A synthesis of N6,N6,N6-trimethyl-L-lysine dioxalate in gram amounts

A procedure is described for the synthesis of crystalline N6,N6,N6-trimethyl-L-lysine dioxalate in gram amounts starting from the commercially available N2-tert-butoxycarbonyl-N6-benzyloxycarbonyl-L-lysine, which is reacted with methyl iodide in methanol in the presence of potassium hydrogen carbonate after deprotection of the side-chain amino group by catalytic hydrogenation. The work-up involves only filtrations and evaporations.     

Synthesis and conformational study of poly(N delta-carbobenzoxy, N delta-benzyl-L-ornithine) and poly(N delta-benzyl-L-ornithine)

Poly(N^δ-carbobenzoxy, N^δ-benzyl-L -ornithine) (PCBLO) was prepared by the standard NCA method. PCBLO was converted into poly(N^δ-benzyl-L -ornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N^δ-benzyl-L -ornithine was synthesized by reacting L -ornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix–coil transition at 12% (v/v) DCA at 25°C, as compared with 36% for poly(N^δ-carbobenzoxy-L -ornithine) (PCLO). Like PCLO, the helix–coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a “coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pH_tr of about 10 for poly-L -ornithine in the same solvent.     

N deposition, N transformation and N leaching in acid forest soils

Nitrogen deposition, mineralisation, uptake and leaching were measured on a monthly basis in the field during 2 years in six forested stands on acidic soils under mountainous climate. Studies were conducted in three Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] plantations (D20: 20 year; D40: 40 yr; D60: 60 yr) on abandoned croplands in the Beaujolais Mounts; and two spruce (Picea abies Karst.) plantations (S45: 45 yr; S90: 90 yr) and an old beech (Fagus sylvatica L.) stand (B150: 150 yr) on ancient forest soils in a small catchment in the Vosges Mountains. N deposition in throughfall varied between 7–8 kg ha^–1 year^–1 (D20, B150, S45) and 15–21 kg ha^–1 yr^–1 (S90, D40, D60). N in annual litterfall varied between 20–29 kg ha^–1 (D40, D60, S90), and 36–43 kg ha^–1 (D20, S45, B150). N leaching below root depth varied among stands within a much larger range, between 1–9 kg ha^–1 yr^–1 (B150, S45, D60) and 28–66 kg ha^–1 yr^–1 (D40, S90, D20), with no simple relationship with N deposition, or N deposition minus N storage in stand biomass. N mineralisation was between 57–121 kg ha^–1 yr^–1 (S45, D40, S90) and between 176–209 kg ha^–1 yr^–1 in (B150, D60 and D20). The amounts of nitrogen annually mineralised and nitrified were positively related. Neither general soil parameters, such as pH, soil type, base saturation and C:N ratio, nor deposition in throughfall or litterfall were simply related to the intensity of mineralisation and/or nitrification. When root uptake was not allowed, nitrate leaching increased by 11 kg ha^–1 yr^–1 at S45, 36 kg ha^–1 yr^–1 at S90 and between 69 and 91 kg ha^–1 yr^–1 at D20, D40, B150 and D60, in relation to the nitrification rates of each plot. From this data set and recent data from the literature, we suggest that: high nitrification and nitrate leaching in Douglas-fir soils was likely related to the former agricultural land use. High nitrification rate but very low nitrate leaching in the old beech soil was related to intense recycling of mineralised N by beech roots. Medium nitrification and nitrate leaching in the old spruce stand was related to the average level of N deposition and to the deposition and declining health of the stand. Very low nitrification and N leaching in the young spruce stand were considered representative of fast growing spruce plantations receiving low N deposition on acidic soils of ancient coniferous forests. Consequently, we suggest that past land use and fine root cycling (which is dependent on to tree species and health) should be taken into account to explain the variability in the relation between N deposition and leaching in forests.     

