Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases

The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin‐2 are endogeneous low‐molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol‐based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin‐2 variant, trappin‐2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin‐2 A62L are tight‐binding inhibitors of all three NSPs with subnanomolar K_is, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane‐bound forms of all three NSPs. The trappin‐2 A62L and elafin‐SLPI chimeras, like wild‐type elafin and trappin‐2, can be covalently cross‐linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti‐inflammatory agents.     

Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.     

Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris.

Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.     

Elafin and its precursor trappin-2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase

Elafin and its precursor trappin-2 (also called pre-elafin) are potent protein inhibitors of neutrophil serine proteases such as leukocyte elastase and proteinase 3. Trappin-2 has unique conserved sequence motifs rich in Gln and Lys residues. These motifs are substrates for transglutaminases that may enable trappin-2 to be cross-linked to extracellular matrix proteins, thus anchoring the inhibitor at its site of action. We have used Western blotting and ELISA-based assays to demonstrate that both elafin and trappin-2 can be conjugated to various extracellular matrix proteins in vitro by a type 2 transglutaminase. Cross-linked elafin and trappin-2 still inhibited their target proteases. Surface plasmon resonance studies allowed the determination of the kinetic constants governing the interaction of fibronectin-bound elafin and trappin-2 with neutrophil elastase and proteinase 3. Both inhibitors were potent inhibitors when cross-linked to fibronectin by transglutamination, with equilibrium dissociation constants K(i) for their interaction with target proteases of 0.3 nM (elastase-elafin), 20 nM (proteinase 3-elafin), 0.3 nM (elastase-trappin-2), and 12 nM (proteinase 3-trappin-2). The conjugated inhibitors reacted more slowly with their target enzymes than did the soluble inhibitors, perhaps due to their immobilization, with association rate constants of 2-7 x 10(5) M(-)(1) s(-)(1) for elastase and 1-4 x 10(4) M(-)(1) s(-)(1) for proteinase 3. We believe this is the first demonstration that transglutaminase-mediated cross-linking of serine protease inhibitors to proteins preserves their inhibitory capacities.     

Alcohol dehydrogenase activity and electron transport in living yeast

1. The excretion of H(+) ions, with practically equivalent uptake of K(+) ions (from 0.1m-potassium chloride), occurs during the aerobic oxidation of ethanol. 2. Acetaldehyde and acetic acid formed at the same time are quantitatively equal to the amount of ethanol oxidized. 3. A slow uptake of K(+) ions occurs during the oxidation of acetaldehyde and a more rapid uptake during the oxidation of d-glyceraldehyde 3-phosphate. 4. The anaerobic reduction of methylene blue is studied, and the inhibitory effect of K(+) and other inorganic cations on the system demonstrated. 5. The cation requirement for equal inhibitory effect is parallel with the reciprocals of the transport affinities for the ;physiological K-carrier' (as taken from Conway & Duggan, 1958). 6. The cation inhibition of methylene blue reduction is reversed by treatment of the yeast with Teepol or by freezing-and-thawing. 7. Azide is shown to inhibit the reduction of methylene blue with intact cells. The inhibition is partially reversed by Teepol treatment and completely by freezing-and-thawing.     

SLPI and trappin-2 as therapeutic agents to target airway serine proteases in inflammatory lung diseases: current and future directions

It is now clear that NSPs (neutrophil serine proteases), including elastase, Pr3 (proteinase 3) and CatG (cathepsin G) are major pathogenic determinants in chronic inflammatory disorders of the lungs. Two unglycosylated natural protease inhibitors, SLPI (secretory leucocyte protease inhibitor) and elafin, and its precursor trappin-2 that are found in the lungs, have therapeutic potential for reducing the protease-induced inflammatory response. This review examines the multifaceted roles of SLPI and elafin/trappin-2 in the context of their possible use as inhaled drugs for treating chronic lung diseases such as CF (cystic fibrosis) and COPD (chronic obstructive pulmonary disease).     

