Characterisation and biological activity of Glu(3) amino acid substituted GIP receptor antagonists

Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release and glucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biological activity of several Glu(3) -substituted analogues of GIP namely; (Ala(3))GIP, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP. Only (Lys(3))- GIP demonstrated moderately enhanced resistance to DPP-IV (p <0.05 to p <0.01) compared to native GIP. All analogues demonstrated a decreased potency in cAMP production (EC50 1.47 to 11.02 nM; p <0.01 to p <0.001) with (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated cAMP production (p <0.05). In BRIN-BD11 cells, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))- GIP did not stimulate insulin secretion with both (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated insulin secretion (p <0.05). Injection of each GIP analogue together with glucose in oblob mice significantly increased the glycaemic excursion compared to control (p <0.05 to p <0.001). This was associated with lack of significant insulin responses. (Ala(3))GIP, (Phe(3))GIP and (Tyr(3))GIP, when administered together with GIP, significantly reduced plasma insulin (p <0.05 top <0.01) and impaired the glucose-lowering ability (p <0.05 to p <0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronounced than (Pro(3))GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala(3)), (Phe(3)), (Tyr(3)) or (Pro(3)) provides a new class of functional GIP receptor antagonists. (C) 2007 Elsevier Inc. All rights reserved.

Comparison of the anti-diabetic effects of GIP- and GLP-1-receptor activation in obese diabetic (ob/ob) mice: studies with DPP IV resistant N-AcGIP and exendin(1-39)amide

Background The two major incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are being actively explored as anti-diabetic agents because they lower blood glucose through multiple mechanisms. The rapid inactivation of GIP and GLP-1 by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) makes their biological actions short-lived, but stable agonists such as N-acetylated GIP (N-AcGIP) and exendin(1-39)amide have been advocated as stable and specific GIP and GLP-1 analogues.

Lys(9) for Glu(9) substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His(7) and Ala(8), can greatly improve resistance to this enzyme. Little research has assessed what effect Glu(9)-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu(9) of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue, (Lys(9))GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys(9))GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor. (C) 2004 Elsevier Inc. All rights reserved.

Enhanced cAMP generation and insulin-releasing potency of two novel Tyr(1)-modified enzyme-resistant forms of glucose-dependent insulinotropic polypeptide is associated with significant antihyperglycaemic activity in spontaneous obesity-diabetes

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin hormone, which potentiates glucose-induced insulin secretion. Antihyperglycaemic actions of GIP provide significant potential in Type 11 diabetes therapy. However, inactivation of GIP by the enzyme dipeptidyl peptidase IV (DPP IV) and its consequent short circulating half-life limit its therapeutic use. Therefore two novel Tyr(1)-Modified analogues of GIP, N-Fmoc-GIP (where Fmoc is 9-fluorenylmethoxycarbonyl) and N-palmitate-GIP, were synthesized and tested for metabolic stability and biological activity. Both GIP analogues were resistant to degradation by DPP IV and human plasma. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, both analogues exhibited a 2-fold increase in cAMP-generating potency compared with native GIP (EC50 values of 9.4, 10.0 and 18.2 nM respectively). Using clonal BRIN-BD11 cells, both analogues demonstrated strong insulinotropic activity compared with native GIP (P <0.01 to P <0.001). In obese diabetic (ob/ob) mice, administration of N-Fmoc-GIP or N-palmitate-GIP (25 nmol/kg) together with glucose (18 mmol/kg) significantly reduced the peak 15 min glucose excursion (1.4- and 1.5-fold respectively; P <0.05 to P <0.01) compared with glucose alone. The area under the curve (AUC) for glucose was significantly lower after administration of either analogue compared with glucose administered alone or in combination with native GIP (1.5-fold; P <0.05). This was associated with a significantly greater AUC for insulin (2.1-fold; P <0.001) for both analogues compared with native GIP. A similar pattern of in vivo responsiveness was evident in lean control mice. These data indicate that novel N-terminal Tyr(1) modification of GIP with an Fmoc or palmitate group confers resistance to degradation by DPP IV in plasma, which is reflected by increased in vitro potency and greater insulinotropic and antihyperglycaemic activities in an animal model of Type 11 diabetes mellitus.

