Amphibian skin secretomics: application of parallel quadrupole time-of-flight mass spectrometry and peptide precursor cDNA cloning to rapidly characterize the skin secretory peptidome of Phyllomedusa hypochondrialis azurea: discovery of a novel peptide family, the hyposins

This study reports the variety of peptides present in the skin secretory peptidome of Phyllomedusa hypochondrialis azurea. Peptide structures, along with post-translational modifications, were elucidated by QTOF MS/MS analysis, cDNA sequencing, or a combination of both. Twenty-two peptides, including 19 novel structures, were identified from six different structural classes, including tryptophyllins, dermorphins, and a novel group of peptides termed hyposins. The study demonstrates the power of this combined approach to mine the rich peptidome compliment of the amphibian defensive skin secretome.     

Cloning of maximakinin precursor cDNAs from Chinese toad,Bombina maxima,venom

Using a novel technique that we have developed for cloning of amphibian skin secretion peptide cDNAs from lyophilized samples, we report here that maximakinin (DLPKINRKGP-bradykinin) is encoded by two different cDNAs, named BMK-1 and BMK-2, containing either four tandem repeat sequences or a single copy. The open reading frames of both precursor cDNAs were found to be 152 and 116 amino acid residues, respectively. These data provide evidence that the structural diversity of peptides in amphibian skin secretions arising from molecular evolutionary events, can be mediated by parallel diversity in encoding mRNAs that in itself may reflect serial gene duplications.     

Dermatoxin and phylloxin from the waxy monkey frog, Phyllomedusa sauvagei: cloning of precursor cDNAs and structural characterization from lyophilized skin secretion

Amphibian skin is a morphologically, biochemically and physiologically complex organ that performs the wide range of functions necessary for amphibian survival. Here we describe the primary structures of representatives of two novel classes of amphibian skin antimicrobials, dermatoxin and phylloxin, from the skin secretion of Phyllomedusa sauvagei, deduced from their respective precursor encoding cDNAs cloned from a lyophilized skin secretion library. A degenerate primer, designed to a highly conserved domain in the 5'-untranslated region of analogous peptide precursor cDNAs from Phyllomedusa bicolor, was employed in a 3'-RACE reaction. Peptides with molecular masses coincident with precursor-deduced mature toxin peptides were identified in LC/MS fractions of skin secretion and primary structures were confirmed by MS/MS fragmentation. This integrated experimental approach can thus rapidly expedite the primary structural characterization of amphibian skin peptides in a manner that circumvents specimen sacrifice whilst preserving robustness of scientific data.     

Peptidomics and genomics analysis of novel antimicrobial peptides from the frog,Rana nigrovittata

Much attention has been paid on amphibian peptides for their wide-ranging pharmacological properties, clinical potential, and gene-encoded origin. More than 300 antimicrobial peptides (AMPs) from amphibians have been studied. Peptidomics and genomics analysis combined with functional test including microorganism killing, histamine-releasing, and mast cell degranulation was used to investigate antimicrobial peptide diversity. Thirty-four novel AMPs from skin secretions of Rana nigrovittata were identified in current work, and they belong to 9 families, including 6 novel families. Other three families are classified into rugosin, gaegurin, and temporin family of amphibian AMP, respectively. These AMPs share highly conserved preproregions including signal peptides and spacer acidic peptides, while greatly diversified on mature peptides structures. In this work, peptidomics combined with genomics analysis was confirmed to be an effective way to identify amphibian AMPs, especially novel families. Some AMPs reported here will provide leading molecules for designing novel antimicrobial agents.     

Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1.

