Chk1 Suppresses a Caspase-2 Apoptotic Response to DNA Damage that Bypasses p53, Bcl-2, and Caspase-3

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore ?-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after ?-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy. © 2008 Elsevier Inc. All rights reserved.

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.

Antagonistic effects of two novel GIP analogs, (Hyp(3))GIP and (Hyp(3)) GIPLys(16)PAL, on the biological actions of GIP and longer-term effects in diabetic ob/ob mice

This study examines the actions of the novel enzyme- resistant, NH2- terminally modified GIP analog ( Hyp(3)) GIP and its fatty acid- derivatized analog ( Hyp(3)) GIPLys(16)PAL. Acute effects are compared with the established GIP receptor antagonist ( Pro(3)) GIP. All three peptides exhibited DPP IV resistance, and significantly inhibited GIP stimulated cAMP formation and insulin secretion in GIP receptor- transfected fibroblasts and in clonal pancreatic BRIN- BD11 cells, respectively. Likewise, in obese diabetic ob/ob mice, intraperitoneal administration of GIP analogs significantly inhibited the acute antihyperglycemic and insulinreleasing effects of native GIP. Administration of once daily injections of ( Hyp(3)) GIP or ( Hyp(3)) GIPLys(16)PAL for 14 days resulted in significantly lower plasma glucose levels ( P <0.05) after ( Hyp3) GIP on days 12 and 14 and enhanced glucose tolerance ( P <0.05) and insulin sensitivity ( P <0.05 to P <0.001) in both groups by day 14. Both ( Hyp(3)) GIP and ( Hyp(3)) GIPLys(16)PAL treatment also reduced pancreatic insulin ( P <0.05 to P <0.01) without affecting islet number. These data indicate that ( Hyp3) GIP and ( Hyp(3)) GIPLys(16)PAL function as GIP receptor antagonists with potential for ameliorating obesity- related diabetes. Acylation of ( Hyp(3)) GIP to extend bioactivity does not appear to be of any additional benefit.

Characterisation and glucoregulatory actions of a novel acylated form of the (Pro(3))GIP receptor antagonist in type 2 diabetes

In this study, we tested the biological activity of a novel acylated form of (Pro(3))glucose-dependent insulinotropic polypetide [(Pro3)GIP] prepared by conjugating palmitic acid to Lys(16) to enhance its efficacy in vivo by promoting binding to albumin and extending its biological actions. Like the parent molecule (Pro(3))GIP, (Pro(3))GIPLys(16)PAL was completely stable to the actions of DPP-IV and significantly (p <0.01 to p <0.001) inhibited GIP-stimulated cAMP production and cellular insulin secretion. Furthermore, acute administration of (Pro(3))GIPLys(16)PAL also significantly (p <0.05 to p <0.001) countered the glucose-lowering and insulin-releasing actions of GIP in ob/ob mice. Daily injection of (Pro(3))GIPLys(16)PAL (25 nmol/kg bw) in 14-18-week-old ob/ob mice over 14 days had no effect on body weight, food intake or non-fasting plasma glucose and insulin concentrations. (Pro(3))GIPLys(16)PAL treatment also failed to significantly alter the glycaemic response to an i.p. glucose load or test meal, but insulin concentrations were significantly reduced (1.5-fold; p <0.05) after the glucose load. Insulin sensitivity was enhanced (1.3-fold; p <0.05) and pancreatic insulin was significantly reduced (p <0.05) in the (Pro(3))GIPLys(16)PAL-treated mice. These data demonstrate that acylation of Lys(16) with palmitic acid in (Pro(3))GIP does not improve its biological effectiveness as a GIP receptor antagonist.

Metabolic effects of sub-chronic ablation of the incretin receptors by daily administration of (Pro(3))GIP and exendin(9-39)amide in obese diabetic (ob/ob) mice

Effects of chemical ablation of the GIP and GLP-1 receptors on metabolic aspects of obesity-diabetes were investigated using the stable receptor antagonists (Pro(3))GIP and exendin(9-39)amide. Ob/ob mice received a daily i.p. injection of saline vehicle, (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides over a 14-day period. Non-fasting plasma glucose levels were significantly (p <0.05) lower in (Pro(3))GIP-treated mice compared to control mice after just 9 days of treatment. (Pro(3))GIP-treated mice also displayed significantly lower plasma glucose concentrations in response to feeding and intraperitoneal administration of either glucose or insulin (p <0.05 to p <0.001). The (Pro(3))GIP-treated group also exhibited significantly (p <0.05) reduced pancreatic insulin content. Acute administration of exendin(9-39) amide immediately prior to re-feeding completely annulled the beneficial effects of sub-chronic (Pro(3))GIP treatment, but non-fasting concentrations of active GLP-1 were unchanged. Combined sub-chronic administration of (Pro(3)GIP) with exendin(9-39)amide revealed no beneficial effects. Similarly, daily administration of exendin(9-39)amide alone had no significant effects on any of the metabolic parameters measured. These studies highlight an important role for GIP in obesity-related forms of diabetes, suggesting the possible involvement of GLP-1 in the beneficial actions of GIP receptor antagonism.

