Cutting edge: Langerin+ dendritic cells in the mesenteric lymph node set the stage for skin and gut immune system cross-talk.

Topical transcutaneous immunization (TCI) presents many clinical advantages, but its underlying mechanism remains unknown. TCI induced Ag-specific IgA Ab-secreting cells expressing CCR9 and CCR10 in the small intestine in a retinoic acid-dependent manner. These intestinal IgA Abs were maintained in Peyer\'s patch-null mice but abolished in the Peyer\'s patch- and lymph node-null mice. The mesenteric lymph node (MLN) was shown to be the site of IgA isotype class switching after TCI. Unexpectedly, langerin(+)CD8alpha(-) dendritic cells emerged in the MLN after TCI; they did not migrate from the skin but rather differentiated rapidly from bone marrow precursors. Depletion of langerin(+) cells impaired intestinal IgA Ab responses after TCI. Taken together, these findings suggest that MLN is indispensable for the induction of intestinal IgA Abs following skin immunization and that cross-talk between the skin and gut immune systems might be mediated by langerin(+) dendritic cells in the MLN.

Skin bradykinin-related peptides (BRPs) and their biosynthetic precusors (kininogens): comparisons between various taxa of Chinese and North American ranid frogs.

Bradykinins and related peptides (BRPs) occur in the defensive skin secretions of many amphibians. Here we report the structures of BRPs and their corresponding biosynthetic precursor cDNAs from the Chinese brown frog, Rana chensinensis, and the North American leopard frog, Lithobates pipiens. R. chensinensis skin contained four transcripts each encoding a different kininogen whose organizations and spectrum of encoded BRPs were similar to those reported for the pickerel frog, Lithobates palustris. In contrast, from L. pipiens, a single skin kininogen was cloned whose structural organization and spectrum of mature BRPs were similar to those reported for the Chinese piebald odorous frog, Huia schmackeri. These data also implied that the endogenous precursor processing proteases in each species pair have identical site-directed specificities, which in part may be dictated by the primary structures of encoded BRPs. Thus the spectra of skin BRPs and the organization of their biosynthetic precursors are not consistent with recent taxonomy. The natural selective pressures that mould the primary structures of amphibian skin secretion peptides are thought to be related to the spectrum of predators encountered within their habitats. Thus similarities and differences in skin bradykinins may be reflective of predator spectra rather than indicative of species relatedness.

Rapid identification of precursor cDNAs encoding five structural classes of antimicrobial peptides from pickerel frog (Rana palustris) skin secretion by single step “shotgun” cloning.

The skin secretion of the North American pickerel frog (Rana palustris) has long been known to have pronounced noxious/toxic properties and to be highly effective in defence against predators and against other sympatric amphibians. As it consists largely of a complex mixture of peptides, it has been subjected to systematic peptidomic study but there has been little focus on molecular cloning of peptide-encoding cDNAs and by deduction, the biosynthetic precursors that they encode. Here, we demonstrate that the cDNAs encoding the five major structural families of antimicrobial peptides can be elucidated by a single step “shotgun” cloning approach using a cDNA library constructed from the source material of the peptidomic studies—the defensive skin secretion itself. Using a degenerate primer pool designed to a highly conserved nucleic acid sequence 5' to the initiation codon of known antimicrobial peptide precursor transcripts, we amplified cDNA sequences representing five major classes of antimicrobial peptides, such as esculentins, brevinins, ranatuerins, palustrins and temporins. Bioinformatic comparisons of precursor open-reading frames and nucleic acid sequences revealed high degrees of structural similarities between analogous peptides of R. palustris and the Chinese bamboo odorous frog, Rana versabilis. This approach thus constitutes a robust technique that can be used either alone or ideally, in parallel with peptidomic analysis of skin secretion, to rapidly extract primary structural information on amphibian skin secretion peptides and their biosynthetic precursors.

Lividins: Novel antimicrobial peptide homologs from the skin secretion of the Chinese Large Odorous frog, Rana (Odorrana) livida: Identification by “shotgun” cDNA cloning and sequence analysis.

