCC 6301 genomic DNA. The pre-tRNA synthesized from the clonedPCR product was efficiently self-spliced in vitro under physiologicalconditions. The gene encoding the tRNA^Leu (UAA), trnL-UAA, wasisolated from a Synechococcus PCC 6301 genomic library and thenucleotide sequence of a 2,167-bp portion was determined. ThetrnL-UAA consists of a 34-bp 5' exon, a 240-bp group I intronand a 50-bp 3' exon. In addition, three open reading frames(ORF1, ORF2 and ORF3) were found in the 5' and 3' flanking regionsof trnL-UAA. The predicted protein sequence of ORF3, which islocated 74-bp upstream from trnL-UAA on the opposite strand,shows 66.2% amino acid identity to that of the SynechocystisPCC 6803 gene encoding subunit L of NADH dehydrogenase (ndhL).     

Effects of BAPTA on force and Ca2+ transient during isometric contraction of frog muscle fibers

The effects of1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid (BAPTA) on force and intracellularCa²⁺ transient were studied duringisometric twitches and tetanuses in single frog muscle fibers. BAPTAwas added to the bathing solution in its permeant AM form (50 and 100 µM). There was no clear correlation between the changes in force andthe changes in Ca²⁺ transient.Thus during twitch stimulation BAPTA did not suppress theCa²⁺ transient until the force hadbeen reduced to <50% of its control value. At the same time, thepeak myoplasmic free Ca²⁺concentration reached during tetanic stimulation was markedly increased, whereas the force was slightlyreduced by BAPTA. The effects of BAPTA were not duplicated by usinganother Ca²⁺ chelator, EGTA,indicating that BAPTA may act differently as aCa²⁺ chelator. Stiffnessmeasurements suggest that the decrease in mechanical performance in thepresence of BAPTA is attributable to a reduced number of active crossbridges. The results could mean that BAPTA, under the conditions used,inhibits the binding of Ca²⁺ totroponin C resulting in a reduced state of activation of the contractile system.

     

Calcium dependence of C-type natriuretic peptide-formed fast K+ channel

The lipid bilayertechnique was used to characterize theCa²⁺ dependence of a fastK⁺ channel formed by a synthetic17-amino acid segment [OaCNP-39-(1-17)] ofa 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchusanatinus) venom (OaV). TheOaCNP-39-(1-17)-formed K⁺ channel was reversiblydependent on1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)Ca²⁺ concentration([Ca²⁺]_cis).The channel was fully active when[Ca²⁺]_ciswas >10⁴ M andtrans (luminal)Ca²⁺ concentration was 1.0 mM, butnot at low[Ca²⁺]_cis.The open probability of single channels increased from zero at1 × 10⁶ McisCa²⁺ to 0.73 ± 0.17 (n = 22) at10³ McisCa²⁺. Channel openings to themaximum conductance of 38 pS were rapidly and reversibly activated when[Ca²⁺]_cis,but not transCa²⁺ concentration(n = 5), was increased to >5 × 10⁴ M(n = 14). Channel openings to thesubmaximal conductance of 10.5 pS were dominant at5 × 10⁴ MCa²⁺.K⁺ channels did not open whencisMg²⁺ orSr²⁺ concentrations were increasedfrom zero to 10³ M or when[Ca²⁺]_ciswas maintained at 10⁶ M(n = 3 and 2). The Hill coefficientand the inhibition constant were 1 and 0.8 × 10⁴ McisCa²⁺, respectively. Thisdependence of the channel on high[Ca²⁺]_cissuggests that it may become active under1) physiological conditions whereCa²⁺ levels are high, e.g., duringcardiac and skeletal muscle contractions, and2) pathological conditions that leadto a Ca²⁺ overload, e.g., ischemicheart and muscle fatigue. The channel could modify a cascade ofphysiological functions that are dependent on theCa²⁺-activatedK⁺ channels, e.g., vasodilationand salt secretion.

     

Isolation and some properties of an aryl acylamidase from red rice, Oryza sativa L., that metabolizes 3',4'-dichloropropionanilide

An aryl acylamidase (aryl-acylamine amidohydrolase, E. C. 3.5.1.a)was isolated from red rice (Oryza sativa L.) seedlings and purifiedapproximately 5-fold with ammonium sulfate precipitation andgel filtration. The enzyme hydrolyzed 3',4'-dichloro- propionanilide(propanil) and several other mono- and dichloro-substitutedpropionanilides and 3',4'-dichloroanilide alkyl analogs. Substratespecificity tests on these analogs indicated the following substratepreference: propanil > 3'-chloropropionanilide > propionanilide> 4'-chloropropionanilide 3',5'-dichloropropionanilide 2'- chioropropionanilide. Tests on a series of 3',4'-dichloroanilidealkyl analogs of propanil revealed: propanil > 3,4-dichloroacetanilide> 3',4'-dichlorovaleranilide. The apparent Km for 3',4'-dichloropropionanilide was determinedfrom a double reciprocal plot of initial velocities at severalsubstrate concentrations. A value of Km=2.5 ? 10^–5 M wasobtained. The temperature optimum, when assayed with propanilas substrate, was 50?C. Although the pH optimum was 8.2, therewas relatively high enzyme activity over a wide range of pHvalues between 7.4–8.7. Several compounds, e.g. p-benzoquinone,pyrocatechol and CuSO₄, were found to effectively inhibit theenzyme activity on propanil. These results indicated that theproperties of this aryl acylamidase from red rice are very similarto those of enzymes from a commercial rice variety and fromother plant species. ¹ Mississippi Agricultural and Forestry Experiment Station cooperating. (Received January 6, 1978; )     

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