Fourier transform infrared (FTIR) spectroscopy was used to monitor secondary structural changes associated with binding of bovine prothrombin and prothrombin fragment 1 to acidic lipid membranes. Prothrombin and prothrombin fragment 1 were examined under four different conditions: in the presence of (a) Na2EDTA, (b) 5 mM CaCl2, and in the presence of CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either (c) bovine brain phosphatidyl-serine (bovPS) or (d) 1,2-dioleoyl-phosphatidylglycerol (DOPG). The widely reported Ca(2+)-induced conformational change in bovine prothrombin fragment 1 was properly detected by our procedures, although Ca(2+)-induced changes in whole prothrombin spectra were too small to be reliably interpreted. Binding of prothrombin in the presence of Ca2+ to procoagulant POPC/bovPS small unilamellar vesicles produced an increase in ordered secondary structures (2% and 3% increases in alpha-helix and beta-sheet, respectively) and a decrease of random structure (5%) as revealed by spectral analysis on both the original and Fourier-self-deconvolved data and by difference spectroscopy with the undeconvolved spectra. Binding to POPC/DOPG membranes, which are less active as procoagulant membranes, produced no detectable changes in secondary structure. In addition, no change in prothrombin fragment 1 secondary structure was detectable upon binding to either POPC/bovPS or POPC/DOPG membranes. This indicates that a membrane-induced conformational change occurs in prothrombin in the nonmembrane-binding portion of the molecule, part of which is activated to form thrombin, rather than in the membrane-binding fragment 1 region. The possible significance of this conformational change is discussed in terms of differences between the procoagulant activities of different acidic lipid membranes.     

Microsatellite variation and population structure of the moor frog (Rana arvalis) in Scandinavia

Several recent studies have found amphibian populations to be genetically highly structured over rather short geographical distances, and that the rate of genetically effective dispersal may differ between the sexes. However, apart from the common frog ( Rana temporaria ) little is known about the genetic structuring and sex-biased dispersal in northern European amphibians. We investigated the patterns of genetic diversity and differentiation within and among Scandinavian populations of the moor frog ( Rana arvalis ) using microsatellite markers. The genetic diversity within local R. arvalis populations was not a simple linear negative function of latitude but a convex one: genetic diversity peaked in mid-latitude populations, and declined thereafter dramatically towards the north. The average degree of genetic differentiation among populations ( F _ST = 0.14) was lower than that observed for the common frog ( F _ST = 0.21), though the pattern of isolation by distance was similar for both species. Contrary to common frogs, no evidence for female-biased dispersal was found. The results reinforce the view that amphibian populations are—in general—highly structured over relatively small geographical distances, even in comparatively recently colonized areas.     

Structure of the C2 domain from novel protein kinase Cepsilon. A membrane binding model for Ca(2+)-independent C2 domains

Protein kinase Cepsilon (PKCepsilon) is a member of the novel PKCs which are activated by acidic phospholipids, diacylglycerol and phorbol esters, but lack the calcium dependence of classical PKC isotypes. The crystal structures of the C2 domain of PKCepsilon, crystallized both in the absence and in the presence of the two acidic phospholipids, 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) and 1,2-dicaproyl-sn-phosphatidic acid (DCPA), have now been determined at 2.1, 1.7 and 2.8 A resolution, respectively. The central feature of the PKCepsilon-C2 domain structure is an eight-stranded, antiparallel, beta-sandwich with a type II topology similar to that of the C2 domains from phospholipase C and from novel PKCdelta. Despite the similar topology, important differences are found between the structures of C2 domains from PKCs delta and epsilon, suggesting they be considered as different PKC subclasses. Site-directed mutagenesis experiments and structural changes in the PKCepsilon-C2 domain from crystals with DCPS or DCPA indicate, though phospholipids were not visible in these structures, that loops joining strands beta1-beta2 and beta5-beta6 participate in the binding to anionic membranes. The different behavior in membrane-binding and activation between PKCepsilon and classical PKCs appears to originate in localized structural changes, which include a major reorganization of the region corresponding to the calcium binding pocket in classical PKCs. A mechanism is proposed for the interaction of the PKCepsilon-C2 domain with model membranes that retains basic features of the docking of C2 domains from classical, calcium-dependent, PKCs.     

Kinestatin: a novel bradykinin B2 receptor antagonist peptide from the skin secretion of the Chinese toad, Bombina maxima

We have isolated a novel bradykinin B(2)-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide. The unmodified sequence, -QIPGLGPLRG-, was located at the C-terminus of a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin.     

Isolation and characterization of four major neutral glycosphingolipids from the liver of the English sole (Parophrys vetulus). Presence of a novel branched lacto-ganglio-iso-globo hybrid structure

We have previously reported on carbohydrate structures of the major acidic glycosphingolipids from the liver of the English sole, Parophrys vetulus (Ostrander, G. K., Levery, S. B., Hakomori, S., and Holmes, E. H. (1988) J. Biol. Chem. 263, 3103-3110). We have now isolated four major neutral glycosphingolipids from English sole liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, and by fast atom bombardment-mass spectrometry. In addition to neutral glycosphingolipids with known structures (CMH, lactosylceramide, and Gg3), a major polar neutral glycosphingolipid was isolated and characterized as having the following novel structure: (formula; see text) The compound represents a novel hybrid of neolacto-, ganglio-, and iso-globo-series glycosphingolipid structures, a combination not previously encountered. Furthermore, the linkage Fuc alpha 1----3GalNac has not been previously reported for a glycosphingolipid. The relationship of these structural elements to known glycoconjugates is discussed.     