Atmospheric N Deposition Increases Organic N Loss from Temperate Forests

Atmospheric deposition of nitrogen (N) resulting from fossil fuel combustion has increased N inputs to temperate forests worldwide with large consequences for forest productivity and water quality. Recent work has illustrated that dissolved organic N (DON) often dominates N loss from unpolluted forests and that the relative magnitude of dissolved inorganic N (DIN) loss increases with atmospheric loading. In contrast to DIN, DON loss is thought to be controlled by soil dynamics that operate independently of N supply and demand and thus should track dissolved organic carbon (DOC) following strict stoichiometric constraints. Conversely, DON loss may shift with N supply if soil (SOM) or dissolved organic matter (DOM) is stoichiometrically altered. Here, we assess these two explanations of DON loss, which we refer to as the Passive Carbon Vehicle and the Stoichiometric Enrichment hypotheses, by analyzing patterns in soil and stream C and N in forest watersheds spanning a broad gradient in atmospheric N loading (5–45 kg N ha⁻¹ y⁻¹). We show that soil N and DON losses are not static but rather increase asymptotically with N loading whereas soil C and DOC do not, resulting in enrichment of organic N expressed as decreased soil C:N and stream DOC:DON ratios. DON losses from unpolluted sites are consistent with conservative dissolution and transport of refractory SOM. As N supply increases, however, N enrichment of organic losses is greater than expected from simple dissolution of enriched soils, suggesting activation of novel pathways of DON production or direct N enrichment of DOM. We suggest that our two hypotheses represent domains of control over forest DON loss as N supply increases but also that stoichiometric enrichment of bulk soils alone cannot fully account for large DON losses in the most N-polluted forests.     

减量施氮对玉米-大豆套作体系土壤氮素残留和氮肥损失的影响

通过田间试验,研究了玉米单作、大豆单作、玉米-豆套作3种种植模式和不施氮、减量施氮(180 kg N·hm^-2)、常量施氮(240 kg N·hm^-2)3种施氮水平对玉米和大豆植株氮素吸收、土壤氮素残留和氮肥损失的影响.结果表明: 玉米-豆套作体系下,施氮提高了玉米土壤中残留的NO₃^--N、NH₄⁺-N含量,但在大豆土壤中则降低.与单作相比,玉米套作的土壤氮素残留量增加,氮肥损失量降低,大豆套作的土壤氮素残留量和氮肥损失量均降低.减量施氮处理下,玉米-豆套作系统的氮肥残留率、损失率和氨挥发损失率分别比玉米单作低17.7%、21.5%和0.4%,比大豆单作高2.0%、19.8%和0.1%.与常量施氮相比,减量施氮降低了玉米-豆套作系统的氮肥残留量、残留率、损失量和损失率,同时还降低了由氨挥发所引起的氮肥损失,其中氮肥残留率、损失率和氨挥发损失率分别降低12.0%、15.4%和1.2%.     

Effects of N fertilization on N2 fixation and N balances of soybean grown after lowland rice

We report a study in northern Thailand to examine the effects of fertilizer N, applied both to paddy rice and to a subsequent soybean crop on symbiotic and yield characteristics of soybean and on the differences between inputs of fixed N₂ and the removal of N as harvested product. Treatments were a factorial arrangement of 0, 100 and 300 kg N ha^-1 applied to the rice (designated R0, R100 and R300, respectively), and 0,25 and 50 kg N ha^-1, applied as starter fertilizer to the soybean (S0, S25 and S50, respectively).Nitrogen applied to the rice increased rice yields by up to 74% but proportions recovered by the rice were low (45% [R100] and 14% [R300]). The rice N treatments had only marginal effects on soybean nodulation (up to 17% reduction in early growth) and above-ground dry matter (up to 9% increase). Effects on soybean seed yield and total N₂ fixed were insignificant. Starter N, applied to the soybean at sowing, also marginally reduced nodulation and enhanced above-ground dry matter. Total N₂ fixed was unaffected but seed yield was increased by up to 6%. For all treatments, total above-ground N ranged from 145 to 179 kg ha^-1 with 72 to 85% (122 and 140 kg ha^-1) derived from N₂ fixation. When harvested product consisted of seed only, differences between inputs of fixed N₂ and removals of seed N were close to zero (-10 to+9 kg N ha^-1) with little effect of fertilizer N. The N balances were reduced by an average of 18 kg N ha^-1 when straw was included as harvested product. We concluded that N applied to the rice and to the following soybean was inefficiently used by those crops and had only marginal effects of symbiotic activity of the soybean. Furthermore, the benefit of the N₂ fixing soybean in this system was to slow the decline of, rather than enhance, the N fertility of the soil     