Raised Trappin2/elafin Protein in Cervico-Vaginal Fluid Is a Potential Predictor of Cervical Shortening and Spontaneous Preterm Birth

Early spontaneous preterm birth is associated with inflammation/infection and shortening of the cervix. We hypothesised that cervico-vaginal production of trappin2/elafin (peptidase inhibitor 3) and cathelicidin antimicrobial peptide (cathelicidin), key components of the innate immune system, are altered in women who have a spontaneous preterm birth. The aim was to determine the relationship between cervico-vaginal fluid (CVF) trappin2/elafin and cathelicidin protein concentrations with cervical length in woman at risk of spontaneous preterm birth. Trappin2/elafin and cathelicidin were measured using ELISA in longitudinal CVF samples (taken between 13 to 30 weeks'' gestation) from 74 asymptomatic high risk women (based on obstetric history) recruited prospectively. Thirty six women developed a short cervix (<25 mm) by 24 weeks'' and 38 women did not. Women who developed a short cervix had 2.71 times higher concentrations of CVF trappin2/elafin from 14 weeks'' versus those who did not (CI 1.94–3.79, p<0.0005). CVF trappin2/elafin before 24 weeks'' was 1.79 times higher in women who had a spontaneous preterm birth <37 weeks'' (CI: 1.05–3.05, p = 0.034). Trappin2/elafin (>200 ng/ml) measured between 14⁺⁰–14⁺⁶ weeks'' of pregnancy predicted women who subsequently developed a short cervix (n = 11, ROC area = 1.00, p = 0.008) within 8 weeks. Cathelicidin was not predictive of spontaneous delivery. Vitamin D status did not correlate with CVF antimicrobial peptide concentrations. Raised CVF trappin2/elafin has potential as an early pregnancy test for prediction of cervical shortening and spontaneous preterm birth. This justifies validation in a larger cohort.     

Inhibition of the respiratory burst by resveratrol in human monocytes: correlation with inhibition of PI3K signaling

trans-Resveratrol (t-RVT) has been shown to have a wide range of anti-inflammatory properties, some of which have been suggested to contribute to the molecular explanation of the French Paradox, a possible reason for the low incidence of heart disease in France. The ability of t-RVT to inhibit the production of reactive oxygen species (ROS) from monocytes (differentiated U937) was investigated using isoluminol, luminol, lucigenin, and 2',7'-dichlorofluorescein (DCF). t-RVT (0.1-50 microM) was found to significantly inhibit cellular ROS production stimulated by f-Met-Leu-Phe (fMLP), 12-phorbol 13-myristate, and arachidonic acid after a 1-h preincubation. The efficacy of t-RVT could be increased if it was added directly into the assay. NADPH-dependent superoxide production was measured in cell homogenates and t-RVT (10-50 microM) was found to have no effect on this activity. The majority of these redox probes require a peroxidase to be oxidized; therefore, the inhibitory effect of t-RVT on ROS measured by these probes is complicated by its ability to be oxidized by peroxidase enzymes and thus compete with the probe. t-RVT, known to be oxidized by the horseradish peroxidase (HRP)/H(2)O(2) system, was found to inhibit the HRP-dependent oxidation of the fluorescent probe DCF and the chemiluminescent probe isoluminol. However, using a redox probe that did not require oxidation by a peroxidase (lucigenin), significant inhibition was still observed. Moreover, the inhibitory effects of t-RVT on fMLP-induced ROS production correlated with significant inhibitory effects on fMLP-induced phosphatidylinositol 3-kinase (PI3K) activity at 50 microM and Akt phosphorylation (10-50 microM). Other known inhibitors of both PI3K and Akt were also found to inhibit this response. Therefore, inhibition of signaling through the PI3K to NADPH oxidase by t-RVT might represent an important anti-inflammatory mechanism.     

