Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants,in pulmonary epithelial cells

The cation channel TRPA1 functions as a chemosensory protein and is directly activated by a number of noxious inhalants. A pulmonary expression of TRPA1 has been described in sensory nerve endings and its stimulation leads to the acceleration of inflammatory responses in the lung. Whereas the function of TRPA1 in neuronal cells is well defined, only few reports exist suggesting a role in epithelial cells. The aim of the present study was therefore (1) to evaluate the expression of TRPA1 in pulmonary epithelial cell lines, (2) to characterize TRPA1-promoted signaling in these cells, and (3) to study the extra-neuronal expression of this channel in lung tissue sections. Our results revealed that the widely used alveolar type II cell line A549 expresses TRPA1 at the mRNA and protein level. Furthermore, stimulating A549 cells with known TRPA1 activators (i.e., allyl isothiocyanate) led to an increase in intracellular calcium levels, which was sensitive to the TRPA1 blocker ruthenium red. Investigating TRPA1 coupled downstream signaling cascades it was found that TRPA1 activation elicited a stimulation of ERK1/2 whereas other MAP kinases were not affected. Finally, using epithelial as well as neuronal markers in immunohistochemical approaches, a non-neuronal TRPA1 protein expression was detected in distal parts of the porcine lung epithelium, which was also found examining human lung sections. TRPA1-positive staining co-localized with both epithelial and neuronal markers underlining the observed epithelial expression pattern. Our findings of a functional expression of TRPA1 in pulmonary epithelial cells provide causal evidence for a non-neuronal TRPA1-mediated control of inflammatory responses elicited upon TRPA1-mediated registration of toxic inhalants in vivo.     

Determination of 3-nitrotyrosine in human urine at the basal state by gas chromatography-tandem mass spectrometry and evaluation of the excretion after oral intake

3-Nitrotyrosine (NO(2)Tyr) is a potential biomarker of reactive-nitrogen species (RNS) including peroxynitrite. 3-Nitrotyrosine occurs in human plasma in its free and protein-associated forms and is excreted in the urine. Measurement of 3-nitrotyrosine in human plasma is invasive and associated with numerous methodological problems. Recently, we have described an accurate method based on gas chromatography (GC)-tandem mass spectrometry (MS) for circulating 3-nitrotyrosine. The present article describes the extension of this method to urinary 3-nitrotyrosine. The method involves separation of urinary 3-nitrotyrosine from nitrite, nitrate and l-tyrosine by HPLC, preparation of the n-propyl-pentafluoropropionyltrimethylsilyl ether derivatives of endogenous 3-nitrotyrosine and the internal standard 3-nitro-l-[(2)H(3)]tyrosine, and GC-tandem MS quantification in the selected-reaction monitoring mode under negative-ion chemical ionization conditions. In urine of ten apparently healthy volunteers (years of age, 36.5+/-7.2) 3-nitrotyrosine levels were determined to be 8.4+/-10.4 nM (range, 1.6-33.2 nM) or 0.46+/-0.49 nmol/mmol creatinine (range, 0.05-1.30 nmol/mmol creatinine). The present GC-tandem MS method provides accurate values of 3-nitrotyrosine in human urine at the basal state. After oral intake of 3-nitro-l-tyrosine by a healthy volunteer (27.6 microg/kg body weight) 3-nitro-l-tyrosine appeared rapidly in the urine and was excreted following a biphasic pharmacokinetic profile. Approximately one third of administered 3-nitro-l-tyrosine was excreted within the first 8 h. The suitability of the non-invasive measurement of urinary 3-nitrotyrosine as a method of assessment of oxidative stress in humans remains to be established.     

Effects of feeding high-quality hay with graded amounts of concentrate on feed intake,performance and blood metabolites of cows in early lactation

Dairy cows are commonly fed energy-dense diets with high proportions of concentrate feedstuffs to meet the increased energy needs of early lactation. However, feeding large amounts of concentrates may cause rumen acidosis and impact cow health. The hypothesis tested was that the energy supply and metabolic health of early-lactation Simmental cows can be maintained when high-quality hay rich in water-soluble carbohydrates (WSC) and crude protein (CP) is fed, despite the proportion of concentrates in the diet being reduced or even excluded. Twenty-four Simmental cows were allocated to one of four feeding groups beginning 10 d before the expected calving date, until 28 d thereafter. The feeding groups were 60CH (60% conventional fibre-rich hay plus 40% concentrate feed), 60HQH (60% high-quality hay plus 40% concentrate feed), 75HQH (75% high-quality hay plus 25% concentrate feed) and 100HQH (100% high-quality hay). The fibre-rich hay and high-quality hay differed in WSC content (110 g vs. 198 g of dry matter (DM)), neutral detergent fibre (646 g vs. 423 g of DM) and CP (65 g vs. 223 g of DM). Individual feed intake and milk production were monitored daily, and blood samples were collected weekly. Dry matter intake (DMI) and milk yield increased post partum, but 4 weeks post partum, the DMI of cows fed 100HQH only reached a daily mean DMI of 18.6 kg, whereas the DMI of the other groups averaged 21.9 kg (p < 0.046). The negative energy balance was less pronounced in cows fed 75HQH since they showed similar milk yields to the cows fed 60CH and 100HQH, but their energy intake was higher. Concentrations of milk components were similar across rations 60CH, 60HQH and 75HQH, as were most of blood parameters. Cows fed 100HQH responded to the energy deficit post partum with a higher ratio of non-esterified fatty acids to cholesterol and a higher concentration of ß-hydroxybutyrate (significant in comparison to cows fed 75HQH, p < 0.05). In conclusion, feeding high-quality hay with a WSC content of 20% in DM has the potential to decrease the proportion of concentrates in dairy cow feeding in early lactation, but cannot fully replace their supplementation due to a limited rumen capacity for forage intake.     