Structure of the acidic N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida.

N-linked carbohydrate chains of the major 55-kDa family, PZP3, of porcine zona pellucida glycoproteins are composed of neutral (28%) and acidic (72%) complex-type chains. The structures of the main components of the neutral chain have been established [Noguchi, S., Hatanaka, Y., Tobita, T. & Nakano, M. (1992) Eur. J. Biochem. 204, 1089-1100]. Here we report the structures of the acidic chains. Only two kinds of acidic fragments were released from PZP3 by endo-beta-galactosidase digestion following beta-elimination of O-linked chains. 500-MHz one-dimensional and two-dimensional 1H-NMR spectroscopy revealed their structures to be Sia alpha(2-3)Gal beta(1-4) [HSO3-6]GlcNAc beta(1-3)Gal and HSO3-6GlcNAc beta(1-3)Gal, showing that the sulfate-containing acidic chains are constructed with non-branched N-acetyllactosamine repeats which have sialic acid(s) at the non-reducing end(s) and sulfate at the C-6 position of GlcNAc residues. The acidic N-linked chains obtained from PZP3 by hydrazinolysis were separated into diantennary chains (34%) and tri- and tetra-antennary chains (66%) by concanavalin-A--agarose gel chromatography. The diantennary chains and their sialidase digests were fractionated by DEAE-HPLC. From the analyses of the endo-beta-galactosidase digests of each fraction, structures of the diantennary acidic chains were determined. They are classified into four groups. The first group is the sialylated chains without the sulfated N-acetyllactosamine repeating unit. The other three groups have the chains of various lengths differing in the number of monosulfated N-acetyllactosamine unit. These chains are extended from the Man alpha(1-3) branch of the trimannosyl core in the second group, from the Man alpha(1-6) branch in the third group, and from both branches in the fourth group. The structural features of the tri- and tetra-antennary acidic chains are also presented.     

Identification of a novel inner-core oligosaccharide structure in Neisseria meningitidis lipopolysaccharide.

The structure of the lipopolysaccharide (LPS) from three Neisseria meningitidis strains was elucidated. These strains were nonreactive with mAbs that recognize common inner-core epitopes from meningococcal LPS. It is well established that the inner core of meningococcal LPS consists of a diheptosyl-N-acetylglucosamine unit, in which the distal heptose unit (Hep II) can carry PEtn at the 3 or 6 position or not at all, and the proximal heptose residue (Hep I) is substituted at the 4 position by a glucose residue. Additional substitution at the 3 position of Hep II with a glucose residue is also a common structural feature in some strains. The structures of the O-deacylated LPSs and core oligosaccharides of the three chosen strains were deduced by a combination of monosaccharide analysis, NMR spectroscopy and MS. These analyses revealed the presence of a structure not previously identified in meningococcal LPS, in which an additional beta-configured glucose residue was found to substitute Hep I at the 2 position. This provided the structural basis for the nonreactivity of LPS with these mAbs. The determination of this novel structural feature identified a further degree of variability within the inner-core oligosaccharide of meningococcal LPS which may contribute to the interaction of meningococcal strains with their host.     

Hantaviruses in small wild living mammals in Czechoslovakia. Results of a 1983-1989 study

During a 7- year period were hunted small wild living mammals and examined by ELISA and RIA techniques for the presence of hantavirus antigen and/or antibodies by MFA. In total 3,050 animals of 16 species caught in 9 out of 10 regions of Czechoslovakia, were examined. The proportion of positive animals was 4.4%. To the positive ones with the serotype 2 (Western type) belonged the following: M. arvalis, C. glareolus, P. subterraneus, A. sylvaticus, A. flavicollis. To the Eastern serotype: A. agrarius in eastern Slovakia, A. flavicollis in North of Bohemia and A. sylvaticus in South of Moravia. The repeatedly examined localities were found to be either repeatedly positive or repeatedly negative. The antigen titres in the lungs of M. arvalis were constant irrespective of sex and season of capture. They were, however, much higher in young animals, whereas the proportion of positivities was higher in adults ones. The titres of antigen in the lungs of C. glareolus never exceeded those of M. arvalis.     

