Heme peroxidase HPX-2 protects Caenorhabditis elegans from pathogens.

Heme-containing peroxidases are important components of innate immunity. Many of them functionally associate with NADPH oxidase (NOX)/dual oxidase (DUOX) enzymes by using the hydrogen peroxide they generate in downstream reactions. Caenorhabditis elegans encodes for several heme peroxidases, and in a previous study we identified the ShkT-containing peroxidase, SKPO-1, as necessary for pathogen resistance. Here, we demonstrated that another peroxidase, HPX-2 (Heme-PeroXidase 2), is required for resistance against some, but not all pathogens. Tissue specific RNA interference (RNAi) revealed that HPX-2 functionally localizes to the hypodermis of the worm. In congruence with this observation, hpx-2 mutant animals possessed a weaker cuticle structure, indicated by higher permeability to a DNA dye, but exhibited no obvious morphological defects. In addition, fluorescent labeling of HPX-2 revealed its expression in the pharynx, an organ in which BLI-3 is also present. Interestingly, loss of HPX-2 increased intestinal colonization of E. faecalis, suggesting its role in the pharynx may limit intestinal colonization. Moreover, disruption of a catalytic residue in the peroxidase domain of HPX-2 resulted in decreased survival on E. faecalis, indicating its peroxidase activity is required for pathogen resistance. Finally, RNA-seq analysis of an hpx-2 mutant revealed changes in genes encoding for cuticle structural components under the non-pathogenic conditions. Under pathogenic conditions, genes involved in infection response were differentially regulated to a greater degree, likely due to increased microbial burden. In conclusion, the characterization of the heme-peroxidase, HPX-2, revealed that it contributes to C. elegans pathogen resistance through a role in generating cuticle material in the hypodermis and pharynx.

The influence of monoamine oxidase variants on the risk of betel quid-associated oral and pharyngeal cancer.

Betel quid (BQ) and areca nut (AN) (major BQ ingredient) are group I human carcinogens illustrated by International Agency for Research on Cancer and are closely associated with an elevated risk of oral potentially malignant disorders (OPMDs) and cancers of the oral cavity and pharynx. The primary alkaloid of AN, arecoline, can be metabolized via the monoamine oxidase (MAO) gene by inducing reactive oxygen species (ROS). The aim of this study was to investigate whether the variants of the susceptible candidate MAO genes are associated with OPMDs and oral and pharyngeal cancer. A significant trend of MAO-A mRNA expression was found in in vitro studies. Using paired human tissues, we confirmed the significantly decreased expression of MAO-A and MAO-B in cancerous tissues when compared with adjacent noncancerous tissues. Moreover, we determined that MAO-A single nucleotide polymorphism variants are significantly linked with oral and pharyngeal cancer patients in comparison to OPMDs patients [rs5953210 risk G-allele, odds ratio = 1.76; 95% confidence interval = 1.02-3.01]. In conclusion, we suggested that susceptible MAO family variants associated with oral and pharyngeal cancer may be implicated in the modulation of MAO gene activity associated with ROS.

Use of MMP-8 and MMP-9 to assess disease severity in children with viral lower respiratory tract infections.

Matrix metalloproteinases (MMPs) play an important role in respiratory inflammatory diseases, such as asthma and chronic obstructive pulmonary disease. It was hypothesized that MMP-8 and MMP-9 may function as biological markers to assess disease severity in viral lower respiratory tract infections in children. MMP-8 and MMP-9 mRNA expression levels in peripheral blood mononuclear cells (PBMCs) and granulocytes obtained in both the acute and recovery phase from 153 children with mild, moderate, and severe viral lower respiratory tract infections were determined using real-time PCR. In addition, MMP-8 and MMP-9 concentrations in blood and nasopharyngeal specimens were determined during acute mild, moderate, and severe infection, and after recovery using ELISA. Furthermore, PBMCs and neutrophils obtained from healthy volunteers were stimulated with RSV, LPS (TLR4 agonist), and Pam3Cys (TLR2 agonist) in vitro. Disease severity of viral lower respiratory tract infections in children is associated with increased expression levels of the MMP-8 and MMP-9 genes in both PBMCs and granulocytes. On the contrary, in vitro experiments showed that MMP-8 and MMP-9 mRNA and protein expression in PBMCs and granulocytes is not induced by stimulation with RSV, the most frequent detected virus in young children with viral lower respiratory tract infections. These data indicate that expression levels of the MMP-8 and MMP-9 genes in both PBMCs and neutrophils are associated with viral lower respiratory tract infections disease severity. These observations justify future validation in independent prospective study cohorts of the usefulness of MMP-8 and MMP-9 as potential markers for disease severity in viral respiratory infections.