Compared action of neutrophil proteinase 3 and elastase on model substrates. Favorable effect of S'-P' interactions on proteinase 3 catalysts

Neutrophil proteinase 3 (Pr3) and elastase (NE) may cause lung tissue destruction in emphysema and cystic fibrosis. These serine proteinases have similar P(1) specificities. We have compared their catalytic activity using acyl-tetrapeptide-p-nitroanilides, which occupy the S(5)-S'(1) subsites of their substrate binding site, and intramolecularly quenched fluorogenic heptapeptides, which bind at S(5)-S'(4). Most p-nitroanilide substrates are turned over slowly by Pr3 as compared with NE. These differences disappear with the fluorogenic heptapeptides, some of which are hydrolyzed even faster by Pr3 than by NE. Elongation of substrates strongly increases the catalytic efficiency of Pr3, whereas it has little effect on NE catalysis. These different sensitivities to S'-P' interactions show that Pr3 and NE are not interchangeable enzymes despite their similar P(1) specificity.     

Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family

Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.     

Kinetics and specificity of guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase towards substituted benzaldehydes

Molybdenum-containing enzymes, aldehyde oxidase and xanthine oxidase, are important in the oxidation of N-heterocyclic xenobiotics. However, the role of these enzymes in the oxidation of drug-derived aldehydes has not been established. The present investigation describes the interaction of eleven structurally related benzaldehydes with guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase, since they have similar substrate specificity to human molybdenum hydroxylases. The compounds under test included mono-hydroxy and mono-methoxy benzaldehydes as well as 3,4-dihydroxy-, 3-hydroxy-4-methoxy-, 4-hydroxy-3-methoxy-, and 3,4-dimethoxy-benzaldehydes. In addition, various amines and catechols were tested with the molybdenum hydroxylases as inhibitors of benzaldehyde oxidation. The kinetic constants have shown that hydroxy-, and methoxy-benzaldehydes are excellent substrates for aldehyde oxidase (Km values 5x10(-6) M to 1x10(-5) M) with lower affinities for xanthine oxidase (Km values around 10(-4) M). Therefore, aldehyde oxidase activity may be a significant factor in the oxidation of the aromatic aldehydes generated from amines and alkyl benzenes during drug metabolism. Compounds with a 3-methoxy group showed relatively high Vmax values with aldehyde oxidase, whereas the presence of a 3-hydroxy group resulted in minimal Vmax values or no reaction. In addition, amines acted as weak inhibitors, whereas catechols had a more pronounced inhibitory effect on the aldehyde oxidase activity. It is therefore possible that aldehyde oxidase may be critical in the oxidation of the analogous phenylacetaldehydes derived from dopamine and noradrenaline.     

Ascorbic acid and copper in linoleate oxidation. 3. Catalysts in combination

In promoting oxidation of 0.02 m potassium linoleate in a buffered (pH 7.0) aqueous dispersion at 37 degrees C, ascorbic acid at low concentrations (1.8 x 10(-6) and 1.8 x 10(-5) m) in combination with copper (1.3 x 10(-7) to 1.3 x 10(-3) m) had greater catalytic activity than the additive activity of the two catalysts individually. Possible explanations for the enhanced catalysis include reduction of copper by ascorbic acid to the cuprous form, increased concentration of semidehydroascorbic acid radical, and formation of a metal-ascorbic acid-oxygen complex. Some combinations of ascorbic acid (1.8 x 10(-4) and 1.8 x 10(-3) m) and copper (1.3 x 10(-6) and 1.3 x 10(-3) m) inhibited the formation of conjugated dienes but not the oxidation of ascorbic acid, and caused rapid loss of part of the conjugated dienes that were already present. It is suggested that free-radical inhibitors formed by the combination of catalysts inhibit initiation of lipid oxidation but not copper-catalyzed oxidation of ascorbic acid. Effects of the inhibitory combinations on changes in UV absorption by conjugated dienes, and absorbance in the TBA test, indicate the presence of at least two conjugated dienes that differ in stability.     

