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Abstract

We demonstrated a facile and environmental-friendly approach to form gold nanoparticles through the reduction of HAuCl4 by aspartame. The single-crystalline structure was illustrated by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The energy-dispersive X-ray spectroscopy (EDS) and Fourier transform infrared (FTIR) results indicated that aspartame played a pivotal role in the reduction and stabilization of the gold crystals. The crystals were stabilized through the successive hydrogen-bonding network constructed between the water and aspartame molecules. Additionally, gold nanoparticles synthesized through aspartame were shown to have good catalytic activity for the reduction of p-nitrophenol to p-aminophenol in the presence of NaBH4.

Synthesis of APM-AuNPs

To obtain APM-AuNPs, 0.4 ml of APM aqueous solution (5 mg/ml) and 200 μl of NaOH (100 mM) were added to 9.3 ml of deionized water and incubated in a water bath (Julabo, Seelbach/Black Forest, Germany) (60°C, 200 rpm) for 10 min. Then, 100 μl of HAuCl4 (100 mM) was added followed by vigorous stirring for 30 s. Finally, the mixture was incubated for an additional 50 min (60°C, 200 rpm). The effects of several parameters, such as APM concentration (3.4 × 10−6 M to 1.7 × 10−4 M), HAuCl4 concentration (1.0 × 10−4 M to 9.0 × 10−4 M), pH (2.0 to 12.0), and temperature (25°C/37°C/60°C/90°C) were systematically investigated.

Rat Mesencephalic TH-Positive Neurons: Primary Culture, Treatment, and Immunocytochemistry

The cultures were performed following the methods described previously (Sun et al. ) with some modifications. Briefly, 1 mm3 of the ventricular mesencephalic region was obtained from Sprague–Dawley rat embryos (embryonic day 15). Two incisions were made, one in the forebrain/mesencephalic limit and the other between the mesencephalon and the hindbrain. Once dissected from the meninges, the tissue was disaggregated using trypsin and DNaseI. Twenty thousand cells were plated in each well of an 8-well chamber slide with B-27/N2 medium containing 2.5 % serum. GDNF, TGF-β, and dcAMP were added once to increase TH-positive cells (Supplementary Fig. 1) and the media were changed every 2 days. The percentage of TH-positive cells achieved was 17–20 %. NPD1 (100 nM) was added to the culture at 7 DIV, 5 min before the addition of 100 nM rotenone, 100 µM MPP+ or 100 µM MPTP. To decide the time of exposure to MPTP and rotenone, 24- and 48-h time points were used (Supplementary Figs. 2, 3). Immunostaining was performed following a previously described protocol (Calandria et al. ). Briefly, cells were fixed with 4 % paraformaldehyde, permeabilized with 1 % Triton and 1 % normal serum in phosphate buffered saline (PBS) for 5 min, and then blocked with 10 % normal serum solution in PBS. The slides were incubated with the primary antibody overnight and then for 1 h with Alexa fluor-conjugated secondary antibodies.

TH-Positive Neuron Cell Death Triggered by Rotenone and MPP+ is Prevented by NPD1

Rotenone and the MPTP metabolite, MPP+, was proposed to induce toxicity primarily by blocking mitochondrial Complex I, which results in the production of superoxide radicals and consequently, oxidative stress (Ramachandiran et al. ). To determine the ability of NPD1 to prevent cell death induced by these three compounds, mesencephalic primary-cultured neurons were exposed to 100 nM rotenone, 100 µM MPTP, or 100 µM MPP+ in the presence and absence of NPD1 for 24 h (Fig. a–c). MPP+ severely decreased the number of TH-positive neurons from 40.3 ± 7.3 in the control to 7.6 ± 1.6; with the addition of NPD1, the number of surviving cells was raised to 14.4 ± 2.7 (Fig. b). MPTP, the precursor of MPP+, also displayed toxicity, promoting the disappearance of 65.7 % of the TH-positive neurons, but this effect was not significantly counteracted by NPD1 (Fig. b). On the other hand, rotenone reduced the TH-positive neuronal population 63 %, and NPD1 reversed that effect almost completely (Fig. c). The NPD1 stereoisomer (10S,17S-diHDHA) had no effect on TH-positive cell survival, indicating that the protection was specifically induced by the S/R stereoisomer (Figs. c, o, p).