Eicosanoid formation by a cytochrome P450 isoform expressed in the pharynx of Caenorhabditis elegans.

Caenorhabditis elegans harbours several CYP (cytochrome P450) genes that are homologous with mammalian CYP isoforms important to the production of physiologically active AA (arachidonic acid) metabolites. We tested the hypothesis that mammals and C. elegans may share similar basic mechanisms of CYP-dependent eicosanoid formation and action. We focused on CYP33E2, an isoform related to the human AA-epoxygenases CYP2C8 and CYP2J2. Co-expression of CYP33E2 with the human NADPH-CYP reductase in insect cells resulted in the reconstitution of an active microsomal mono-oxygenase system that metabolized EPA (eicosapentaenoic acid) and, with lower activity, also AA to specific sets of regioisomeric epoxy- and hydroxy-derivatives. The main products included 17,18-epoxyeicosatetraenoic acid from EPA and 19-hydroxyeicosatetraenoic acid from AA. Using nematode worms carrying a pCYP33E2::GFP reporter construct, we found that CYP33E2 is exclusively expressed in the pharynx, where it is predominantly localized in the marginal cells. RNAi (RNA interference)-mediated CYP33E2 expression silencing as well as treatments with inhibitors of mammalian AA-metabolizing CYP enzymes, significantly reduced the pharyngeal pumping frequency of adult C. elegans. These results demonstrate that EPA and AA are efficient CYP33E2 substrates and suggest that CYP-eicosanoids, influencing in mammals the contractility of cardiomyocytes and vascular smooth muscle cells, may function in C. elegans as regulators of the pharyngeal pumping activity.

Mapping out starvation responses.

What are the pathways that underlie the coordinated responses of an organism to well-fed and food-deprived states? A report in this issue of Cell Metabolism suggests that starvation functions via a muscarinic acetylcholine receptor to activate MAP kinase signaling in the pharyngeal muscle of C. elegans (You et al., 2006).

Starvation activates MAP kinase through the muscarinic acetylcholine pathway in Caenorhabditis elegans pharynx.

Starvation activates MAPK in the pharyngeal muscles of C. elegans through a muscarinic acetylcholine receptor, Gqalpha, and nPKC as shown by the following results: (1) Starvation causes phosphorylation of MAPK in pharyngeal muscle. (2) In a sensitized genetic background in which Gqalpha signaling cannot be downregulated, activation of the pathway by a muscarinic agonist causes lethal changes in pharyngeal muscle function. Starvation has identical effects. (3) A muscarinic antagonist blocks the effects of starvation on sensitized muscle. (4) Mutations and drugs that block any step of signaling from the muscarinic receptor to MAPK also block the effects of starvation on sensitized muscle. (5) Overexpression of MAPK in wild-type pharyngeal muscle mimics the effects of muscarinic agonist and of starvation on sensitized muscle. We suggest that, during starvation, the muscarinic pathway to MAPK is activated to change the pharyngeal muscle physiology to enhance ingestion of food when food becomes available.

Higher pharyngeal epithelial gene expression of angiotensin-converting Enzyme-2 in patients with upper respiratory infection.

We analyzed the expression of ACE2 in the pharyngeal epithelium and examined its relationship with clinical features and serological parameters in patients with upper respiratory infection (URI). The expression level of the ACE2 gene was significantly higher in patients with URI (n = 125) than in healthy control (HC) individuals (n = 52) (p < 0.0001). The ACE2 gene expression level was significantly and positively correlated with age (r=0.1799, p = 0.0447) and body temperature (r=0.1927, p = 0.0427), which may help explain increasing coinfections with SARS-CoV-2 and other respiratory pathogens.

Molecular characterization, expression and localization of a peroxiredoxin from the sheep scab mite, Psoroptes ovis.