Histamine-releasing and antimicrobial peptides from the skin secretions of the dusky gopher frog, Rana sevosa

Seven novel peptides were isolated from the skin secretions of the North American dusky gopher frog, Rana sevosa, on the basis of antimicrobial activity and histamine release from rat peritoneal mast cells. The peptides were purified to homogeneity using HPLC and characterized by electrospray ion-trap mass spectrometry, MALDI-TOF mass spectrometry and Edman sequencing. Bioinformatic analysis of primary structures revealed that the novel peptides could be assigned to four established families of ranid frog antimicrobial peptides, namely esculentin-1, esculentin-2, brevinin-1 and ranatuerin-2. The peptides were named in accordance with accepted terminology as ranatuerin 2SEa, etc., reflecting the peptide family name, the species of origin (SE for sevosa) and the isotype (a). Of major interest was the fact that brevinin 1SE displayed significant structural similarity to ponericin W5, an antibacterial venom peptide from the ant, Pachyconyla goeldii. This is a further example of amphibian skin defensive peptides showing striking structural similarities to peptides from insects. These data may shed some light on the functional biological relevance of defensive peptides that possess both antimicrobial and histamine-releasing activities.     

A sensitive method based on fluorescence-detected circular dichroism for protein local structure analysis

We report an improved fluorescence-detected circular dichroism (FDCD)-based analytical method that is useful for probing protein three-dimensional structures. The method uses a novel FDCD device with an ellipsoidal mirror that functions on a standard circular dichroism (CD) spectrometer and eliminates all artifacts. Our experiments demonstrated three important findings. First, the method is applicable to any proteins either by using intrinsic fluorescence derived from tryptophan residues or by introducing a fluorescent label onto nonfluorescent proteins. Second, by using intrinsic fluorescence, FDCD spectroscopy can detect a structural change in the tertiary structure of metmyoglobin due to stepwise denaturation on a change in pH. Such changes could not be detected by conventional CD spectroscopy. Third, based on the typical advantages of fluorescence-based analyses, FDCD measurements enable observation of only the target proteins in a solution even in the presence of other peptides. Using our ellipsoidal mirror FDCD device, we could observe structural changes of fluorescently labeled calmodulin on binding with Ca²⁺ and/or interacting with binding peptides. Because FDCD appears to reflect the protein’s local structure around the fluorophore, it may provide a useful means for “pinpoint analysis” of protein structures.     

The solution structure of a locked nucleic acid (LNA) hybridized to DNA

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2'-O, 4'-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32A. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3 A. The average twist over the sequence are 35.9 degrees +/- 0.3 degrees. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5'-CT(L)AG-3' site than in the unmodified 5'-CTAG-3' site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.     

Morphological and histochemical study of supragingival human calculus and dental plaque using ruthenium hexammine trichloride and acridine orange.

Ruthenium hexammine trichloride (RHT) and acridine orange were used to preserve and visualize anionic groups in human plaque and dental calculus. RHT-reacting material was present on the membrane of micro-organisms and in intermicrobial spaces of the calcifying areas, and seems to correspond to, and derive from, acidic glyco- and phospholipids of the plasma membrane of the micro-organisms. However, the presence of acidic salivary peptidoglycans cannot be ruled out. Two types of calcification were found: extramicrobial and intramicrobial. The former consisted of calcified deposits irregularly scattered in the intermicrobial matrix. They were in close relationship with RHT-reacting material, or were placed inside vesicular structures delimited by a membrane. Intramicrobial calcification consisted of small aggregates of needle-shaped crystals and/or of granular deposits; in both cases, they either masked the whole cytoplasm of the micro-organisms, or were located only over the plasma membrane. These results suggest that mineral deposition occurs in connection with acidic components of intermicrobial matrix, microbial plasma membranes, and cytoplasms. The addition of RHT and acridine orange to fixing and decalcifying solutions yields satisfactory preservation of dental calculus and plaque, and apparently reduces loss of their anionic organic components and increases their electron density. However, these substances are not sufficient to preserve all ultrastructural details in decalcified areas, probably because the inorganic substance prevents reaction of acridine orange and RHT with the organic components of the calcified matrix.