Neurons That Survived with the Addition of NPD1 Showed Better Dendritic Morphology on Average

Mitochondrial damage and depletion leads to neurite retraction or shortening (Cherra et al. ). We analyzed the dendritic arbors of surviving TH-positive neurons to evaluate whether NPD1 addition affected neurite integrity. General features were assessed, including maximum dendritic order reached for each TH-positive neuron, number of dendrite segments and branches, the sum of all the segment lengths for each neuron, and the mean for the dendrite diameters. MPTP and MPP+ only altered the parameters related to the length of the dendrites and not related to their thickness, as shown in Fig. d–m. Overall, the integrity of the dendritic arbor was best preserved in cells treated with NPD1 in comparison with the ones that survived without NPD1 treatment (Fig. d–m). Although more TH-positive neurons survived the rotenone treatment with the addition of NPD1 (Fig. c), there was no difference in the maximum dendritic order, the number of segments, or the combined length of all branches and segments when neurons were treated with the lipid mediator (Fig. n, o, q). Intriguingly, the number of branches (Fig. p) on average did not show any significant changes in rotenone-treated TH-positive neurons when compared to controls, suggesting a different mechanism of toxicity. On the contrary, MPP+ induced a reduction in length and number of branches (Fig. d, f, h, j, l), and these effects were reversed almost completely by NPD1. MPTP produced an outcome similar to MPP+, although NPD1 did not provide the same level of improvement (Fig. e, g, i, k, m).

Discussion

NPD1 is a docosahexaenoic acid derivative shown to be protective in neurons and RPE cells undergoing proteotoxic and oxidative stress damage (Mukherjee et al. ; Calandria et al. ) as well as in other dysruptions of homeostasis (Bazan et al. ). Here we report specific NPD1 effects on the survival and dendrite architecture of TH-positive cells exposed to MPTP, its metabolite MPP+, or rotenone. NPD1 increased the survival of TH-positive cells treated with MPP+ (Fig. b) or rotenone (Fig. c). MPTP treatment did not reduce the neuron population to the same extent, and NPD1-mediated neuroprotection was not as strong as with the other two toxins (Fig. b). Interestingly, MPTP toxicity was not reversed by NPD1 to the same extent as MPP+, suggesting that both actions are executed by different mechanisms. MPTP conversion to MPP+ and release occurs in glial cells (Ransom et al. ). It may be possible that this processing of MPTP delays and decreases the amount of MPP+ that reaches neurons, thus causing a weaker but sustained damage compared to the acute insult produced by MPP+. This may explain the differences in TH-positive neurons survival and the protection exerted by NPD1 (Fig. b).

Co-crystallization and Soaking Experiments

For ygiF-PPPi-Mg2+/Mn2+, the substrate-bound complex was obtained by soaking (20–30 min) ygiF apo crystals in their crystallization buffer supplemented with 20% (v/v) glycerol, 10 mm sodium tripolyphosphate and 5 mm MgCl2 or MnCl2. ThTPase-ThTP-Mg2+. Form B crystals were soaked in crystallization buffer containing 20% (v/v) glycerol, 10 mm thiamine triphosphate and 10 mm MgCl2.