The sheep scab mite, Psoroptes ovis, induces an intensely pruritic exudative dermatitis which is responsible for restlessness, loss of appetite and weight loss. Within the first 24 h of infection, there is a rapid inflammatory influx of eosinophils and apoptosis of the keratinocytes at the site of infection. The former cell type is capable of a sustained respiratory burst, toxic products of which may directly damage the mite and also contribute to lesion formation. Analysis of a P. ovis expressed sequence tag (EST) database identified a number of antioxidant enzyme-encoding sequences, including peroxiredoxin (thioredoxin peroxidase EC 1.11.1.15), all of which may help the mite endure the potentially toxic skin environment. A full length sequence encoding Po-TPx, a protein of 206 amino acids which showed high homology to a peroxiredoxin from the salivary gland of the tick Ixodes scapularis, was amplified from P. ovis cDNA. Recombinant Po-TPx was expressed in bacteria and antiserum to this protein was used to localize native Po-TPx in mite sections. Peroxiredoxin was localized, amongst other sites, to a subpharyngeal region in mite sections. The recombinant protein was recognized by sera from sheep infested with the mite suggesting that it may be secreted or excreted by the mite and interact with the host immune response.

Telomerase Expression in Medaka ( Oryzias melastigma) Pharyngeal Teeth.

Nonmammalian vertebrates have the capacity of lifelong tooth replacement. In all vertebrates, tooth formation requires contact and interaction between the oral or pharyngeal epithelium and the underlying mesenchyme. To secure lifelong replacement, the presence of odontogenic stem cells has been postulated, particularly in the epithelial compartment. This study uses an advanced teleost fish species, the marine medaka Oryzias melastigma, a close relative to Oryzias latipes, to examine the expression and distribution of telomerase reverse transcriptase (Tert), the catalytic unit of telomerase, in developing pharyngeal teeth and to relate these data to the proliferative activity of the cells. The data are complemented by expression analysis of the pluripotency marker oct4 and bona fide stem cell marker lgr5. Tert distribution and tert expression in developing tooth germs show a dynamic spatiotemporal pattern. Tert is present first in the mesenchyme but is downregulated as the odontoblasts differentiate. In contrast, in the epithelial enamel organ, Tert is absent during early stages of tooth formation and upregulated first in ameloblasts. Later, Tert is expressed and immunolocalized throughout the entire inner enamel epithelium. The pattern of Tert distribution is largely mutually exclusive with that of proliferating cell nuclear antigen (PCNA) immunoreactivity: highly proliferative cells, as revealed by PCNA staining, are negative for Tert; conversely, PCNA-negative cells are Tert-positive. Only the early condensed mesenchyme is both Tert- and PCNA-positive. The absence of tert-positive cells in the epithelial compartment of early tooth germs is underscored by the absence of oct4- and lgr5-positive cells, suggesting ways other than stem cell involvement to secure continuous renewal.

COX-2 inhibitors celecoxib and rofecoxib prevent oxidative DNA fragmentation.

BACKGROUND: Cyclooxygenase (COX) is the key regulatory enzyme in prostaglandin (PG) synthesis and is up-regulated in many premalignant and malignant lesions. The aim of this study was to investigate the in vitro DNA protective or damaging effects of COX-2 inhibitors using the single-cell gel electrophoresis (Comet) assay. MATERIALS AND METHODS: Cells from miniorgan cultures of pharyngeal mucosa from 30 patients were incubated once or five times with the COX-2 inhibitors celecoxib and rofecoxib. After treatment with H2O2, DNA fragmentation was determined. RESULTS: DNA strand-breaks were significantly reduced in cells pre-incubated with COX-2 inhibitors. Repeated incubation with celecoxib showed the strongest effect. This direct influence on DNA repair could be excluded by implementing DNA repair steps into the Comet assay. CONCLUSION: The findings suggest that, in addition to the known influence of COX-2 inhibitors on immune surveillance, neo-angiogenesis and cell proliferation, these substances may express a direct antimutagenic effect in conditions of oxidative stress.

Purification and molecular characterization of lamb pregastric lipase.