Control of proteolysis by norepinephrine and insulin in brown adipocytes: role of ATP,phosphatidylinositol 3-kinase,and p70 S6K

The objective of this study was to evaluate some of the mechanisms by which norepinephrine (NE) and insulin may influence protein degradation in mouse brown adipocytes differentiated in cultures. The effects of NE and insulin, alone or in combination, on three factors known to influence proteolysis (maintenance of cell ATP and 1-phosphatidylinositol 3-kinase (PI 3-kinase) and p70 ribosomal S6-kinase (p70 S6K) activities) were examined. It was proposed that NE affects proteolysis indirectly by decreasing cell ATP from activation of uncoupling protein-1 (UCP1)-dependent mitochondrial respiration. This was tested by comparing the effects of NE and fatty acids (which directly activate UCP1) on proteolysis in brown adipocytes, as well as in pre-adipocytes and 3T3-L1 adipocytes, which do not express UCP1. An inhibitory effect of insulin on proteolysis is observed in both pre-adipocytes and differentiated cells, whereas NE and exogenously added fatty acids inhibit proteolysis only in brown adipocytes. There is a linear relationship between reductions in cell ATP and proteolysis in response to increasing concentrations of NE or fatty acids. PI 3-kinase activity is required for proteolysis, because two selective inhibitors (wortmannin and LY294002) reduce proteolysis in both pre-adipocytes and differentiated cells. This effect is not additive to that of NE, which suggests they affect the same proteolytic pathway. In contrast to NE, insulin increases PI 3-kinase activity and phosphorylation of p70 S6K. Rapamycin, which prevented insulin-dependent increase in phosphorylation of p70 S6K, increases proteolysis in brown adipocytes and antagonizes the inhibitory effect of insulin on proteolysis, but not the inhibitory effect of NE. Thus, insulin inhibits proteolysis via rapamycin-sensitive activation of p70 S6K, whereas the effect of NE appears largely to be a function of decreasing cell ATP content.     

Fatty acid-glucose interaction in the control of protein synthesis by isolated rat lung cells

(1) The addition of long chain fatty acids to the incubation medium of isolated rat lung cells produced a dose-dependent inhibition of protein labelling from L-[3H]valine. Maximal rate changes were observed at fatty acids levels within the range of their physiological concentration. (2) The effect of fatty acids on protein labelling does not seem to be mediated by their oxidation. The following observations seem to support this conclusion: (a) the rate of fatty acid oxidation by lung cells was remarkably low, so that no significant variations in the state of reduction of the NAD system were detected; (b) there was no correspondence in the dose-response patterns of fatty acid oxidation and inhibition of protein labelling; (c) octanoate was much more actively oxidized than oleate, however the latter was more effective in decreasing protein labelling. (3) An apparent relationship between the length of the fatty chain and its ability to inhibit protein labelling seems to exist. The longer the chain the stronger the inhibitory effect observed. (4) The effect of fatty acid on protein labelling seems to be mediated by a cellular energy depletion secondary to an inhibition of the respiratory chain. Their ability to decrease oxygen uptake and adenine nucleotide content was also proportional to the chain length. (5) Glucose, which apparently acted by increasing energy production at substrate level phosphorylation, partially prevented the inhibitory effect of fatty acid on protein labelling. This observation supports the point of view that fatty acids do not act in decreasing protein labelling by perturbing directly the protein synthesis machinery but decreasing the phosphorylation potential.     

Kinetic analysis of heparin and glucan sulfates binding to P-selectin and its impact on the general understanding of selectin inhibition