Abstract

Abnormal metabolism is another cancer hallmark. The two most characterized altered metabolic pathways are high rates of glycolysis and glutaminolysis, which are natural targets for cancer therapy. Currently, a number of newer compounds to block glycolysis and glutaminolysis are being developed; nevertheless, lonidamine and 6-diazo-5-oxo-L-norleucine (DON) are two old drugs well characterized as inhibitors of glycolysis and glutaminolysis, respectively, whose clinical development was abandoned years ago when the importance of cancer metabolism was not fully appreciated and clinical trial methodology was less developed. In this review, a PubMed search using the words lonidamine and 6-diazo-5-oxo-L-norleucine (DON) was undertaken to analyse existing information on the preclinical and clinical studies of these drugs for cancer treatment. Data show that they exhibit antitumor effects; besides there is also the suggestion that they are synergistic. We conclude that lonidamine and DON are safe and potentially effective drugs that need to be reevaluated in combination as metabolic therapy of cancer.

2. Glycolytic and Glutaminolytic Inhibitors in Development

Regarding glutaminolytic agents, these are fewer. Among these oldest ones are the (i) glutamine analogs: acivicin, 6-diazo-5-oxo-L-norleucine (DON), azaserine, and azotomycin and (ii) miscellaneous more selective and potent inhibitors: bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and its analogs. Other agents are ebselen, chelerythrine, apomorphine, and CB-839 [–]. Apart from the oldest analogs of glutamine such as acivicin, DON, azaserine, and azotomycin which were clinically evaluated several decades ago and their development abandoned, only CB-839 among the newest analogs has recently reached to phase I clinical trials.

4.1. Chemistry

6-Diazo-5-oxo-L-norleucine also known as DON was initially described as an antitumor antibiotic isolated from an unidentified streptomyces strain. It has a molecular weight of 171.15 and its empirical formula is C6H9N3O3. DON is very sensitive to heat and pH, and the optimal pH at room temperature ranges from 4.5 to 6.5. DON is a light yellow powder very soluble in water and aqueous solutions of methanol, acetone, or ethanol []. As an analog of glutamine, DON has been used as an inhibitor of glutamine utilizing enzymes such as carbamoyl phosphate synthase (CAD), CTP synthase (CTPS), FGAR amidotransferase, guanosine monophosphate synthetase (GMPS), PRPP amidotransferase, NAD synthase, asparagine synthase, and glutaminase. These enzymes are used in several important metabolic pathways such as the purine, pyrimidine, and amino acid synthesis as well as a coenzyme of the electron transport chain and in the first step of glutaminolysis [–].

4.5. Safety and Tolerability

From the experience with DON for cancer treatment it can be concluded that it is a safe drug and that the dose-limiting toxicity is nausea and vomiting which varied according to the schedule and dose. Divided doses (every four or six hours) caused more nausea and vomiting than the same dose in single daily administration. Despite the fact that DON in addition to its inhibitory action upon GSL1 has a number of targets involved in DNA synthesis, the resulting myelotoxicity is mild with fast recovery, manifested by leucopenia and thrombocytopenia and never life threatening.

Abstract

Arachidonic acid (ARA) metabolites produced by cyclo-oxygenase and lipoxygenase are important mediators maintaining physiological renal function. However, the effects of exogenous ARA on kidney function in vivo remain unknown. This study examined the effects of long-term oral ARA administration on normal renal function as well as inflammation and oxidative stress in aged rats. In addition, we measured levels of renal eicosanoids and docosanoids using liquid chromatography–tandem mass spectrometry. Control or ARA oil (240 mg/kg body weight/day) was orally administered to 21-month-old Wistar rats for 13 weeks. Levels of plasma creatinine, blood urea nitrogen, inflammatory and anti-inflammatory cytokines, reactive oxygen species, and lipid peroxidation were not significantly different between the two groups. The ARA concentration in the plasma, kidney, and liver increased in the ARA-administered group. In addition, levels of free-form ARA, prostaglandin E2, and 12- and 15-hydroxyeicosatetraenoic acid increased in the ARA-administered group, whereas renal concentration of docosahexaenoic acid and eicosapentaenoic acid decreased in the ARA-administered group. Levels of docosahexaenoic acid-derived protectin D1, eicosapentaenoic acid-derived 5-, and 18-hydroxyeicosapentaenoic acids, and resolvin E2 and E3 decreased in the ARA-administered group. Our results indicate that long-term ARA administration led to no serious adverse reactions under normal conditions and to a decrease in anti-inflammatory docosahexaenoic acid- and eicosapentaenoic acid-derived metabolites in the kidneys of aged rats. These results indicate that there is a possibility of ARA administration having a reducing anti-inflammatory effect on the kidney.