Odorous frogs of the sub-genus Odorrana are of oriental distribution, and are so called due to the foul smell of their defensive skin secretions released from specialized skin glands following stress or predator attack. Here we report the application of a “shotgun” skin secretion cDNA library cloning technique which can rapidly expedite identification of secretion bioactive peptides. From a library constructed from the skin secretion of the Large Chinese Odorous frog, Rana (Odorrana) livida, we have identified four novel peptides whose primary structures were deduced initially from cloned precursors. Subsequently, mature peptides were located in and structurally characterized from reverse phase HPLC fractions of skin secretion. Named lividins 1–4, these were found to be structural homologs of known antimicrobial peptide families from Rana frogs. Rapid identification of novel peptides can thus be rapidly achieved using this non-invasive, non-destructive technology and the extensive similarities revealed between antimicrobial peptide precursor organization and nucleic acid sequences would lend support to the hypothesis that they have a common ancestral origin.

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and North American Rana frogs share the same families of skin antimicrobial peptides.

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and the North American pickerel frog (Rana palustris) occupy different ecological niches on two different continents with no overlap in geographical distribution. R. palustris skin secretions contain a formidable array of antimicrobial peptides including homologs of brevinin-1, esculentin-1, esculentin-2, ranatuerin-2, a temporin and a family of peptides considered of unique structural attributes when isolated, palustrins 1–3. Here we describe the structures of mature peptides and precursors of eight putative antimicrobial peptides from the skin secretion of the Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis). Each peptide represents a structural homolog of respective peptide families isolated from R. palustris, including two peptides identical in primary structure to palustrin 1c and palustrin 3b. Additionally, two peptides were found to be structural homologs of ranatuerin 2B and ranatuerin 2P from the closely-related North American species, Rana berlandieri (the Rio Grande leopard frog) and Rana pipiens (the Northern leopard frog), respectively. Both palustrins and ranatuerins have hitherto been considered unique to North American ranid frogs. The use of primary structures of amphibian skin antimicrobial peptides is thus questionable as a taxonomic device or alternatively, the micro-evolution and/or ancestry of ranid frogs is more highly complex than previously thought.

Identification and molecular cloning of novel trypsin inhibitors from the dermal venom of the Oriental fire-bellied toad (Bombina orientalis) and the European yellow-bellied toad (Bombina variegata).

The structural diversity of polypeptides in amphibian skin secretion probably reflects different roles in dermal regulation or in defense against predators. Here we report the structures of two novel trypsin inhibitor analogs, BOTI and BVTI, from the dermal venom of the toads, Bombina orientalis and Bombina variegata. Cloning of their respective precursors was achieved from lyophilized venom cDNA libraries for the first time. Amino acid alignment revealed that both deduced peptides, consisting of 60 amino acid residues, including 10 cysteines and the reactive center motif, -CDKKC-, can be affirmed as structural homologs of the trypsin inhibitor from Bombina bombina skin.

An investigation of possible mechanisms of the water-gas shift reaction over a ZrO2-supported Pt catalyst

The present work investigates the reactivity of the surface species observable by in situ DRIFTS formed over a Pt/ZrO2 during the water-gas shift (WGS) reaction. A DRIFTS cell/mass spectrometer system was operated at the chemical steady state during isotopic transients to yield information about the true nature (i.e., main reaction intermediate or spectators) of adsorbates. Only carbonyl and formate species were observed by DRIFTS under reaction conditions; the surface coverage of carbonate species was negligible. Isotopic transient kinetic analyses revealed that formates exchanged uniformly according to a first-order law, suggesting that most formates observed by DRIFTS were of the same reactivity. In addition, the time scale of the exchange of the reaction product CO2 was significantly shorter than that of the surface formates. Therefore, a formate route based on the formates as detected by DRIFTS can be ruled out as the main reaction pathway in the present case. The number of precursors of the reaction product CO2 was smaller than the number of surface Pt atoms, suggesting that carbonyl species or some \

Aromatic dioxygenases: Molecular biocatalysis and applications

Aromatic dioxygenases have been found to catalyse single and tandem oxidation reactions of conjugated polyenes. Rational selection and design of dioxygenases, allied to substrate shape, size and substitution pattern, has been used to control regiochemistry and stereochemistry during the oxygenation process. The resulting enantiopure bioproducts have been increasingly utilised as precursors for new and alternative routes in chiral synthesis.