N,N,N-三甲基壳聚糖甲基硫酸盐的合成及理化性质的测定

壳聚糖与甲醛、甲酸反应得到N,N-二甲基壳聚糖,然后以硫酸二甲酯为季铵化试剂反应得到N,N,N-三甲基壳聚糖甲基硫酸盐(TMCMS),用IR1、H NMR和元素分析对其结构进行了表征。元素分析结果表明其季铵化度为74.6%,差示扫描量热法和热重分析法结果表明其热稳定性比壳聚糖差,但其水溶性明显优于壳聚糖,25℃时在水中的溶解度可达20 mg/mL,浓度为2 mg/mL时在pH 3～12范围内无沉淀产生。     

Box-modeling of 15N/14N in mammals

The ¹⁵N/¹⁴N signature of animal proteins is now commonly used to understand their physiology and quantify the flows of nutrient in trophic webs. These studies assume that animals are predictably ¹⁵N-enriched relative to their food, but the isotopic mechanism which accounts for this enrichment remains unknown. We developed a box model of the nitrogen isotope cycle in mammals in order to predict the ¹⁵N/¹⁴N ratios of body reservoirs as a function of time, N intake and body mass. Results of modeling show that a combination of kinetic isotope fractionation during the N transfer between amines and equilibrium fractionation related to the reversible conversion of N-amine into ammonia is required to account for the well-established ≈4‰ ¹⁵N-enrichment of body proteins relative to the diet. This isotopic enrichment observed in proteins is due to the partial recycling of ¹⁵N-enriched urea and the urinary excretion of a fraction of the strongly ¹⁵N-depleted ammonia reservoir. For a given body mass and diet δ¹⁵N, the isotopic compositions are mainly controlled by the N intake. Increase of the urea turnover combined with a decrease of the N intake lead to calculate a δ¹⁵N increase of the proteins, in agreement with the observed increase of collagen δ¹⁵N of herbivorous animals with aridity. We further show that the low δ¹⁵N collagen values of cave bears cannot be attributed to the dormancy periods as it is commonly thought, but inversely to the hyperphagia behavior. This model highlights the need for experimental investigations performed with large mammals in order to improve our understanding of natural variations of δ¹⁵N collagen.     

Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine,N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine methyl ester, and reductively methylated concanavalin A

Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine (TML) and N epsilon,N epsilon,N alpha,N alpha-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl alpha-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the alpha-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2 approximately equal to 1-10 M-1 s-1, k3 approximately equal to 10(6) M-1 s-1 and ki, the rate constant for nitrogen inversion was about 10(8)-10(9) s-1. Upper limits of 10(12) and 10(3) M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 X 10(4) s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reductively methylated peptides or proteins.     

脲酶／硝化抑制剂对土壤有效态氮、微生物量氮和小麦氮吸收的影响

研究了脲酶抑制剂(NBPT)、硝化抑制剂(DCD)及二者组合在草甸棕壤上施用对尿素态N转化及土壤总有效态N、微生物量N的影响．结果表明,尿素配施NBPT、DCD及抑制剂组合能够增加尿素水解后土壤NH4^+含量2％-53％。显著降低了氧化态N的浓度,抑制了土壤中铵态N的氧化,增加土壤总有效N34％-44％,小麦吸N量增加0．26％-6．79％。其中以脲酶抑制剂与硝化抑制剂组合的效果最明显．抑制剂施用增加了微生物在小麦生长初期对有效态N固持,有利于后期土壤有效态N的矿化．     