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine Ib) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylsae (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

Benzothiazole bipyridine complexes of ruthenium(II) with cytotoxic activity

A series of benzothiazole-substituted trisbipyridine ruthenium(II) analogues {[Ru(bpy)(2)(4,5'-bbtb)](2+), [Ru(bpy)(2)(5,5'-bbtb)](2+) and [Ru(bpy)(2)(5-mbtb)](2+) [bpy is 2,2'-bipyridine, bbtb is bis(benzothiazol-2-yl)-2,2'-bipyridine, 5-mbtb is 5-(benzothiazol-2-yl),5'-methyl-2,2'-bipyridine]} have been prepared and compared with the complex [Ru(bpy)(2)(4,4'-bbtb)](2+) reported previously. From the UV-vis spectral studies, substitution at the 5-position of the bpy causes the ligand-centred transitions to occur at considerably lower energy than for those with the functionality at the 4-position, while at the same time causing the emission to be effectively quenched. However, substitution at the 4-position causes the metal-to-ligand charge transfer to occur at lower energies. Fluorescent intercalator displacement studies indicate that the doubly substituted complexes displace ethidium bromide from a range of oligonucleotides, with the greater preference shown for bulge and hairpin sequences by the Lambda enantiomer. Since the complexes only show small variation in the UV-vis spectra on the introduction of calf thymus DNA and a small increase in fluorescence they do not appear to be intercalators, but appear to associate within one of the grooves. All of the reported bisbenzothiazole complexes show reasonable cytotoxicity against a range of human cancer cell lines.

Probing warm dense lithium by inelastic X-ray scattering

One of the grand challenges of contemporary physics is understanding strongly interacting quantum systems comprising such diverse examples as ultracold atoms in traps, electrons in high-temperature superconductors and nuclear matter. Warm dense matter, defined by temperatures of a few electron volts and densities comparable with solids, is a complex state of such interacting matter. Moreover, the study of warm dense matter states has practical applications for controlled thermonuclear fusion, where it is encountered during the implosion phase, and it also represents laboratory analogues of astrophysical environments found in the core of planets and the crusts of old stars, Here we demonstrate how warm dense matter states can be diagnosed and structural properties can be obtained by inelastic X-ray scattering measurements on a compressed lithium sample. Combining experiments and ab initio simulations enables us to determine its microscopic state and to evaluate more approximate theoretical models for the ionic structure.

FMRFamide-related peptides (FaRPs): A new family of peptides from amphibian defensive skin secretions

Amphibian defensive skin secretions are known to contain a plethora of biologically-active peptides that are often structural and functional analogues of vertebrate neuropeptides. Here we report the structures of two invertebrate neuropeptide analogues, IPPQFMRF amide (IF-8 amide) and EGDEDEFLRF amide (EF-10 amide), from the defensive skin secretions of two different species of African hyperoliid frogs, Kassina maculata and Phylictimantis verrucosus, respectively. These represent the first canonical FMRF amide-related peptides (FaRPs) from a vertebrate source. The cDNA encoding IF-8 amide was cloned from a skin secretion library and found to contain a single copy of the peptide located at the C-terminus of a 58 amino acid residue open-reading frame. These data extend the potential targets of the defensive arsenal of amphibian tegumental secretions to parasitic/predatory invertebrates and the novel peptides described may represent the first vertebrate peptidic endectocides.

The construction of a whole-cell biosensor for phosphonoacetate, based on the LysR-like transcriptional regulator PhnR from Pseudomonas fluorescens 23F

The phnA gene that encodes the carbon-phosphorus bond cleavage enzyme phosphonoacetate hydrolase is widely distributed in the environment, suggesting that its phosphonate substrate may play a significant role in biogeochemical phosphorus cycling. Surprisingly, however, no biogenic origin for phosphonoacetate has yet been established. To facilitate the search for its natural source we have constructed a whole-cell phosphonoacetate biosensor. The gene encoding the LysR-type transcriptional activator PhnR, which controls expression of the phosphonoacetate degradative operon in Pseudomonas fluorescens 23F, was inserted in the broad-host-range promoter probe vector pPROBE-NT, together with the promoter region of the structural genes. Cells of Escherichia coli DH5a that contained the resultant construct, pPANT3, exhibited phosphonoacetate-dependent green fluorescent protein fluorescence in response to threshold concentrations of as little as 0.5 µM phosphonoacetate, some 100 times lower than the detection limit of currently available non-biological analytical methods; the pPANT3 biosensor construct in Pseudomonas putida KT2440 was less sensitive, although with shorter response times. From a range of other phosphonates and phosphonoacetate analogues tested, only phosphonoacetaldehyde and arsonoacetate induced green fluorescent protein fluorescence in the E. coli DH5a (pPANT3) biosensor, although at much-reduced sensitivities (50 µM phosphonoacetaldehyde and 500 µM arsonoacetate).