Beyond the Matrix-Assisted Laser Desorption Ionization (MALDI) Biotyping Workflow: in Search of Microorganism-Specific Tryptic Peptides Enabling Discrimination of Subspecies

A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspecies-specific biomarker peptides for three Salmonella subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level.     

Effects of the replacement of concentrate and fibre-rich hay by high-quality hay on chewing,rumination and nutrient digestibility in non-lactating Holstein cows

The aim of this study was to investigate the effects of high-quality hay with an elevated sugar content alone or with graded amounts of concentrate feed on chewing and ruminating activity, apparent total tract digestibility (ATTD) and ruminal pH at different time points after feeding in the free ruminal liquid (FRL) and the particle-associated ruminal liquid (PARL). Eight rumen cannulated non-lactating Holstein cows were arranged in a Latin square design in four experimental runs lasting 25 d each. The four diets tested were 60NQ (60% normal-quality hay + 40% concentrate), 60HQ (60% high-quality hay + 40% concentrate), 75HQ (75% high-quality hay + 25% concentrate) and 100HQ (100% high-quality hay). Normal and high-quality hays differed in sugar contents (11.3% vs. 18.7% in dry matter [DM]), neutral detergent fibre (NDF; 57.7% vs. 46.3% in DM), acid detergent fibre (ADF, 35.0% vs. 23.5% in DM) and crude protein (CP, 11.3% vs. 23.5% in DM). Data showed that ATTD of DM, CP, NDF and ADF was higher with the high-quality hay diets. Time spent eating was reduced with high-quality hay. However, time spent ruminating was longest in Group 100HQ. In all groups, ruminal pH of FRL and PARL decreased with time after the morning feeding. But 10 h later, pH of Group 100HQ was higher again compared with the other groups. Considering the average pH in FRL over all measured time points, cows in Groups 60NQ and 100HQ had higher pH values of 6.85 and 6.83, respectively. Regarding pH values in PARL, animals of Group 60NQ displayed the highest pH value (6.68), whereas the lowest value of 6.21 was found in Group 60HQ. Overall, results suggest that high-quality hay maintains the diet’s structural effectiveness by stimulating rumination and stabilising ruminal pH while greatly improving ATTD. However, the structural effectiveness of the high-quality hay gets impaired with increasing proportion of concentrate feed in the diet.     

Bronchial epithelial cell-derived prostaglandin E2 dampens the reactivity of dendritic cells

Airway epithelial cells regulate immune reactivity of local dendritic cells (DCs), thus contributing to microenvironment homeostasis. In this study, we set out to identify factors that mediate this regulatory interaction. We show that tracheal epithelial cells secrete soluble factors that downregulate TNF-α and IL-12p40 secretion by bone marrow-derived DCs but upregulate IL-10 and arginase-1. Size exclusion chromatography identified small secreted molecules having high modulatory activity on DCs. We observed that airway tracheal epithelial cells constitutively release the lipid mediator PGE(2). Blocking the synthesis of PGs within airway epithelial cells relieved DCs from inhibition. Cyclooxygenase-2 was found to be expressed in primary tracheal epithelial cell cultures in vitro and in vivo as shown by microdissection of epithelial cells followed by real-time PCR. Paralleling these findings we observed that DCs treated with an antagonist for E-prostanoid 4 receptor as well as DCs lacking E-prostanoid 4 receptor showed reduced inhibition by airway epithelial cells with respect to secretion of proinflammatory cytokines measured by ELISA. Furthermore, PGE(2) mimicked the effects of epithelial cells on DCs. The results indicate that airway epithelial cell-derived PGE(2) contributes to the modulation of DCs under homeostatic conditions.     

Glutathione promotes prostaglandin H synthase (cyclooxygenase)-dependent formation of malondialdehyde and 15(S)-8-iso-prostaglandin F2α

Prostaglandin (PG) H synthases (PGHS) or cyclooxygenases (COX) catalyse the peroxidation of arachidonic acid (AA) to PGG₂ and PGH₂ which are further converted to a series of prostaglandins and thromboxane A₂. Here, we report that GSH promotes concomitant formation of the current oxidative stress biomarkers malondialdehyde (MDA) and 15(S)-8-iso-prostaglandin F_2α from AA via PGHS. This illustrates an uncommon interplay of enzymatic and chemical reactions to produce species that are considered to be exclusively produced by free-radical-catalysed reactions. We propose mechanisms for the PGHS/AA/GSH-dependent formation of MDA, 15(S)-8-iso-prostaglandin F_2α and other F₂-isoprostanes. These mechanisms are supported by clinical observations.