氮和钙交互作用对番茄氮素吸收的影响

以沈阳草甸土为栽培基质,采用二次D-饱和最优设计,进行番茄N、Ca两因素效应的盆栽试验,结果表明,在本试验条件下,番茄茎、叶、果实中氮素含量随生育期的进展而下降,氮肥对番茄果实中NO3^--N含量影响呈直线正效应,但各处理番茄果实中NO3^--N含量均不超过可生食标准．适量施用钙肥有利于控制果实中NO3^--N含量,氮肥对番茄植株地上部分和果实中N含量影响均符合报酬递减律．氮肥和钙肥对番茄N素积累符合报酬递减律;番茄从开花期到果实膨大期属N素最大效应期．番茄叶片硝酸还原酶活性受氮素影响较大．N、Ca配合施用,有利于提高番茄叶片硝酸还原酶活性,钙素可降低硝酸还原酶活性,土壤中NO3^--N含量和电导率EC值均随氮肥施用增加而提高。     

Interaction of N,N,N-trialkylammonioundecahydro-closo-dodecaborates with dipalmitoyl phosphatidylcholine liposomes

N,N,N-Trialkylammonioundecahydrododecaborates (1-), a novel class of compounds of interest for use as anions in ionic liquids, interact with DPPC liposomes. Increasing compound concentration causes an increasing negative ζ potential. Dissociation constants demonstrate that the binding capacity increases strongly with longer chain length. N,N,N-Trialkylammonioundecahydrododecaborates with longer alkyl chains show a detergent-like behavior: the compounds incorporate into the liposome membrane and differential scanning calorimetric experiment show already low concentrations cause a complete disappearance of the peak representing the gel-to-liquid crystalline phase transition. In contrast, compounds with shorter alkyl chains only interact with the headgroups of the lipids. Investigations by means of cryo-TEM reveal that all derivatives induce significant morphological changes of the liposomes. N,N,N-Trialkylammonioundecahydrododecaborates with short alkyl chains produce large bilayer sheets, whereas those with longer alkyl chains tend to induce the formation of open or multi-layered liposomes. We propose that the binding of N,N,N-trialkylammonioundecahydrododecaborates is mainly due to electrostatic interactions between the doubly negatively charged cluster unit and the positively charged choline headgroup; the positively charged ammonium group might be in contact with the deeper-lying negatively charged phosphate. For N,N,N-trialkylammonioundecahydrododecaborates with longer alkyl chains hydrophobic interactions with the non-polar hydrocarbon part of the membrane constitute an additional important driving force for the association of the compounds to the lipid bilayer.     

Studies on the availability of Azolla N and urea N for rice growth using 15N

Field experiments (20 m² plots) were conducted to compare Azolla and urea as N sources for rice (Oryza sativa L.) in both the wet and dry seasons. Parallel microplot (1 m²) experiments were conducted using ¹⁵N. A total of approximately 60 kg N ha^-1 was applied as urea, Azolla, or urea plus Azolla. Urea or Azolla applied with equal applications of 30 kg N ha^-1 at transplanting (T) and at maximum tillering (MT) were equally effective for increasing rice grain yields in both seasons. Urea at 30 kg N ha^-1 at T and Azolla 30 kg N ha^-1 at MT was also equally effective. Urea applied by the locally recommended best split (40 kg at T and 20 kg at MT) gave a higher yield in the wet season, but an equal yield in the dry season. The average yield increase was 23% in the wet season, and 95% in the dry season. The proportion of the N taken up by the rice plants which was derived from urea (%NdfU) or Azolla (%NdfAz) was essentially identical for the treatments receiving the same N split. Recovery of ¹⁵N in the grain plus straw was also very similar. Positive yield responses to residual N were observed in the succeeding rice crop following both the wet and dry seasons, but the increases were not always statistically significant. Recovery of residual ¹⁵N ranged from 5.5 to 8.9% for both crops in succeeding seasons. Residual recovery from the urea applications was significantly higher than from Azolla in the crop succeeding the dry season crop. Azolla was equally effective as urea as an N source for rice production on a per kg N basis.     

