The purification and characterization of phosphonopyruvate hydrolase, a novel carbon-phosphorus bond cleavage enzyme from Variovorax sp. Pal2.

Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co2+-dependent and showed a pH optimum of 6.7–7.0. The enzyme had a Km of 0.53 mM for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond.

Novel frenatins from the skin of the Australasian Giant White-lipped tree frog, Litoria infrafrenata: cloning of precursor cDNAs and identification in defensive skin secretion.

The Australasian anuran amphibian genus Litoria, contains many phenotypically-diverse species as a result of radial evolution of an ancestral species into different biotopes much in the manner of the indigenous marsupial mammals. In common with members of the Central/South American genus Phyllomedusa, their specialized skin granular glands are factories for the production of a plethora of biologically-active peptides. Here we report a more detailed study of those present in the defensive skin secretion of the Australasian giant white-lipped tree frog, Litoria infrafrenata, and, for the first time, we have identified three novel frenatins by deduction of primary structures from cDNAs that were cloned from a library constructed from lyophilized skin secretion using a recently-developed technique. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of a frenatin peptide. Processed peptides corresponding in molecular mass to the deduced molecular masses of frenatins (named 1.1, 3, 3.1 and 4.1) were identified in the same secretion sample using HPLC and mass spectroscopy. The application of this technique thus permits parallel peptidomic and transcriptomic analyzes on the same lyophilized skin secretion sample circumventing sacrifice of specimens from endangered herpetofauna.

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

We have isolated a novel bradykinin B2-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.

First report of a novel plant lysozyme with both antifungal and antibacterial activities.

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4 kDa in SDS–polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355 U/mg at pH 5.5 and 30 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 °C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers.

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

The structural organization of aurein precursor cDNAs from the skin secretion of the Australian green and golden bell frog, Litoria aurea.

Aureins are a family of peptides (13-25 residues), some of which possess potent antimicrobial and anti-cancer properties, which have been classified into 5 subgroups based upon primary structural similarities. They were originally isolated from the defensive skin secretions of the closely related Australian bell frogs, Litoria aurea and Litoria raniformis, and of the 23 aurein peptides identified, 10 are common to both species. Using a recently developed technique, we have constructed a cDNA library from the defensive secretion of the green and golden bell frog, L. aurea, and successfully cloned a range of aurein precursor transcripts containing entire open-reading frames. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of aurein. The deduced precursor structures for the most active aureins (2.2 and 3.1) confirmed the presence of a C-terminal amidation motif whereas that of aurein 5.3 did not. Processed peptides corresponding in molecular mass to aureins 2.2, 2.3, 2.5, 3.1 and 5.3 were identified in the same secretion sample using LC/MS. The application of this technique thus permits parallel peptidomic and transcriptomic analyses on the same lyophilized skin secretion sample circumventing sacrifice of specimens of endangered herpetofauna.

The 67 kDa laminin receptor: structure, function and role in disease

The 67LR (67 kDa laminin receptor) is a cell-surface receptor with high affinity for its primary ligand. Its role as a laminin receptor makes it an important molecule both in cell adhesion to the basement membrane and in signalling transduction following this binding event. The protein also plays critical roles in the metastasis of tumour cells. Isolation of the protein from either normal or cancerous cells results in a product with an approx. molecular mass of 67 kDa. This protein is believed to be derived from a smaller precursor, the 37LRP (37 kDa laminin receptor precursor). However, the precise mechanism by which cytoplasmic 37LRP becomes cell-membrane-embedded 67LR is unclear. The process may involve post-translational fatty acylation of the protein combined with either homo- or hetero-dimerization, possibly with a galectin-3-epitope-containing partner. Furthermore, it has become clear that acting as a receptor for laminin is not the only function of this protein. 67LR also acts as a receptor for viruses, such as Sindbis virus and dengue virus, and is involved with internalization of the prion protein. Interestingly, unmodified 37LRP is a ribosomal component and homologues of this protein are found in all five kingdoms. In addition, it appears to be strongly associated with histones in the eukaryotic cell nucleus, although the precise role of these interactions is not clear. Here we review the current understanding of the structure and function of this molecule, as well as highlighting areas requiring further research.

Critical stages of the brewing process for changes in antioxidant activity and levels of phenolic compounds in ale

Samples were taken at each stage of brewing (malt, milling, mashing, wort separation, hop addition, boiling, whirlpool, dilution, fermentation, warm rest, chill-lagering, beer filtration, carbonation and bottling, pasteurization, and storage). The level of antioxidant activity of unfractionated, low-molecular-mass (LMM) and high-molecular-mass (HMM) fractions was measured by the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfortic acid) radical cation (ABTS(.+)) and ferric-reducing antioxidant power (FRAP) procedures. Polyphenol levels were assessed by HPLC. The LMM fraction ( 0.001) in catechin and ferulic acid levels. Increases in antioxidant activity levels were observed after mashing, boiling, fermentation, chill-lagering, and pasteurization, in line with previous studies on lager. Additionally, increases in the level of antioxidant activity occurred after wort separation and carbonation and bottling and were accompanied by increases in levels of most monitored polyphenols. Data from the ABTS(.-) and FRAP assays indicated that the compounds contributing to the levels of antioxidant activity responded differently in the two procedures. Levels of ferulic, vanillic, and chlorogenic acids and catechin accounted for 45-61% of the variation in antioxidant activity levels.