Regio- and stereo-selective dioxygenase-catalysed cis-dihydroxylation of fjord-region polycyclic arenes

Bacterial dioxygenase-catalysed cis-dihydroxylation of the tetracyclic arenes benzo[c]phenanthrene 2, and the isosteric compounds benzo[b]naphtho[1,2-d]furan 8, and benzo[b]naphtho[1,2-d]thiophene 9, has been found to occur exclusively at fjord-region bonds. The resulting cis-dihydrodiols 7, 10 and 11 were found to be enantiopure and of similar absolute configuration. cis-Dihydroxylation was also observed in the pseudo-fjord region of the 8,9,10,11-tetrahydro-precursors (12 and 13) of benzo[b]naphtho[1,2-d]furan 8, and benzo[b]naphtho[1,2-d]thiophene 9, to yield the corresponding enantiopure hexahydro cis-diols 14 and 15. A novel tandem cis-dihydroxylation and bis-desaturation of the tetrahydro-substrate, tetrahydrobenzo[b]naphtho[1,2-d]thiophene 13, catalysed by biphenyl dioxygenase, was found to yield the fjord-region cis-dihydrodiol 17 of benzo[b]naphtho[1,2-d]thiophene 9.

syn-Benzene dioxides: chemoenzymatic synthesis from 2,3-cis-dihydrodiol derivatives of monosubstituted benzenes and their application in the synthesis of regioisomeric 1,2- and 3,4-cis-dihydrodiols and 1,4-dioxocins

cis-2,3-Dihydrodiol metabolites of monosubstituted halobenzenes and toluene have been used as synthetic precursors of the corresponding 3,4-cis-dihydrodiols. Enantiopure syn-benzene dioxide intermediates were reduced to the 3,4-cis-dihydrodiols and thermally racemised via the corresponding 1,4-dioxocins. The syn-benzene dioxide-1,4-dioxocin valence tautomeric equilibrium ratio was found to be dependent on the substituent position. The methodology has also been applied to the synthesis of both enantiomers of the 1,2-(ipso)- and 3,4-cis-dihydrodiols of toluene. This chemoenzymatic approach thus makes available, for the first time, all three possible cis-dihydrodiol regioisomers of a monosubstituted benzene.

Neuroactive substances and associated major muscle systems in Bucephaloides gracilescens (Trematoda : Digenea) metacercaria and adult

Cholinergic, serotoninergic and neuropeptidergic components of the nervous system were examined and compared in the progenetic metacercaria and adult gasterostome trematode, Bucephaloides gracilescens in order to provide baseline information on neuronal control of the musculature involved in egg-assembly. Enzyme cytochemistry and indirect immunocytochemical techniques interfaced with confocal scanning laser microscopy demonstrated all three classes of neuroactive substance throughout the central and peripheral nervous systems. A comparable orthogonal arrangement of the central nervous system (CNS) and peripheral array of nerve plexuses was observed in both metacercaria and adult. Staining patterns for cholinergic and peptidergic substances showed significant overlap, while the serotoninergic system was confined to a separate set of neurons. Immunostaining for FMRFamide-related peptides (FaRPs) was strong in the CNS and peripheral innervation to the attachment apparatus of metacercaria and adult but was only found in the innervation of the ootype in ovigerous adults, implicating FaRPs in neuronal control of the muscle of the female reproductive tract during egg-assembly.