Electrosprayed polyelectrolyte complexes between mucoadhesive N,N,N,-trimethylchitosan-homocysteine thiolactone and alginate/carrageenan for camptothecin delivery

Novel hydrogel polyelectrolyte complexes (PECs) between the N,N,N,-trimethylchitosan-homocysteine thiolactone (TM-HT-chitosan) and two anionic polymers were investigated. The particles of pure thiolated chitosan and its PECs with alginate and carrageenan were fabricated using the electrospray ionization technique. The hydrogel PEC particles were characterized by scanning electron microscopy, dynamic light scattering, Fourier transform infrared microscopy, thermogravimetric analysis, encapsulation efficiency (EE), mucoadhesive property and in vitro drug release behavior. TM-HT-chitosan/alginate particles could be loaded with camptothecin (CPT), employed as a model anti-cancer drug, at an over 70% EE, and revealed both a reduced burst effect and a prolonged release of CPT over 3 days. The resultant TM-HT-chitosan/alginate PEC particles displayed a 5.60-, 1.86- and 1.55-fold stronger mucoadhesive property compared to that of the unmodified chitosan/alginate PEC at pH 1.2, 4.0 and 6.4, respectively, and this was not affected by the CPT loading level.     

Synthesis of tritium-labelled isopenicillin N, penicillin N and 6-aminopenicillanic acid.

1. Phenoxymethylpenicillin sulphoxide 4-methoxybenzyl ester was labelled with 3H in its 2-beta-methyl group. Its specific radioactivity was 362 mCi/mmol. 2. Removal of the side chain of this compound yielded the corresponding ester of 6-aminopenicillanic acid sulphoxide and coupling of the latter with the appropriate protected alpha-aminoadipic acid gave 4-methoxybenzyloxycarbonylisopenicillin N sulphoxide di-4-methoxybenzyl ester or the corresponding derivative of penicillin N. 3. Removal of the protective groups by hydrogenolysis and reduction of the sulphoxide group yielded 3H-labelled isopenicillin N or penicillin N. 4. 3H-labelled phenoxymethylpenicillin sulphoxide was obtained by hydrogenolysis from its 4-methoxybenzyl ester. Reduction of its sulphoxide group and subsequent removal of the side chain gave 3H-labelled 6-aminopenicillanic acid.     

Regioselective fluorescent labeling of N,N,N-trimethyl chitosan via oxime formation

Fluorescent labeling of chitosan and its derivatives is widely used for in vitro visualization and is accomplished by random introduction of the fluorophore to the polymer backbone, conceivably altering the bioactivity of the polymer. Here, we report for the first time the regioselective conjugation of a fluorophore to the reducing end of a fully N,N,N-trimethylated chitosan (TMC) by oxime formation. End-labeled conjugation of 5-(2-((aminooxyacetyl)amino)ethylamino)naphthalene-1-sulfonic acid (EDANS-O-NH(2)) fluorophore to TMC to form TMC-oxime-EDANS (f-TMC) was confirmed by (1)H NMR and fluorescence spectroscopy. Average molecular weight calculations of f-TMC with (1)H NMR and fluorescence spectroscopy gave similar results or ～7.7kDa. f-TMC in human bronchial epithelial cells was both cell membrane bound as well as intracellularly localized. This demonstrates the proof-of-concept for selective oxime formation at the reducing end of a chitosan derivative, which can be used for tracking chitosan in gene and drug delivery purposes and gives rise to further modifications with other functional groups.     

Symbiotic N2 fixation by soybean in organic and conventional cropping systems estimated by 15N dilution and 15N natural abundance

