BACE1 Polymorphisms Do Not Influence Platelet Membrane β-secretase Activity or Genetic Susceptibility for Alzheimer’s Disease in the Northern Irish Population

ß-Site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a biological and positional candidate gene for Alzheimer’s disease (AD). BACE1 is a protease that catalyses APP cleavage at the ß-secretase site. We evaluated all common and putatively functional polymorphisms in the genomic region encompassing BACE1 for an association with AD, and for functional effects on platelet ß-secretase activity. Tag SNPs (n = 10) derived from phase II of the International HapMap Project, and a nonsynonymous variant, were successfully genotyped in 901 Caucasian individuals from Northern Ireland using Sequenom iPLEX and TaqMan technologies. APOE genotyping was performed by PCR-RFLP. Platelet membrane ß-secretase activity was assayed in a subset of individuals (n = 311). Hardy–Weinberg equilibrium was observed for all variants. Evidence for an association with AD was observed with multi-marker haplotype analyses (P = 0.01), and with rs676134 when stratified for APOE genotype (P = 0.02), however adjusting for multiple testing negated the evidence for association of this variant with AD. ?2 analysis of genotype and allele frequencies in cases versus controls for individual SNPs revealed no evidence for association (5% level). No genetic factors were observed that significantly influenced platelet membrane ß-secretase activity. We have selected an appropriate subset of variants suitable for comprehensive genetic investigation of the BACE1 gene. Our results suggest that common BACE1 polymorphisms and putatively functional variants have no significant influence on genetic susceptibility to AD, or platelet ß-secretase activity, in this Caucasian Northern Irish population.

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.

Elements of the granular gland peptidome and transcriptome persist in air-dried skin of the South American orange-legged leaf frog, Phyllomedusa hypocondrialis.

The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin—a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

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.

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.

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.

Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine 0-sulfation motif.

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position -1 and the basic amino acid residue (Arg) at position -2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.

Bradykinin-related peptides, including a novel structural variant, (Val1)-bradykinin, from the skin secretion of Guenther's frog, Hylarana guentheri and their molecular precursors.

Multiple bradykinin-related peptides including a novel bradykinin structural variant, (Val1)-bradykinin, have been identified from the defensive skin secretion of Guenther's frog, Hylarana guentheri by a tandem mass spectrometry method. Subsequently, four different preprobradykinin cDNAs, which encoded multiple bradykinin copies and its structural variants, were consistently cloned from a skin derived cDNA library. These preprobradykinin cDNAs showed little structural similarity with mammalian kininogens and the kininogens from the skin of toads, but have regions that are highly conserved in the kininogens from another ranid frog, Odorrana schmackeri. Alignment of these preprobradykinins revealed that preprobradykinin 1, 2 and 3 may derive from a single gene by alternative exon splicing.

Helokinestatin: a new bradykinin B2 receptor antagonist decapeptide from lizard venom.

Synthetic bradykinin antagonist peptides/peptoids have been powerful tools for delineating the roles of kinins in both normal physiology and in pathological states. Here, we report the identification of a novel, naturally occurring bradykinin B2 receptor antagonist peptide, helokinestatin, isolated and structurally characterized from the venoms of helodermatid lizards—the Gila monster (Heloderma suspectum) and the Mexican beaded lizard (Heloderma horridum). The primary structure of the peptide was established by a combination of microsequencing and mass spectroscopy as Gly-Pro-Pro-Tyr-Gln-Pro-Leu-Val-Pro-Arg (Mr 1122.62). A synthetic replicate of helokinestatin was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle, mediated by the B2 receptor sub-type, in a dose-dependent manner. Natural selection, that generates functional optimization of predatory reptile venom peptides, can potentially provide new insights for drug lead design or for normal physiological or pathophysiological processes.