Lamb pregastric lipase (LPGL) was purified from pharyngeal tissues. The purification procedure was based on an aqueous extract containing 0.7% Tween 80 which was chromatographed on DEAE-cellulose anion-exchanger and adsorbed on HA-Ultrogel followed by gel filtration on Ultrogel AcA-54. The final enzymatic preparation, where the overall activity recovery was 3%, showed a single protein band on SDS-PAGE with a molecular mass of 50 kDa. LPGL is a glycoprotein containing approx. 14% (w/w) of carbohydrate. Extensive deglycosylation using peptide N-glycosidase F yielded a protein with an apparent molecular mass of 43 kDa. An uncontrolled proteolysis of LPGL during the purification lead to a 45 kDa form which was previously observed in human lysosomal acid lipase (HLAL) and rabbit gastric lipase (RGL). The labile bond X54-Leu55 was identified. Isoelectric focusing of LPGL reveals a major band corresponding to an isoelectric point of 4.8. The pure enzyme displayed specific activities of 950 U mg-1, 300 U mg-1 and 30 U mg-1 at pH 6.0, using tributyroylglycerol, trioctanoylglycerol and trioleoylglycerol as substrates, respectively. Using Western blot analysis, a cross-immunoreactivity of LPGL was observed with purified anti-human gastric lipase polyclonal antibodies. Determination of the amino-acid sequence of 62 residues revealed a high degree of homology with other known preduodenal lipases.

Effect of beta-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk.

The interaction of bovine beta-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine beta-lactoglobulin displays only one high affinity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 x 10(6) and 2.3 x 10(6) M-1, respectively. However, other binding sites with low affinity are also present. The existence of one high affinity binding site is in accordance with the amount of fatty acids naturally bound to beta-lactoglobulin isolated from milk. The effect of beta-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of beta-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that beta-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.

Drug-metabolizing enzymes in pharyngeal mucosa and in oropharyngeal cancer tissue.

Cytochrome P4501A1 (CYP1A1) and the UDP-glucuronosyltransferase isoform UGT1A6 were studied in pharyngeal mucosa and squamous cancer tissue obtained from 27 male subjects (10 healthy nonsmoking volunteers, 10 smokers, and 7 smokers with pharyngeal cancer). CYP1A activity (7-ethoxyresorufin O-deethylase) was significantly induced in smokers as compared to nonsmokers (2.3 +/- 1.1 and 0.8 +/- 0.4 pmol x min[-1] x mg protein[-1], respectively). Immunoblot analysis demonstrated enhanced CYP1A1 protein in smokers. UGT activity towards 4-methylumbelliferone and 1-naphthol was also detectable in oropharyngeal mucosa. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis indicated that UGT activity was at least in part due to the expression of UGT1A6. In cancer tissue, CYP1A activity was decreased in comparison with surrounding healthy mucosa (1.2 +/- 0.9 in tumor tissue vs. 2.2 +/- 0.7 pmol x min[-1] x mg protein[-1], respectively), whereas means and medians of UGT activity were unchanged. The results suggest that phase I and II drug-metabolizing enzymes are detectable in oropharyngeal mucosa and that CYP1A activity is inducible by constituents of cigarette smoke.

Embryonic expression of cholesterogenic genes is restricted to distinct domains and colocalizes with apoptotic regions in mice.

Cholesterol biosynthesis has been assumed to be an ubiquitous process in vertebrate organisms. Here we present data demonstrating that expression of key enzymes of cholesterol biosynthesis is restricted to specific tissues during embryonic development. Distinct expression starts in the dorsal neural tube at embryonic day 8 and is later detected in dorsal root and cephalic ganglia, in the pharyngeal pouches and limb buds. In the limb, expression becomes progressively restricted to interdigital regions during differentiation. Caspase3 whole mount immunostaining revealed that cholesterol biosynthesis colocalizes with apoptotic regions that are targets of the morphogenic signal Sonic hedgehog. This expression pattern correlates closely with the shared phenotypic features of cholesterol biosynthesis and hedgehog mutants.

Change in the expression of hypopharyngeal-gland proteins of the worker honeybees (Apis mellifera L.) with age and/or role.

The roles of adult worker honeybees change with age; young workers nurse brood by secreting bee milk (royal jelly), and older workers forage for nectar and pollen and process nectar into honey. The electrophoretic profile of worker hypopharyngeal-gland proteins changes with age and/or role. Immunoblotting analysis using affinity-purified antibodies against three major proteins (50, 56, and 64 kDa) of the nurse-bee gland showed that they are synthesized selectively and secreted as bee-milk proteins. Immunofluorescence study showed that the proteins condense in the duct after secretion from acini. However, a major 70-kDa protein synthesized specifically in the forager-bee hypopharyngeal gland was identified as an alpha-glucosidase. Therefore, the hypopharyngeal gland seems to have two distinct states differentiated by synthesizing of different major proteins depending on the age-dependent role change.