Nitrogen (N) is often the most limiting nutrient in organic cropping systems. N₂ fixing crops present an important option to improve N supply and to maintain soil fertility. In a field experiment, we investigated whether the lower N fertilization level and higher soil microbial activity in organic than conventional systems affected symbiotic N₂ fixation by soybean (Glycine max, var. Maple Arrow) growing in 2004 in plots that were since 1978 under the following systems: bio-dynamic (DYN); bio-organic (ORG); conventional with organic and mineral fertilizers (CON); CON with exclusively mineral fertilizers (MIN); non-fertilized control (NON). We estimated the percentage of legume N derived from the atmosphere (%Ndfa) by the natural abundance (NA) method. For ORG and MIN we additionally applied the enriched ¹⁵N isotope dilution method (ID) based on residual mineral and organic ¹⁵N labeled fertilizers that were applied in 2003 in microplots installed in ORG and MIN plots. These different enrichment treatments resulted in equal %Ndfa values. The %Ndfa obtained by NA for ORG and MIN was confirmed by the ID method, with similar variation. However, as plant growth was restricted by the microplot frames the NA technique provided more accurate estimates of the quantities of symbiotically fixed N₂ (Nfix). At maturity of soybean the %Ndfa ranged from 24 to 54%. It decreased in the order ORG > CON > DYN > NON > MIN, with significantly lowest value for MIN. Corresponding Nfix in above ground plant material ranged from 15 to 26 g N m^-2, with a decreasing trend in the order DYN = ORG > CON > MIN > NON. For all treatments, the N withdrawal by harvested grains was greater than Nfix. This shows that at the low to medium %Ndfa, soybeans did not improve the N supply to any system but removed significant amounts of soil N. High-soil N mineralization and/or low-soil P availability may have limited symbiotic N₂ fixation.     

Seasonal N2 fixation by cool-season pulses based on several15N methods

Summary Accurate estimates of N₂ fixation by legumes are requisite to determine their net contribution of fixed N₂ to the soil N pool. However, estimates of N₂ fixation derived with the traditional¹⁵N methods of isotope dilution and A_N value are costly.Field experiments utilizing¹⁵N-enriched (NH₄)₂SO₄ were conducted to evaluate a modified difference method for determining N₂ fixation by fababean, lentil, Alaska pea, Austrian winter pea, blue lupin and chickpea, and to quantify their net contribution of fixed N₂ to the soil N pool. Spring wheat and non-nodulated chickpea, each fertilized with two N rates, were utilized as non-fixing controls.Estimates of N₂ fixation based on the two control crops were similar. Increasing the N rate to the controls reduced A_N values 32, 18 and 43% respectively in 1981, 1982 and 1983 resulting in greater N₂ fixation estimates. Mean seasonal N₂ fixation by fababean, lentil and Austrian winter pea was near 80 kg N ha^–1, pea and blue lupin near 60 kg N ha^–1, and chickpea less than 10 kg N ha^–1. The net effects of the legume crops on the soil N pool ranged from a 70 kg N ha^–1 input by lentil in 1982, to a removal of 48 kg N ha^–1 by chickpea in 1983.Estimates of N₂ fixation obtained by the proposed modified difference method approximate those derived by the isotope dilution technique, are determined with less cost, and are more reliable than the total plant N procedure.Scientific paper No. 6605. College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA 99164, U.S.A.     

Activation of guinea-pig and bovine neutrophil NADPH oxidase by N,N′-dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) is a potent stimulant of superoxide generation in guinea-pig peritoneal and bovine blood neutrophils. The dependence of DCCD-elicited respiratory burst on the compositon of the medium was investigated. At 37°C, the superoxide generation was short-lived and a rapid losses of enzymatic activity was observed; at 0°C, the activity could be preserved for hours. Superoxide generation by whole cells was accompanied by exocytic degranulation. Prolonged incubation with DCCD at 37°C resulted also in a progressive loss of cellular integrity evidenced by the release of a fraction of lactate dehydrogenase. K_m values of the particulate NADPH oxidase isolated from DCCD-triggered guinea-pig and bovine cells were 31.7 and 50.0 μM, respectively. Cells pre-equilibrated with the potential sensitive dye Di-S-C₃-(5) exhibited changes in the transmembrane potential upon stimulation. Stimulation with DCCD resulted also in the release of membrane-associated calcium, indicated by quenching of the fluorescence of chlortetracyclineloaded neutrophils. Both effects were observed also in human neutrophils which did not generate superoxide upon exposure to DCCD. The mechanism of DCCD-induced responses is discussed.