Effect of leukotriene C4 exposure on ciliated cells of the nasal mucosa

To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10⁻⁶M to 10⁻⁹M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10⁻⁸M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a γ-glutamyl transpeptidase (γ-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by γ-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Differential sensitivity of C3 and C4 turfgrass species to increasing atmospheric vapor pressure deficit

Temperature and vapor pressure deficit (VPD) effects on turfgrass growth are almost always confounded in experiments because VPD commonly is substantially increased in elevated-temperature treatments. The objective of this study as to examine specifically the influence of VPD on transpiration response of four ‘warm-season’ (C₄) and four ‘cool-season’ (C₃) turfgrasses to increasing VPD at a stable temperature (29.3 ± 1.5 °C). Although transpiration rates were noticeably lower in C₄ grasses, transpiration rates increased linearly in response to increasing VPD across the range of 0.8–3.0 kPa. In contrast, transpiration rates of C₃ increased sharply with increasing VPD across the range of low VPDs, but became constrained at higher VPDs (>1.35 kPa). Restricted transpiration rate at elevated VPD was most evident in Agrostis palustris and Lolium perenne. Assuming restricted transpiration rates reflect a limitation on leaf CO₂ uptake, these results indicate that the commonly observed decline in growth of C₃ (and success of C₄) grasses at elevated temperature may include a sensitivity to elevated VPD.     

Crystal structure of leukotriene A4 hydrolase in complex with kelatorphan, implications for design of zinc metallopeptidase inhibitors

Leukotriene A₄ hydrolase (LTA4H) is a key enzyme in the inflammatory process of mammals. It is an epoxide hydrolase and an aminopeptidase of the M1 family of the MA clan of Zn-metallopeptidases. We have solved the crystal structure of LTA4H in complex with N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]-L-alanine, a potent inhibitor of several Zn-metalloenzymes, both endopeptidases and aminopeptidases. The inhibitor binds along the sequence signature for M1 aminopeptidases, GXMEN. It exhibits bidentate chelation of the catalytic zinc and binds to LTA4H’s enzymatically essential carboxylate recognition site. The structure gives clues to the binding of this inhibitor to related enzymes and thereby identifies residues of their S1′ sub sites as well as strategies for design of inhibitors.     

Relationship between allocation of absorbed light energy in PSII and photosynthetic rates of C3 and C4 plants

Two C₃ dicotyledonous crops and five C₄ monocotyledons treated with three levels of nitrogen were used to evaluate quantitatively the relationship between the allocation of absorbed light energy in PSII and photosynthetic rates (P _N) in a warm condition (25–26°C) at four to five levels [200, 400, 800, 1,200 (both C₃ and C₄) and 2,000 (C₄ only) μmol m⁻² s⁻¹] of photosynthetic photon flux density (PPFD). For plants of the same type (C₃ or C₄), there was a linear positive correlation between the fraction of absorbed light energy that was utilized in PSII photochemistry (P) and P _N, regardless of the broad range of their photosynthetic rates due to species-specific effect and/or nitrogen application; meanwhile, the fraction of absorbed light energy that was dissipated through non-photochemical quenching (D) showed a negative linear regression with P _N for each level of PPFD. The intercept of regression lines between P and P _N of C₃ and C₄ plants decreased, and that between D and P _N increased with increasing PPFD. With P and D as the main components of energy dissipation and complementary to each other, the fraction of excess absorbed light energy (E) was unchanged by P _N under the same level of PPFD. At the same level of P _N, C₄ plants had lower P and higher D than C₃ plants, due to the fact that C₄ plants with little or no photorespiration is considered a limited energy sink for electrons. Nevertheless there was a significant negative linear correlation between D and P when data from both C₃ and C₄ plants at varied PPFD levels was merged. The slope of regression lines between P and D was 0.85, indicating that in plants of both types, most of the unnecessary absorbed energy (ca. 85%) could dissipate through non-photochemical quenching, when P was inhibited by low P _N due to species-specific effect and nitrogen limitation at all levels of illumination used in the experiment.     