P-Selectin, expressed on activated endothelial cells and platelets, is a high kinetic adhesion receptor involved in leukocyte rolling of the inflammatory response, or in tumor cell binding in the course of metastasis. Thus, P-selectin inhibition is a promising therapeutic target. The anti-inflammatory and anti-metastatic activities of heparin have partly been related to the inhibition of P-selectin binding. Here we apply a quartz crystal microbalance (QCM) biosensor to determine the kinetic constants of heparin and other sulfated polysaccharides binding to immobilized P-selectin. Binding kinetics of the derivatives were correlated with their inhibitory capacity in a P-selectin cell rolling assay. Three commercial heparins differ in cell rolling inhibition and display slightly different affinities (KD 1.21 x 10(-6) M to 5.86 x 10(-7) M). Inhibitory capacity appears to be mainly driven by a slow off-rate from the receptor (2.27 x 10(-3) s-1 to 1.23 x 10(-3) s-1). To correlate the impact of binding kinetics on inhibitory capacity structurally, we analyzed six semisynthetic glucan sulfates. They display different degrees of sulfation (DS), which has a strong influence on inhibitory activity. Kinetic data illustrate that the inhibitory capacity correlates excellently with the off-rate of these polysaccharides (R = 0.99), while the association (on-rate) affects activity to a lesser extent. In general, the consideration of binding kinetics sheds new light on the mechanism of selectin inhibition. A much slower dissociation of the inhibitors from the receptor than the physiological ligands is key for inhibitory capacity. Structurally, highly charged compounds with a slow off-rate, such as heparin or glucan sulfates, appear as potent candidates for P-selectin inhibition.     

Differences between human proteinase 3 and neutrophil elastase and their murine homologues are relevant for murine model experiments

Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3-related autoimmune processes investigated in wild-type-, mNE- and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico-chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc-AAPV-pNA and Suc-AAPV-pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by alpha(1)-antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom-designed hNE inhibitor, Val(15)-aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species-specific assessment of neutrophil protease function and inhibition.     

Aza-boronic acids as non-beta-lactam inhibitors of AmpC-beta-lactamase

With the aim of improving the ability of non-beta-lactam inhibitors to inhibit AmpC-beta-lactamase, a series of 3-aza-phenyl-boronic acid derivatives was obtained using in parallel synthesis. The molecules were tested against Escherichia coli AmpC-beta-lactamase. The best inhibitors, 3-(2-hydroxy-naphthalen-1-ylazo)-phenyl-boronic acid (12) and 3-(2,4-dihydroxy-naphthalen-1-ylazo)-phenyl-boronic acid (14), showed apparent inhibition constant values (K(i)) of 0.3 and 0.45 microM and increased the potency of the semi-synthetic cephalosporin antibiotic, ceftazidime, lowering its minimum inhibitory concentration (MIC) value of 50%, against Gram-negative bacteria strains, producing high levels of AmpC-beta-lactamase.     

脲酶-硝化抑制剂对减缓尿素转化产物氧化及淋溶的作用

利用原状土柱模拟试验,研究了脲酶抑制剂氢醌(HQ),硝化抑制剂包被碳化钙(ECC)和双氰胺(DCD)以及它们的不同组合对尿素转化产物土壤持留、氧化以及淋溶的影响.结果表明,与其它抑制剂处理相比,HQ+DCD组合能有效抑制尿素水解产物的氧化,使其以交换态NH₄⁺的形式在土壤中长时间持留;氧化作用的抑制不仅减少了氧化产物NO₃^-的累积,也降低了NO₃^-淋溶潜势,使其淋入下层土壤的深度仅限在5～10 cm范围内,且淋溶量显著降低.     

Cardiolipins are 'in vitro' inhibitors of rat brain (Na+ + K+)-dependent ATPases. A probable mechanism of action

Cardiolipins were found to potentiate the 'in vitro' inhibitory activity of (-)-delta 9-tetrahydrocannabinol on (Na+ + K+)-dependent rat brain ATPases. The compounds were found to be powerful inhibitors by themselves. At optimal concentrations of cations (Na+, K+, Mg2+), the compounds were found to be noncompetitive inhibitors of ATP (Ki = 3.5 x 10(-6) M) and 'uncompetitive' inhibitors of Na+. From gas-liquid chromatographic analysis of the cardiolipin preparations it can be inferred that their effectiveness as inhibitors is related to the linoleic acid contents. The preliminary data presented here suggest that cardiolipins inhibit the Na+-dependent phosphorylation step in the hydrolysis of ATP. Based on the observations reported in this work, a hypothesis is presented suggesting that there may be a functional or evolutionary explanation for the paucity of cardiolipins in cell plasma membranes.