Animals and treatments

Rats (Jcl: Wistar) purchased from CLEA Japan (Osaka, Japan) were housed in a room under controlled temperature (23 ± 2°C), humidity (50 ± 10%), and light–dark cycle (light: 07:00–19:00; dark: 19:00–07:00). They were fed a fish oil-deficient diet (F-1™ Funabashi Farm, Funabashi, Japan) and water ad libitum. Inbred second-generation male rats, fed the same F-1 diet, were used for our study. Fatty acid composition in the F-1 diet was described previously []. ARA oil was obtained from Cargill Alking Bioengineering (Wuhan and Hubei, China) [,]. The fatty acid compositions of the ARA and control oils are shown in . Twenty one-month-old male rats were divided into two groups: the control group was orally administrated with the control oil and the ARA group was administrated ARA oil (240 mg ARA/kg body weight/day) for 13 weeks. After a 16-h fast, rats were anesthetized with intraperitoneal sodium pentobarbital (65 mg/kg body weight), following which their plasma, kidneys, and liver were removed, immediately frozen in liquid nitrogen, and stored at −30°C until further use. The kidneys and liver were homogenized in phosphate buffer (pH, 7.4) using a Teflon homogenizer (AGC techno glass Co., Ltd. Shizuoka, Japan). The homogenates were immediately frozen in liquid nitrogen and stored at −30°C until use. Concentrations of creatinine and blood urea nitrogen (BUN) were determined in plasma samples using an automatic analyzer (BiOLiS 24i; Tokyo Boeki Medical System Ltd., Tokyo, Japan).

Levels of eicosanoids and docosanoids in the kidney

Kidney analyses revealed an increase in renal formation of PGE2, 12-HETE, and 15-HETE in the ARA-administered group (). Moreover, endogenous formation of DHA-derived PD1 and EPA-derived 5-HEPE, 18-HEPE, RvE2, and RvE3 decreased significantly in the ARA-administered group. Nonesterified ARA levels in the kidney increased significantly in the ARA-administered group, whereas levels of nonesterified EPA and DHA were not significantly different between the two groups.

Discussion

Concentrations of non-esterified EPA and DHA increased slightly, but not significantly, in the ARA-administered group compared with those in the control group, although total EPA and DHA concentrations decreased in the kidney. Levels of EPA-derived eicosanoids 5-HEPE, 18-HEPE, RvE2, and RvE3 and the DHA-derived docosanoids PD1 decreased significantly in the ARA-administered group. These results suggest that ARA directly competes with the storage of EPA and DHA at the sn-2 position in phospholipids and blocks the production of EPA- and DHA-derived metabolites []. We have reported that the DHA-derived docosanoids RvDs and PD1 protect renal damage progression induced by metabolic syndrome []. EPA-derived eicosanoids and DHA-derived docosanoids are endogenous mediators with potent anti-inflammatory actions in the kidneys []. Administration of RvDs or PD1 to mice prior to ischemia results in a reduction in functional and morphological renal injury []. Hong and Lu demonstrated that RvDs and PD1 repress renal interstitial fibrosis, and PD1 inhibits the inflammatory response and promotes renoprotective heme-oxygenase-1 expression during acute kidney injury []. Heme-oxygenase-1 decreases acute tubular necrosis and significantly reduces COX-2 and microsomal PGE synthase expression []. Our study demonstrated that RvE2, RvE3, and PD1 levels decreased significantly in the ARA-administered group, indicating that anti-inflammatory defense may be attenuated by ARA administration.