Comparative study of in vitro lipogenesis in various adipose tissues in the growing domestic pig (Sus domesticus).

Activities of acetyl-CoA-carboxylase, malic enzyme and glucose-6-phosphate-dehydrogenase were measured in seven different anatomical sites in the growing pig (20-120 kg weight). The three enzyme activities increased up to 40-60 kg weight and then decreased, malic enzyme becoming the main producer of NADPH, irrespective of the adipose tissue. Subcutaneous adipose tissue of the neck area was much thicker and exhibited much lower lipogenic enzyme activities than backfat. Subcutaneous adipose tissue is heterogeneous in the pig with some areas exhibiting very low lipogenesis and high lipid deposition importing triglycerides from other areas with high lipogenesis. However, these conclusions based on the measurement of enzyme activity potentials need to be confirmed with measurements of actual activities.

Plasma membrane Ca(2+) ATPase Atp2b1a regulates bone mineralization in zebrafish.

The zebrafish transgenic lines provide a possibility to observe the development of tissues and organs in real time. Using the reporter line for the zebrafish plasma membrane Ca(2+) ATPase (SqET4), we detected its expression in the epithelium of pharyngeal teeth and analyzed its role in their calcification and that of cranial bones. atp2b1a's expression in the pharyngeal epithelium is faithfully recapitulated in the SqET4 transgenics by GFP expression. We showed by morpholino knockdown of Atp2b1a translations as well as chemical inhibition of Atp2b1a pump activity using carboxyeosin, that its activity is required to facilitate calcification of the developing pharyngeal teeth by the dental epithelium. Atp2b1a could be required during calcification of endochondral bones, where it acts at two levels: 1) by exporting Ca(2+) from ameloblasts, it provides raw material for calcifying the pharyngeal teeth by adjacent odontoblasts; and 2) by regulating terminal differentiation of pharyngeal epithelial cells, including ameloblasts required for tissue hyper-mineralization. atp2b1a's expression in the pharyngeal epithelium is regulated by the homeodomain transcription factor dlx2b.

Pharyngeal polysaccharide deacetylases affect development in the nematode C. elegans and deacetylate chitin in vitro.

Chitin (ß-1,4-linked-N-acetylglucosamine) provides structural integrity to the nematode eggshell and pharyngeal lining. Chitin is synthesized in nematodes, but not in plants and vertebrates, which are often hosts to parasitic roundworms; hence, the chitin metabolism pathway is considered a potential target for selective interventions. Polysaccharide deacetylases (PDAs), including those that convert chitin to chitosan, have been previously demonstrated in protists, fungi and insects. We show that genes encoding PDAs are distributed throughout the phylum Nematoda, with the two paralogs F48E3.8 and C54G7.3 found in C. elegans. We confirm that the genes are somatically expressed and show that RNAi knockdown of these genes retards C. elegans development. Additionally, we show that proteins from the nematode deacetylate chitin in vitro, we quantify the substrate available in vivo as targets of these enzymes, and we show that Eosin Y (which specifically stains chitosan in fungal cells walls) stains the C. elegans pharynx. Our results suggest that one function of PDAs in nematodes may be deacetylation of the chitinous pharyngeal lining.

An organic-solvent-tolerant esterase from turkey pharyngeal tissue.

Stability is a crucial factor for the application of enzymes in biotechnology. Investigation of esterase activity in the pharyngeal tissue of turkey (Meleagris gallopavo), showed that optimum catalytic conditions of pure enzyme were 50 degrees C and pH 8.5. Turkey pharyngeal esterase (TPE) retained 75% of its maximum activity after incubation for 1h at 50 degrees C. Thermostability of the esterase was enhanced in the presence of an analogous substrate: phosphatidylcholine. TPE had a wide pH range of stability (pH 4.0-10.0). Esterase activity was compatible with the presence of organic solvents. Furthermore, the hydrolysis was found to be slightly activated by Ca(2+), but drastically reduced by Zn(2+) and Cu(2+). Phenylmethanesulphonyl fluoride (PMSF) a serine-specific inhibitor, strongly inhibited the esterase activity, whereas beta-mercaptoethanol, a thiol group inhibitor, did not show any effect on the activity. Esterase activity in the presence of organic solvents, as well as in acidic and alkaline pHs and at high temperatures makes it a good candidate for its application in non-aqueous biocatalysis.