Effects of sub-chronic exposure to naturally occurring N-terminally truncated metabolites of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), GIP(3-42) and GLP-1 (9-36)amide, on insulin secretion and glucose homeostasis in ob/ob mice

Glucose-dependent insulinotrophic polypepticle (GIP) and glucagon-like peptide-1 (GLP-1) are important enteroendocrine hormones that are rapidly degraded by an ubiquitous enzyme dipeptidyl peptidase IV to yield truncated metabolites GIP(3-42) and GLP-1 (9-36)amide. In this study, we investigated the effects of sub-chronic exposure to these major circulating forms of GIP and GLP-1 on blood glucose control and endocrine pancreatic function in obese diabetic (ob/ob) mice. A once daily injection of either peptide for 14 days had no effect on body weight, food intake or pancreatic insulin content or islet morphology. GLP-1(9-36)amide also had no effect on plasma glucose homeostasis or insulin secretion. Mice receiving GIP(3-42) exhibited small but significant improvements in non-fasting plasma glucose, glucose tolerance and glycaemic response to feeding. Accordingly, plasma insulin responses were unchanged suggesting that the observed enhancement of insulin sensitivity was responsible for the improvement in glycaemic control. These data indicate that sub-chronic exposure to GIP and GLP-1 metabolites does not result in physiological impairment of insulin secretion or blood glucose control. GIP(3-42) might exert an overall beneficial effect by improving insulin sensitivity through extrapancreatic action.

Synthesis of 3-(4-tert-butylphenyl)-2-propen-1-one, a precursor to Lilial (R), via an aldol condensation in an ionic liquid

An efficient synthesis of a precursor to Lilial(R), based on an aldol condensation in an ionic liquid, is described, utilising piperidine as the base catalyst. The yields obtained with this methodology are significantly increased in comparison with those reported in organic solvents to date. In the ionic liquid, the self-aldol condensation of propanal is suppressed and leads to an increased selectivity with respect to the cross-aldol condensation product without the need to use an excess of 4-tert-butylbenzaldehyde to obtain high selectivities.

Liquid and solid-state structures of 1,3-dimethylimidazolium salts

Using neutron and single crystal X-ray diffraction the structures of 1,3-dimethylimidazolim chloride and hexafluorophosphate salts have been determined in the liquid and the solid-state. The relative hydrogen bonding characteristics and sizes of the two anions force the ions to pack differently. In each case, a strong correlation between the crystal structure and liquid structure is found.

Ultrasound-assisted green synthesis of nanocrystalline ZnO in the ionic liquid [hmim][NTf2]

For the first time, zinc oxide nanoparticles have been synthesized by the sonochemical method in an ionic liquid, 1-hexyl-3-methylimidazolium his (trifluoromethylsulfonyl) imide, liquid [hmim][NTf2] as a solvent. The morphology and structure of ZnO nanoparticles have been characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). A possible mechanism is proposed to explain the formation of ZnO nanostructures. (C) 2008 Elsevier B.V. All rights reserved.

Kinetic model for the hydrolysis of lignocellulosic biomass in the ionic liquid, 1-ethyl-3-methyl-imidazolium chloride†

The kinetics of the acid-catalysed hydrolysis of cellobiose in the ionic liquid 1-ethyl-3-methylimidazolium chloride, [C(2)mim]Cl, was studied as a model for general lignocellulosic biomass hydrolysis in ionic liquid systems. The results show that the rate of the two competing reactions, polysaccharide hydrolysis and sugar decomposition, vary with acid strength, and that for acids with an aqueous pK(a) below approximately zero, the hydrolysis reaction is significantly faster than the degradation of glucose, thus allowing hydrolysis to be performed with a high selectivity in glucose. In tests with soluble cellulose, hemicellulose (xylan), and lignocellulosic biomass (Miscanthus grass), comparable hydrolysis rates were observed with bond scission occurring randomly along the biopolymer chains, in contrast to end-group hydrolysis observed with aqueous acids.

Ionic liquids via reaction of the zwitterionic 1,3-dimethylimidazolium-2-carboxylate with protic acids. Overcoming synthetic limitations and establishing new halide free protocols for the formation of ILs

The previously reported preparation of 1,3-dimethylimidazolium salts by the reaction of 1,3-dialkylimidazolium-2-carboxylate zwitterions with protic acids has been reinvestigated in detail, leading to the identification of two competing reactions: isomerisation and decarboxylation. The ability to control both pathways allows this methodology to be used as an effective, green, waste-free approach to readily prepare a wide range of ionic liquids in high yields. Additionally, this reaction protocol opens new possibilities in the formation of other imidazolium salts, whose syntheses were previously either very expensive (due to ion exchange protocols involving metals like Ag) or difficult to achieve (due to multiple extractions and large quantities of hard to remove inorganic by-products).

A simple colorimetric method for the quality control of 1-alkyl-3-methylimidazolium ionic liquid precursors

A simple colorimetric method to monitor the production of ionic liquid precursors is developed, which is based on the determination of 1-methylimidazole with copper(II) chloride. The synthesis of 1-ethyl-3-methylimidazolium chloride, an industrially important ionic liquid precursor, can be followed and the purity of the final product can be readily assessed in a quick and convenient manner.