Abstract

Metabolomic analysis revealed that Methylobacterium cells accumulate a large amount of ergothioneine (EGT), which is a sulfur-containing, non-proteinogenic, antioxidative amino acid derived from histidine. EGT biosynthesis and its role in methylotrophy and physiology for plant surface-symbiotic Methylobacterium species were investigated in this study. Almost all Methylobacterium type strains can synthesize EGT. We selected one of the most productive strains (M. aquaticum strain 22A isolated from a moss), and investigated the feasibility of fermentative EGT production through optimization of the culture condition. Methanol as a carbon source served as the best substrate for production. The productivity reached up to 1000 μg/100 ml culture (1200 μg/g wet weight cells, 6.3 mg/g dry weight) in 38 days. Next, we identified the genes (egtBD) responsible for EGT synthesis, and generated a deletion mutant defective in EGT production. Compared to the wild type, the mutant showed better growth on methanol and on the plant surface as well as severe susceptibility to heat treatment and irradiation of ultraviolet (UV) and sunlight. These results suggested that EGT is not involved in methylotrophy, but is involved in their phyllospheric lifestyle fitness of the genus in natural conditions.

Chemical treatments

The following compounds were tested in root growth assays using the indicated concentrations. 9-HOT (9(S)-hydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid), 9-KOT (9-keto-10(E),12(Z),15(Z)-octadecatrienoic acid) and OPDA (12-Oxo-Phytodienoic Acid) were prepared as described in [,], respectively. TIBA (2,3,5-triiodobenzoic acid), Naphthalene-Acetic Acid (NAA) and Jasmonic Acid (JA) were obtained from Sigma (Munich, Germany).

Screening for transcription factors involved in oxylipin signaling and detoxification

Aiming at TF function involved in the responses to 9-LOX-oxylipins, we assayed the root growth of 2-week-old Arabidopsis seedlings on MS-Agar supplemented with 25 μM 9-HOT. In contrast to [], a “wavy” root growth phenotype was not reproducibly observed under our experimental conditions, but root apical growth was severely impaired (). Based on the hypothesis that oxylipin signaling or detoxification programs are transcriptionally controlled, a screen based on overexpression of TFs should identify crucial regulators involved in these processes. Here, we established a screen using the AtTORF-Ex resource [], a collection of GATEWAY®-derived Arabidopsis TF overexpression lines employing the 35S promoter and scored plants showing wildtype (wt) root growth after 9-HOT application to the media. Screening of more than 6000 seedlings recovered 201 individuals (3%) which display tolerance to 9-HOT (). According to the calculations in [], the sample size should be statistically sufficient to cover all possible TFs in the screen with a probability of 99%. Due to the conserved GATEWAY®-att sequences flanking the TF orfs in the AtTORF-Ex transgenic lines, the respective overexpressed TF genes can easily be recovered by PCR (). As summarized in , several TF genes have been isolated up to 5 times, and hence their overexpression leads reproducibly to 9-HOT tolerant root growth (e.g. ERF106, ERF107, MADS-box factor AGL86). Members of the bZIP family are clearly overrepresented among the TFs leading to 9-HOT tolerance. This is particularly interesting, as bZIP members have been described as regulators of protective pathways towards endogenous or exogenous oxylipin molecules in plant and non-plant species [,,,–]. However, evolutionary conservation is not supported by homology outside the bZIP domain. Frequently, we observed that expression of related family members resulted in the same growth phenotype. Presumably, these TFs share related binding properties and functional redundancies. This was particularly true for bZIP factors of the group S [], which overall were found 18 times in the screen. The most abundant bZIP members are the highly related TFs bZIP11 and bZIP2 (overall 7 times). Furthermore, CRFs are a group of TFs which modulate cytokinine signaling [,]. Although single CRFs were found only twice, overall 3 related group members were detected. Cytokinine has a crucial function in controlling root meristem size [] and hence, CRF overexpression may overcome the block in root growth caused by 9-HOT. In contrast, TFs identified only once or twice are less likely candidates. Nevertheless, less abundant TFs may also be important but might have missed for technical reasons. We have selected several TF candidates to study their impact in the tolerance mechanism to 9-HOT in more detail.