New catanionic surfactants based on 1-alkyl-3-methylimidazolium alkylsulfonates, [C(n)H(2n+1)mim][CmH2m+1SO3]: mesomorphism and aggregation

Anionic and cationic alkyl-chain effects on the self-aggregation of both neat and aqueous solutions of 1-alkyl-3-methylimidazolium alkylsulfonate salts ([C(n)H(2n+ 1)mim][CmH2m+1SO3]; n = 8, 10 or 12; m = 1 and n = 4 or 8; m = 4 or 8) have been investigated. Some of these salts constitute a novel family of pure catanionic surfactants in aqueous solution. Examples of this class of materials are rare; they are distinct from both mixed cationic-anionic surfactants (obtained by mixing two salts) and gemini surfactants (with two or more amphiphilic groups bound by a covalent linker). Fluorescence spectroscopy and interfacial tension measurements have been used to determine critical micelle concentrations (CMCs), surface activity, and to compare the effects of the alkyl-substitution patterns in both the cation and anion on the surfactant properties of these salts. With relatively small methylsulfonate anions (n = 8, 10 and 12, m = 1), the salts behave as conventional single chain cationic surfactants, showing a decrease of the CMC upon increase of the alkyl chain length (n) in the cation. When the amphiphilic character is present in both the cation and anion (n = 4 and 8, m = 4 and 8), novel catanionic surfactants with CMC values lower than those of the corresponding cationic analogues, and which exhibited an unanticipated enhanced reduction of surface tension, were obtained. In addition, the thermotropic phase behaviour of [C(8)H(18)mim][C8H18SO3] (n = m = 8) was investigated using variable temperature X-ray scattering, polarising optical microscopy and differential scanning calorimetry; formation of a smectic liquid crystalline phase with a broad temperature range was observed.

Nigrocin-2 peptides from Chinese Odorrana frogs – integration of UPLC/MS/MS with molecular cloning in amphibian skin peptidome analysis

Peptidomics is a powerful set of tools for the identification, structural elucidation and discovery of novel regulatory peptides and for monitoring the degradation pathways of structurally and catalytically important proteins. Amphibian skin secretions, arising from specialized granular glands, often contain complex peptidomes containing many components of entirely novel structure and unique site-substituted analogues of known peptide families. Following the discovery that the granular gland transcriptome is present in such secretions in a PCR-amenable form, we designed a strategy for peptide structural characterization involving the integration of ‘shotgun’ cloning of cDNAs encoding peptide precursors, deduction of putative bioactive peptide structures, and confirmation of these structures using tandem MS/MS sequencing. Here, we illustrate this strategy by means of elucidation of the primary structures of nigrocin-2 homologues from the defensive skin secretions of four species of Chinese Odorrana frogs, O. schmackeri, O. livida, O. hejiangensis and O. versabilis. Synthetic replicates of the peptides were found to possess antimicrobial activity. Nigrocin-2 peptides occur widely in the skin secretions of Asian ranid frogs and in those of the Odorrana group, and are particularly well-represented and of diverse structure in some species. Integration of the molecular analytical technologies described provides a means for rapid structural characterization of novel peptides from complex natural libraries in the absence of systematic online database information.

4 ◦C and beyond: what did this mean for biodiversity in the past?