Fermentation of heated gluten systems by gut microflora

The Maillard reaction causes changes to protein structure and occurs in foods mainly during thermal treatment. Melanoidins, the final products of the Maillard reaction, may enter the gastrointestinal tract, which is populated by different species of bacteria. In this study, melanoidins were prepared from gluten and glucose. Their effect on the growth of faecal bacteria was determined in culture with genotype and phenotype probes to identify the different species involved. Analysis of peptic and tryptic digests showed that low molecular mass products are formed from the degradation of melanoidins. Results showed a change in the growth of bacteria. This in vitro study demonstrated that melanoidins, prepared from gluten and glucose, affect the growth of the gut microflora.

Effect of roasting on the antioxidant activity of coffee brews

Colombian Arabica coffee beans were roasted to give light, medium, and dark samples. Their aqueous extracts were analyzed by gel filtration chromatography, UV-visible spectrophotometry, capillary electrophoresis, and the ABTS(.+) assay. A progressive decrease in antioxidant activity (associated mainly with chlorogenic acids in the green beans) with degree of roasting was observed with the simultaneous generation of high (HMM) and low molecular mass (LMM) compounds possessing antioxidant activity. Maximum antioxidant activity was observed for the medium-roasted coffee; the dark coffee had a lower antioxidant activity despite the increase in color. Analysis of the gel filtration chromatography fractions showed that the LMM fraction made a greater contribution to total antioxidant activity than the HMM components.

Analysis of malts by capillary electrophoresis

A range of malts, as well as their high- and low-molecular-mass fractions, has been examined by capillary electrophoresis in phosphate buffer, pH 2.5, and in carbonate buffer, pH 9.5, and the results have been compared with those for roasted barley and for caramels. The malts fall into two categories: (i) the lightly roasted malts, where the high-molecular-mass coloured fraction is negatively charged at pH 9.5 and positively charged at pH 2.5; and (ii) the highly roasted malts (and the roasted barley), where the high-molecular-mass fraction migrates close to the electro-osmotic flow at both pH 9.5 and 2.5, implying that it carries little or no charge. The former category shows migration patterns similar to Class III caramels, whereas the latter migrates differently from Class I, III and IV caramels as well as from the former. Capillary electrophoresis therefore has considerable potential for differentiating between malts and between malts and caramels and roasted barley. (C) 2002 Society of Chemical Industry.

Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

Relaxometric study of copper [15]metallacrown-5 gadolinium complexes derived from alpha-aminohydroxamic acids

Proton nuclear magnetic relaxation dispersion (NMRD) profiles were recorded between 0.24 mT and 1.4 T for lanthanum(III)- and gadolinium(III)-containing [15]metallacrown-5 complexes derived from alpha-aminohydroxamic acids and with copper(n) as the ring metal. The influence of the different R-groups on the proton relaxivity was investigated, and a linear relationship between the relaxivity and the molecular mass of the metallacrown complex was found. The selectivity of the metallacrown complexes was tested by transmetalation experiments with zinc(n) ions. The crystal structure of the copper [15]metallacrown-5 gadolinium complex with glycine hydroximate ligands is reported.

Upregulation of Oxidative Stress Markers in Human Microvascular Endothelial Cells by Complexes of Serum Albumin and Digestion Products of Glycated Casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95 degrees C for 5 h to give AGE-casein (AGE-Cas). Simulated stomach and small intestine digestion of AGE-Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)-LMM-AGE-Cas complexes. Stimulation of human microvascular endothelial cells with BSA-LMM-AGE-Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin-3 (AGE-113), tumor necrosis factor alpha, and a marker of the mitogen-activated protein kinase pathway (MAPK-1), as well as p65NF-kappa B activation. Cells treated with LMM digestion products of AGE-Cas significantly increased AGE-R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA-LMM-AGE-Cas and LMM-AGE-Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and down-stream inflammatory pathways. AGE-R3 may protect against these effects.

Peptide DV-28 amide: Prototype of a novel class of bradykinin receptor antagonist isolated from the defensive skin secretion of bombinid toads

Kinestatin, isolated from the skin of the Chinese toad, Bombina maxima, was the first bradykinin B2 receptor antagonist identified in amphibians. Molecular cloning established that it is co-encoded with the bradykinin-related peptide, maximakinin, within one of several skin kininogens. To examine other species within the genus Bombina for the presence of structural homologues of kinestatin, we subjected skin secretion of the toad, Bombina orientalis, to HPLC fractionation with subsequent bioassay of fractions for antagonism of bradykinin activity using an isolated rat tail artery smooth muscle preparation. A single fraction was located that inhibited bradykinin-induced relaxation of rat arterial smooth muscle and MALDI-TOF analysis of this fraction revealed that it contained a single peptide of molecular mass 3198.5 Da. Further primary structural analysis of this peptide showed that it was a 28-mer with an N-terminal Asp (D) residue and a C-terminal Val (V) residue that was amidated. The peptide was named DV-28 amide in accordance with these primary structural attributes. Synthetic DV-28 amide replicated the observed bradykinin antagonistic effect within the smooth muscle bioassay in a dose-dependent manner. In addition, it was observed to inhibit the proliferation of human microvessel endothelial cells (HMECs) as assessed by MTT assay. Bioinformatic analysis revealed that DV-28 amide was, like kinestatin, co-encoded with a bradykinin receptor agonist on one of two skin kininogens identified in B. orientalis. DV-28 amide thus represents a novel class of bradykinin antagonist from skin secretions of bombinid toads that appear to be a rich source of such novel peptides.