干旱胁迫对2种光合类型C4荒漠植物叶片光合特征酶和抗氧化酶活性的影响

以荒漠C₄草本植物蔷薇猪毛菜(NADP苹果酸酶型,NADP-ME）和粗枝猪毛菜（NAD苹果酸酶型,NAD-ME）为研究对象,采用盆栽控水试验设置正常供水和轻度、中度、重度干旱处理（土壤含水量分别为田间持水量80%、60%、45%和35%）,通过测定不同程度干旱胁迫下叶片含水量、C₄光合特征酶和抗氧化酶活性等指标,探讨不同类型C₄荒漠植物光合特征酶和抗氧化系统对干旱逆境的适应机制。结果显示：（1）2种植物叶片含水量均随干旱胁迫的加剧不同程度降低。（2）叶片磷酸烯醇式丙酮酸羧化酶（PEPC）活性在中度干旱胁迫下显著增加而在重度干旱胁迫下急剧下降;蔷薇猪毛菜NAD-ME活性和粗枝猪毛菜NADP-ME活性都很低,且它们基本不受干旱胁迫的影响;随干旱胁迫的加剧,蔷薇猪毛菜NADP-ME活性呈下降趋势,而粗枝猪毛菜NAD-ME活性先显著增加而在重度干旱胁迫下显著降低。（3）随着干旱胁迫的加剧,叶片超氧化物歧化酶（SOD）活性呈下降趋势,过氧化物酶（POD）活性在不同程度干旱胁迫下均有不同程度增加;过氧化氢酶（CAT）活性在中度干旱胁迫下均有不同程度的增加,但在重度干旱胁迫下蔷薇猪毛菜CAT活性降低,而粗枝猪毛菜CAT活性显著增加;丙二醛（MDA）含量随干旱胁迫的加剧均有不同程度的增加。研究认为,一定程度干旱胁迫下,2种荒漠植物的PEPC活性均有增加;不同光合类型C₄植物叶片脱羧酶（NADP-ME和NAD-ME）对干旱胁迫的响应有明显的差异。POD和CAT是这两种C₄植物适应干旱胁迫的主要抗氧化酶,但蔷薇猪毛菜CAT在重度干旱胁迫下没有起到积极保护作用。     

Structures and mechanisms of enzymes in the leukotriene cascade

Leukotrienes are a family of proinflammatory lipid mediators of the innate immune response and are important signaling molecules in inflammatory and allergic conditions. The leukotrienes are formed from arachidonic acid, which is released from membranes by cPLA₂, and further converted by 5-lipoxygenase to form the labile epoxide leukotriene (LT) A₄. This intermediate is converted by either of the two enzymes, LTA₄ hydrolase or LTC₄ synthase, to form LTB₄ or LTC₄, respectively. In order for 5-lipoxygenase to work efficiently in cells, five-lipoxygenase-activating protein needs to be present. LTB₄ is one of the most powerful chemotactic agents whereas LTC₄ induces smooth muscle contractions, for example in the airways causing bronchoconstriction in asthmatic patients. The leukotrienes and the five enzymes/proteins involved in their formation have been subject to intense studies including drug design programs. Compounds blocking the formation or action of leukotrienes are potentially beneficial in treatment of several acute and chronic inflammatory diseases of the cardiovascular and respiratory systems. In order to succeed with drug development studies, knowledge of the molecular characteristics of the targets is indispensable. This chapter reviews the biochemistry, catalytic, and structural properties of the enzymes in the leukotriene cascade.     