Novel brevinins from Chinese piebald odorous frog (Huia schmackeri) skin deduced from cloned biosynthetic precursors

Antimicrobial peptides represent the most characterized and diverse class of peptides within the defensive skin secretions of anuran amphibians. With an ever expanding database of primary structures, the current accepted rules for nomenclature have become increasingly difficult to apply to peptides whose primary structural attributes are either unique or that fall between those that define existing groups. An additional factor that adds to the confusion is the regular re-classification or revision of existing taxa. In the present study, we have identified five new antimicrobial peptide homologs in the defensive skin secretion of the Chinese piebald odorous frog, Huia schmackeri (formerly Rana (Odorrana) schmackeri), by cloning of their respective biosynthetic precursors. As these peptides are obvious homologs of the brevinin-1 and brevinin-2 families we have named these in accordance: (1) brevinin-1HS1, (2) brevinin-2HS1, (3) brevinin-2HS2, (4) brevinin-2HS3 and (5) brevinin-1HS2. The reasons for adopting these names are discussed. It is clear that with an ever-increasing number of amphibian skin antimicrobial peptides appearing in the literature that a consistent nomenclature scheme needs to be established.

Advanced glycation of fibronectin impairs vascular repair by endothelial progenitor cells: Implications for vasodegeneration in diabetic retinopathy

PURPOSE. Vascular repair by marrow-derived endothelial progenitor cells (EPCs) is impaired during diabetes, although the precise mechanism of this dysfunction remains unknown. The hypothesis for the study was that progressive basement membrane (BM) modification by advanced glycation end products (AGEs) contributes to impairment of EPC reparative function after diabetes-related endothelial injury.

METHODS. EPCs isolated from peripheral blood were characterized by immunocytochemistry and flow cytometry. EPC interactions on native or AGE-modified fibronectin (AGE-FN) were studied for attachment and spreading, whereas chemotaxis to SDF-1 was assessed with the Dunn chamber assay. In addition, photoreactive agent-treated monolayers of retinal microvascular endothelial cells (RMECs) produced circumscribed areas of apoptosis and the ability of EPCs to “endothelialize” these wounds was evaluated.

RESULTS. EPC attachment and spreading on AGE-FN was reduced compared with control cells (P < 0.05–0.01) but was significantly restored by pretreatment with Arg-Gly-Asp (RGD). Chemotaxis of EPCs was abolished on AGE-FN but was reversed by treatment with exogenous RGD. On wounded RMEC monolayers, EPCs showed clustering at the wound site, compared with untreated regions (P < 0.001); AGE-FN significantly reduced this targeting response (P < 0.05). RGD supplementation enhanced EPC incorporation in the monolayer, as determined by EPC participation in tight junction formation and restoration of transendothelial electric resistance (TEER).

CONCLUSIONS. AGE-modification of vascular substrates impairs EPC adhesion, spreading, and migration; and alteration of the RGD integrin recognition motif plays a key role in these responses. The presence of AGE adducts on BM compromises repair by EPC with implications for vasodegeneration during diabetic microvasculopathy.

Neuroprotective actions of a histidine analogue in models of ischemic stroke.

Histidine is a naturally occurring amino acid with antioxidant properties, which is present in low amounts in tissues throughout the body. We recently synthesized and characterized histidine analogues related to the natural dipeptide carnosine, which selectively scavenge the toxic lipid peroxidation product 4-hydroxynonenal (HNE). We now report that the histidine analogue histidyl hydrazide is effective in reducing brain damage and improving functional outcome in a mouse model of focal ischemic stroke when administered intravenously at a dose of 20 mg/kg, either 30 min before or 60 min and 3 h after the onset of middle cerebral artery occlusion. The histidine analogue also protected cultured rat primary neurons against death induced by HNE, chemical hypoxia, glucose deprivation, and combined oxygen and glucose deprivation. The histidine analogue prevented neuronal apoptosis as indicated by decreased production of cleaved caspase-3 protein. These findings suggest a therapeutic potential for HNE-scavenging histidine analogues in the treatment of stroke and related neurodegenerative conditions.

Prediction- and simulation-error based perceptron training: Solution space analysis and a novel combined training scheme

Previous papers have noted the difficulty in obtaining neural models which are stable under simulation when trained using prediction-error-based methods. Here the differences between series-parallel and parallel identification structures for training neural models are investigated. The effect of the error surface shape on training convergence and simulation performance is analysed using a standard algorithm operating in both training modes. A combined series-parallel/parallel training scheme is proposed, aiming to provide a more effective means of obtaining accurate neural simulation models. Simulation examples show the combined scheme is advantageous in circumstances where the solution space is known or suspected to be complex. (c) 2006 Elsevier B.V. All rights reserved.