Expression of the bZIP transcription factor TGA5 and its target gene CYP81D11 provoke tolerance to 9-HOT

TGAs have been proposed to be involved in controlling detoxification pathways with respect to oxylipins or xenobiotics. The cytochrome P450 monoxygenase gene CYP81D11 is transcriptionally induced by lipid peroxidation products like phytoprostanes, OPDA and JA as well as electrophilic xenobiotics (e.g. 2,4-dichlorophenoxyacetic acid, benoxacor, or the auxin-transport inhibitor TIBA (2,3,5-triiodobenzoic acid) [,]. Along this line, plants constitutively expressing CYP81D11 show significantly increased root growth on 9-HOT containing media (), demonstrating that the CYP81D11 enzyme is sufficient to provide full tolerance to 9-HOT. Hence, although the enzymatic function of CYP81D11 is not well-defined yet, these and previous data propose a function in metabolizing both 9- and 13-LOX-derived oxylipins. However, in comparison to wt, root growth of the cyp81d11 mutant on 9-HOT is slightly but not significantly reduced. Most likely, other CYP450 proteins may share redundant functions. Further studies are needed to define the substrate specificity of CYP81D11 with respect to xenobiotics and endogenous compounds.

Abstract

In order to explore the possibilities of using zinc (Zn) stable isotope ratios as dietary indicators, we report here on the measurements of the ratio of stable isotopes of zinc (66Zn/64Zn, expressed here as δ66Zn) in bioapatite (bone and dental enamel) of animals from a modern food web in the Koobi Fora region of the Turkana Basin in Kenya. We demonstrate that δ66Zn values in both bone and enamel allow a clear distinction between carnivores and herbivores from this food web. Differences were also observed between browsers and grazers as well as between carnivores that consumed bone (i.e. hyenas) compared to those that largely consume flesh (i.e. lions). We conclude that Zn isotope ratio measurements of bone and teeth are a new and promising dietary indicator.

Conclusions

The results of this study show that the Zn isotopic composition of both bone and dental enamel is strongly affected by diet. Carnivore mineralized tissues are typically depleted in the heavier Zn isotope, and are approximately 0.4–0.5‰ lower in their 66Zn/64Zn ratio compared to herbivores from the same location and food web. We showed that Zn isotopes of both bone and enamel distinguished between carnivores and herbivores within the modern Koobi Fora food web. Further work is needed to confirm if variations between types of consumers are applicable to regions with various underlying bedrock and soil Zn isotope ratio. Additionally, the preservation of dietary Zn isotope signatures in fossil teeth needs to be tested to explore the potential for palaeodietary reconstructions.

Methods

Utilizing myriocin, a potent inhibitor of sphingolipid synthesis, we evaluated the immune regulatory role of de novo ceramide generation in vitro and in vivo. Intratracheal myriocin was administered alone or during house dust mite sensitization (HDM) of BALB/C mice and airway hyper‐responsiveness (AHR) was evaluated by invasive plethysmography followed by bronchial lavage (BAL) cytology and cytokine quantification.

Results

Myriocin inhibits and HDM exposure activates de novo ceramide synthesis in bone marrow‐derived dendritic cells. Mice receiving intratracheal myriocin developed a mild airway neutrophilic infiltrate without inducing a significant increase in AHR. CXCL1 was elevated in the BAL fluid of myriocin‐treated mice while the neutrophilic chemotactic factors anaphylatoxin C5a, leukotriene B4, and IL‐17 were unaffected. HDM treatment combined with myriocin led to a dramatic enhancement of AHR (63% increase over HDM alone, p < 0.001) and increased granulocyte pulmonary infiltrates versus HDM or myriocin alone. Elevated Th2 T cell counts and Th2 cytokines/chemokines (IL5, IL13, CCL17) were observed in mice treated with combined HDM/myriocin compared to HDM alone. Myriocin‐treated pulmonary CD11c+ cells stimulated with HDM secreted significantly more CXCL1 than cells stimulated with HDM alone while HDM stimulated airway epithelial cells showed no change in CXCL1 secretion following myriocin treatment.