Carboxypeptidase A-catalyzed direct conversion of leukotriene C4 to leukotriene F4

Leukotrienes (LTs) are 5-lipoxygenase (5-LO)-derived arachidonic metabolites that constitute a potent set of lipid mediators produced by inflammatory cells. Leukotriene A(4), a labile allylic epoxide formed from arachidonic acid by dual 5-LO activity, is the precursor for LTB(4) and LTC(4) synthesis. LTC(4) is further transformed enzymatically by the sequential action of gamma-glutamyltranspeptidase and dipeptidase to LTD(4) and LTE(4), respectively. In this report, we present evidence that bovine pancreatic carboxypeptidase A (CPA), which shares significant sequence homology with CPA in mast cell granules, catalyzes the conversion of LTC(4) to LTF(4) via the hydrolysis of an amide bond. The identity of CPA-catalyzed LTC(4) hydrolysis product as LTF(4) was confirmed by several analytical criteria, including enzymatic conversion to conjugated tetraene by soybean LO, conversion to LTE(4) by gamma-glutamyltranspeptidase, cochromatography with the standard LTF(4) and positive-ion fast-atom bombardment mass spectral analysis. Thus, it appears that the physiological significance of this single-step transformation may point toward a major cellular homeostatic mechanism of metabolizing LTC(4), a potent bronco- and vasoconstrictor, to a less potent form of cysteinyl LTs.     

Role of Janus kinase-2 in IgE receptor-mediated leukotriene C4 production by mast cells

We have previously shown that Janus kinase 3, a member of the family of non-receptor protein tyrosine kinases, plays a critical role in the regulation of FcεRI-mediated mast cell responses. In the current study, we investigated the role of another JAK family member, JAK2, in these responses. Our results show that the treatment of IgE-sensitized mouse mast cells with an inhibitor of JAK2 (AG490) blocked the release of leukotriene C₄ in a dose-dependent fashion after antigen challenge. However, prostaglandin PG D₂ production and degranulation were not affected under identical experimental conditions. Transfection of RBL-2H3 mast cells with JAK-2 specific small interfering RNA resulted in a 50% reduction of LTC₄ release in response to FcεRI crosslinking, but did not inhibit mast cell degranulation or calcium ionophore-induced LTC₄ release, indicating involvement of JAK2 in IgE receptor-mediated leukotriene release. Taken together, these data suggest that JAK2 is a critical regulator of IgE/antigen-induced production of LTC₄ in mast cells.     

Direct and selective small-molecule inhibition of photosynthetic PEP carboxylase: New approach to combat C4 weeds in arable crops

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C₄ photosynthesis. Besides, non-photosynthetic isoforms of PEPC are found in bacteria and all types of plants, although not in animals or fungi. A single residue in the allosteric feedback inhibitor site of PEPC was shown to adjust the affinity of the photosynthetic and non-photosynthetic isoforms for feedback inhibition by metabolites of the C₄ pathway. Here, we applied computational screening and biochemical analyses to identify molecules that selectively inhibit C₄ PEPC, but have no effect on the activity of non-photosynthetic PEPCs. We found two types of selective inhibitors, catechins and quinoxalines. Binding constants in the lower μM range and a strong preference for C₄ PEPC qualify the quinoxaline compounds as potential selective herbicides to combat C₄ weeds.     

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.     

Fatty acids induce leukotriene C4 synthesis in macrophages in a fatty acid binding protein-dependent manner

Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C₄ (LTC₄) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC₄ and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC₄ and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC₄ precursor leukotriene A₄ (LTA₄) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC₄ synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC₄ secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC₄ and contribute to the insulin resistant state.     

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D4 in intestinal epithelial cells

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT₁R and CysLT₂R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF-α), and leukotriene D₄ (LTD₄), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC₄ synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF-α and LTD₄, to a lesser extent, up-regulate the CysLT₁R levels. Interestingly, TNF-α also reduced CysLT₂R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression.     