How do the predicted climatic changes (IPCC, 2007) for the next century compare in magnitude and rate to those that Earth has previously encountered? Are there comparable intervals of rapid rates of temperature change, sea-level rise and levels of atmospheric CO2 that can be used as analogues to assess possible biotic responses to future change? Or are we stepping into the great unknown? This perspective article focuses on intervals in time in the fossil record when atmospheric CO2 concentrations increased up to 1200 ppmv, temperatures in mid- to high-latitudes increased by greater than 4 ?C within 60 years, and sea levels rose by up to 3 m higher than present. For these intervals in time, case studies of past biotic responses are presented to demonstrate the scale and impact of the magnitude and rate of such climate changes on biodiversity. We argue that although the underlying mechanisms responsible for these past changes in climate were very different (i.e. natural processes rather than anthropogenic), the rates and magnitude of climate change are similar to those predicted for the future and therefore potentially relevant to understanding future biotic response. What emerges from these past records is evidence for rapid community turnover, migrations, development of novel ecosystems and thresholds from one stable ecosystem state to another, but there is very little evidence for broad-scale extinctions due to a warming world. Based on this evidence from the fossil record, we make four recommendations for future climate-change integrated conservation strategies.

Construction of cDNA libraries from trifluoroacetic acid-solvated amphibian skin secretions: molecular cloning of multiple bombinin-like peptide precursor transcripts from a library of yellow-bellied toad (Bombina variegata) secretion

Amphibian skin secretions are renowned as complex mixtures of bioactive peptides many of which are analogues of endogenous regulatory peptides. While skin secretions can be obtained non-invasively for peptidome analysis, parallel studies on the granular gland transcriptome required specimen sacrifice. The aim of the present study was to analyse archived skin secretions to determine the robustness of bioactive peptide precursor-encoding polyadenylated mRNAs in an attempt to extract maximum molecular information from rare samples. A range of solvated skin secretion samples were examined after lyophilisation for their potential to generate viable and comprehensive cDNA libraries based upon polyadenylated mRNA capture and amplification/cloning using appropriate commercial kits. Here we present unequivocal data that the granular gland transcriptome persists in a PCR amenable format even after storage for as long as 12 years in 0.1%(v/v) aqueous trifluoroacetic acid (TFA). We used a pooled skin secretion sample (2 ml) from the yellow-bellied toad, Bombina variegata (n = 14), containing the equivalent of 5 mg/ml of lyophilised skin secretion, that had been used in part for peptide isolation purposes in 1998 and had been stored at - 20 °C since that time. In the first cloning experiment, 12 different bombinin-like peptide precursor cDNAs were cloned encoding 17 different bombinins, the majority of which were novel. Subsequently, bombesin and bradykinin-related peptide precursor transcripts have been cloned successfully. These data illustrate the unexpected stability/longevity of the transcriptome in these secretions — a finding with implications for both this field of research and for the wider field of molecular biology.

beta-1,2,3-Triazolyl-Nucleosides as Nicotinamide Riboside Mimics

The synthesis of a series of pyridine- and piperidine-substituted 1,2,3-triazolides linked to a riboside moiety is described. The presence of a triazolide substituent on the pyridine moiety permitted the facile reduction of the latter under mild hydrogenation conditions. These analogues were modelled as to define their similarity to nicotinamide riboside and quantify their ability to bind NAD-dependent protein deacetylases.

Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology

Paralytic shellfish poisoning (PSP) toxin monitoring in shellfish is currently performed using the internationally accredited AOAC mouse bioassay. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. The feasibility of using a surface plasmon resonance optical biosensor to detect PSP toxins in shellfish tissue below regulatory levels was examined. Three different PSP toxin protein binders were investigated: a sodium channel receptor (SCR) preparation derived from rat brains, a monoclonal antibody (GT13-A) raised to gonyautoxin 2/3, and a rabbit polyclonal antibody (R895) raised to saxitoxin (STX). Inhibition assay formats were used throughout. Immobilization of STX to the biosensor chip surface was achieved via amino-coupling. Specific binding and inhibition of binding to this surface was achieved using all proteins tested. For STX calibration curves, 0 - 1000 ng/mL, IC50 values for each binder were as follows: SCR 8.11 ng/mL; GT13-A 5.77 ng/mL; and R895 1.56 ng/mL. Each binder demonstrated a different cross-reactivity profile against a range of STX analogues. R895 delivered a profile that was most likely to detect the widest range of PSP toxins at or below the internationally adopted regulatory limits.