Animals, immunizations, and materials

Seven‐ to eight‐week‐old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME), maintained in a specific pathogen free facility at Cincinnati Children's Hospital Medical Center (CCHMC) and handled under Institutional Animal Care and Use Committee‐approved procedures. Following isoflourane anesthesia, animals underwent allergen immunization as previously described with the following modifications: mice were serially immunized intratracheally every 48 h for 2 weeks with low‐endotoxin, low‐dose HDM extract (D. pteronyssinus, Greer Labs; Lenoir, NC) (3 μg total protein/dose in 50 μL of sterile phosphate‐buffered saline plus vehicle [PBS]). Myriocin (Cayman Chemical Company; Ann Arbor, MI) stock solutions (5 mM) were prepared in methanol, and aqueous solutions were prepared fresh before each experiment (1.2 μg/intratracheal dose). Ceramide C6 (N‐hexanoyl‐D‐erythro‐sphingosine) was obtained from Avanti Polar Lipids (Alabaster, AL), mouse GM‐CSF was acquired from PeproTech(Rocky Hill, NJ), and RPMI and serine‐free MEM were purchased from Gibco (Carsbad, CA). Normal human bronchial epithelial cells (NHBE) were obtained from Lonza (Walkerville, MD) and cultured per the manufacturer's instructions. Murine bone marrow‐derived dendritic cells (BMDCs) were generated as previously described .

Pulmonary SPT inhibition enhances airway hyper‐responsiveness

Allergen‐induced AHR is a robust physiologic end‐point for quantitating the influence of bioactive compounds on the pathophysiology of allergic asthma. Using a well‐characterized murine model of HDM‐induced AHR, we investigated the biochemical and physiologic effects of intratracheal myriocin on lung lipid content, lung function, and airway structure (treatment protocol outlined in Fig. A). Similar to other investigators, we administered a low dose of intratracheal HDM extract, which sub‐maximally induces AHR and airway inflammation, thus increasing our ability to detect additive and synergistic effects , , , . First, we performed AHR measurement on mice after eight serial doses of intratracheal myriocin and did not detect a significant rise in AHR compared with vehicle‐treated control mice (Fig. B) despite a sustained reduction in lung ceramide levels (Fig. C). Next, we performed histologic examination of lung tissue from myriocin‐treated mice, which showed normal bronchial and alveolar architecture, similar to vehicle‐treated mice (Fig. A vs. D).

Pulmonary SPT inhibition enhances airway hyper‐responsiveness

After establishing that intratracheal myriocin does not induce significant AHR in our system, we compared the effect of HDM/myriocin co‐administration with that of HDM alone and found that co‐administration significantly enhanced AHR (p < 0.001) (Fig. B). Myriocin's effect on lung ceramide levels, however, was undetectable in animals co‐treated with HDM/myriocin (Fig. C,Supplemental Fig. S1B). Lung histology from both HDM only and HDM/myriocin‐treated mice revealed similar levels of peribronchial inflammatory infiltrates (Fig. B vs. E) and goblet cell metaplasia (Fig. C vs. F, Supplementary Fig. S1C). Goblet cell gene expression analysis revealed that HDM exposure induced multiple asthma‐associated transcripts, including chloride channel calcium‐activated 1 (Clca1/Gob‐5), mucin 5, subtypes A and C, tracheobronchial/gastric (Muc5ac), intelectin 1 (Itln1), and intelectin 2 (Itln2/B). Compared to HDM alone, HDM/myriocin co‐administration enhanced Itln1 and Itln2 expression, had no effect on Clca1, and significantly decreased Muc5ac expression. Therefore, in an allergic model of asthma, myriocin‐treatment is associated with enhanced HDM‐induced AHR and an altered goblet cell gene expression pattern.