Flatworm nerve-muscle: structural and functional analysis

Platyhelminthes occupy a unique position in nerve-muscle evolution, being the most primitive of metazoan phyla. Essentially, their nervous system consists of an archaic brain and associated pairs of longitudinal nerve cords cross-linked as an orthogon by transverse commissures. Confocal imaging reveals that these central nervous system elements are in continuity with an array of peripheral nerve plexuses which innervate a well-differentiated grid work of somatic muscle as well as a complexity of myofibres associated with organs of attachment, feeding, and reproduction. Electrophysiological studies of flatworm muscles have exposed a diversity of voltage-activated ion channels that influence muscle contractile events. Neuronal cell types are mainly multi- and bi-polar and highly secretory in nature, producing a heterogeneity of vesicular inclusions whose contents have been identified cytochemically to include all three major types of cholinergic, aminergic, and peptidergic messenger molecules. A landmark discovery in flatworm neurobiology was the biochemical isolation and amino acid sequencing of two groups of native neuropeptides: neuropeptide F and FMRFamide-related peptides (FaRPs). Both families of neuropeptide are abundant and broadly distributed in platyhelminths, occurring in neuronal vesicles in representatives of all major flatworm taxa. Dual localization studies have revealed that peptidergic and cholinergic substances occupy neuronal sets separate from those of serotoninergic components. The physiological actions of neuronal messengers in flatworms are beginning to be established, and where examined, FaRPs and 5-HT are myoexcitatory, while cholinomimetic substances are generally inhibitory. There is immunocytochemical evidence that FaRPs and 5-HT have a regulatory role in the mechanism of egg assembly. Use of muscle strips and (or) muscle fibres from free-living and parasitic flatworms has provided baseline information to indicate that muscle responses to FaRPs are mediated by a G-protein-coupled receptor, and that the signal transduction pathway for contraction involves the second messengers cAMP and protein kinase C.

Microscopical evaluation of neural connectivity between paired stages of Eudiplozoon nipponicum (Monogenea : Diplozoidae)

Diplozoidae monogeneans are fish-gill ectoparasites comprising 2 individuals fused in so-called permanent copula. This unique situation occurs when 2 larvae (diporpae) make contact on the host gill, such that their union triggers maturation into an individual adult worm. The present study examined paired stages of Eudiplozoon nipponicum microscopically to ascertain whether somatic fusion involves neural connectivity between these 2 heterogenic larvae. Neuronal pathways were demonstrated in whole-mount preparations of the worm, using indirect immunocytochemical techniques interfaced with confocal scanning laser microscopy for peptidergic and serotoninergic innervations and enzyme cytochemical methodology and light microscopy for cholinergic components. Elements of the central nervous systems of paired worms are connected by commissures the region of fusion so that the 2 systems are in structural continuity. Interindividual connections were most apparent between corresponding ventral nerve cords. All 3 classes of neuronal mediators were identified throughout both central and peripheral connections of the 2 nervous systems. The anatomical complexity and apparent plasticity of the diplozoon nervous system suggest that it has a pivotal role not only in motility, feeding, and reproductive behaviors but also in the events of larval pairing and somatic fusion.

Effect of cultivar and storage time on the volatile flavor components of baked potato

Tubers of five cultivars of potato were stored at 4 degreesC for 2 3 and 8 months and baked in a conventional oven The flavor compounds from the baked potato flesh were isolated by headspace adsorption onto Tenax and analyzed by gas chromatography-mass spectrometry On a quantitative basis compounds derived from lipid and Maillard reaction/sugar degradation dominated the flavor isolates with sulfur compounds, methoxypyrazines, and terpenes making smaller contributions Levels of 37 of the > 150 detected compounds were monitored in each cultivar with time of storage Many significant differences were found in levels of individual compounds compound classes and total monitored compounds for the individual effects of cultivar and storage time and for their two way interaction Differences may be explained by variations in levels of flavor precursors and activities of enzymes mediating flavor compound formation among cultivars and storage times In addition differences in agronomic conditions may partly account for variations among cultivars Overall of the compounds monitored those most likely having the greatest flavor impact were 2-isopropyl 3 methyoxypyrazine 2 isobutyl 3-methoxypyrazine dimethyl trisulfide, decanal and 3 methylbutanal, with methylpropanal, 2 methylbutanal methional, and nonanal also being probable important contributors to flavor.

Comparison of the volatile components of eight cultivars of potato after microwave baking

Eight cultivars of potato were baked in a microwave oven. The flavour components of the flesh were isolated by headspace trapping onto Tenax and analysed by gas chromatography-mass spectrometry. Lipid degradation and the Maillard reaction and/or sugar degradation were the main sources of the 80 flavour components identified. It is suggested that total levels of compounds and variations among their profiles may be attributed to differences in activities of lipid enzymes and levels of flavour precursors in the range of cultivars investigated. (C) 2002 Elsevier Science Ltd.