Drought stress increases the production of 5-hydroxynorvaline in two C4 grasses

Plants produce various compounds in response to water deficit. Here, the presence and identification of a drought-inducible non-protein amino acid in the leaves of two C₄ grasses is first reported. The soluble amino acids extracted from the leaves of three different species were measured by high-performance liquid chromatography of derivatives formed with o-phthaldialdehyde and β-mercaptoethanol. One amino acid that increased in amount with drought stress had a retention time not corresponding to any common amino acid. Its identity was determined by metabolite profiling, using ¹H NMR and GC-MS. This unusual amino acid was present in the dehydrated leaves of Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel, but was absent from Paspalum dilatatum Poir. Its identity as 2-amino-5-hydroxypentanoic acid (5-hydroxynorvaline, 5-HNV) was confirmed by synthesis and co-chromatography of synthetic and naturally occurring compounds. The amount of 5-HNV in leaves of the more drought tolerant C₄ grasses, C. dactylon and Z. japonica, increased with increasing water deficit; therefore, any benefits from this unusual non-protein amino acid for drought resistance should be further explored.     

Synthesis of N-alkyl glycine amides as potent inhibitors of leukotriene A4 hydrolase

The synthesis and biological evaluation of a series of N-alkyl glycine amide analogs as LTA₄-h inhibitors and the importance of the introduction of a benzoic acid group to the potency and pharmacokinetic parameters of our analogs are described. The lead compound in the series, 4q, has excellent potency and oral bioavailability.     

Discovery of novel and potent aryl diamines as leukotriene A4 hydrolase inhibitors

The synthesis and biological evaluation of a series of aryl diamines as inhibitors of LTA₄-h inhibitors are described. The optimization which led to the identification of the optimal para-substitution on the diphenyl ether moiety and diamine spacer is discussed. The resulting compounds such as 3l have excellent enzyme and cellular potency as well as desirable pharmacokinetic properties.     

Infection of maize leaves with Ustilago maydis prevents establishment of C4 photosynthesis

The Basidiomycete fungus Ustilago maydis is the common agent of corn smut and is capable of inducing gall growth on infected tissue of the C₄ plant maize (Zea mays). While U. maydis is very well characterized on the genetic level, the physiological changes in the host plant in response to U. maydis infection have not been studied in detail, yet.Therefore, we examined the influence of U. maydis infection on photosynthetic performance and carbon metabolism in maize leaf galls.At all stages of development, U. maydis-induced leaf galls exhibited carbon dioxide response curves, CO₂ compensation points and enzymatic activities that are characteristic of C₃ photosynthesis, demonstrating that the establishment of C₄ metabolism is prevented in infected tissue. Hexose contents and hexose/sucrose ratio of leaf galls remained high at 6 days post infection, while a shift in free sugar metabolism was observed in the uninfected controls at that time point. Concomitantly, transitory starch production and sucrose accumulation during the light period remained low in leaf galls. Given that U. maydis is infectious on young developing tissue, the observed changes in carbohydrate metabolism suggest that the pathogen manipulates the developing leaf tissue to arrest sink-to-source transition in favor of maintaining sink metabolism in the host cells.Furthermore, evidence is presented that carbohydrate supply during the biotrophic phase of the pathogen is assured by a fungal invertase.     

Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris

A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterative induction. P. pastoris was found to express several proteases; however, adjustment of medium pH to limit their activity did not correlate with increased scFv recovery. Induction medium pH values of 6.5-8.0 were most conducive to scFv production, despite significant differences in cell growth rates. Increasing inoculum density limited growth potential but gave rise to higher levels of scFv production. Three factors, medium composition, pre-induction osmotic stress, and temperature, had the greatest effects on protein production. Supplementation of the induction medium with arganine, casamino acids, or EDTA increased scFv production several fold, as did cultivation under osmotic stress conditions during pre-induction biomass accumulation. Incubation at 15 versus 30 degrees C extended the period whereby cells were capable of producing scFv from 1 to 7 days. Under optimal conditions, yeast cultures yielded 25 mg/L of functional scFv and could be subject to five reiterative inductions.