Tissue transglutaminase (TG2) - a wound response enzyme

Repair of tissue after injury depends on a series of concerted but overlapping events including, inflammation, re-epithelialization, neovascularization and synthesis and stabilization of a fibrous extracellular matrix (ECM) that is remodeled to emulate normal tissue over time. Particular members of the transglutaminase (TG) family are upregulated during wound healing and act as a novel class of wound-healing mediators during the repair process. This group of enzymes which crosslink proteins via epsilon(gamma-glutamyl) lysine bridges are involved in wound healing through their ability to stabilize proteins and also by regulating the behavior of a wide variety of cell types that are recruited to the damaged area in order to carry out tissue repair. In this article we discuss the function of the most widely expressed member of the TG family "tissue transglutaminase" (TG2) in wound repair. Using both early and recent evidence from the literature we demonstrate how the multifunctional TG2 affects the stability of the ECM, cell-ECM interactions and as a consequence cell behavior within the different phases of wound healing, and highlight how TG2 itself might be exploited for therapeutic use.

Carbon monoxide inhibits sprouting angiogenesis and vascular endothelial growth factor receptor-2 phosphorylation

Carbon monoxide (CO) is a gaseous autacoid known to positively regulate vascular tone; however, its role in angiogenesis is unknown. The aim of this study was to investigate the effect of CO on angiogenesis and vascular endothelial growth factor (VEGF) receptor-2 phosphorylation. Human umbilical vein endothelial cells (HUVECs) were cultured on growth factor- reduced Matrigel and treated with a CO-releasing molecule (CORM-2) or exposed to CO gas (250 ppm). Here, we report the surprising finding that exposure to CO inhibits vascular endothelial growth factor (VEGF)-induced endothelial cell actin reorganisation, cell proliferation, migration and capillary-like tube formation. Similarly, CO suppressed VEGF-mediated phosphorylation of VEGFR-2 at tyrosine residue 1175 and 1214 and basic fibroblast growth factor- (FGF-2) and VEGF-mediated Akt phosphorylation. Consistent with these data, mice exposed to 250 ppm CO (1h/day for 14 days) exhibited a marked decrease in FGF-2-induced Matrigel plug angiogenesis (p<0.05). These data establish a new biological function for CO in angiogenesis and point to a potential therapeutic use for CO as an anti-angiogenic agent in tumour suppression.

Towards a 21st-century roadmap for biomedical research and drug discovery: consensus report and recommendations

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and nongovernmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathway-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this.

Oxidation of organic compounds in molten salts

The objective of this research was to investigate the oxidation of organic compounds in molten alkali metal hydroxides containing manganates. It has been shown that controlled oxidation can be readily achieved with high specificity to give products in high yield with very short reaction times. The concurrent changes in the melt were monitored using a vibrating platinum indicator electrode with a quazi-reference electrode which was successfully developed for use in molten (Na-K)OH eutectic at 523K. Henry's Law constants for water in the molten eutectic system (Na-K)OH have been measured and used to calculate the water concentration in the melt. The electrochemistry of manganates in molten (Na-K)OH eutectic at 523K has been studied using the vibrating platinum electrode, and the existence of the species Mn(II), Mn(II!), Mn(IV), Mn(V) and Mn(VI) in such melts has been investigated at various water concentrations. The half-wave potentials of the voltammetric waves were measured versus the cathodic limit of the melt. The stability of Mn(V) or Mn(VI) in the melt was achieved by varying the water concentration. A range of organic chemicals has been passed through molten (Na-K)OH at 523K and the reactions of these chemicals with the melt have been studied. The same organics were then passed through molten (Na-K)OH containing stabilized Mn(V) or Mn{VI) without violent reaction. Methanol, allyl alcohol, propane 1, 2 diol, I-heptene and acetone were oxidized by Mn(V) and Mn(VI). Ethanol was only oxidized by Mn(VI), isopropanol and benzyl alcohol were only oxidized by Mn(V). Npropanol, butanol, 2 methyl propan-2-ol, n-hexane, n-heptane toluene and cyclohexane were unchanged by both Mn(V) and Mn(VI). Detailed experiments have been performed on the reactions of ethanol, iso-propanol and methanol in molten (Na-K)OH containing stabilized Mrt(V) or Mn(VI), and reaction mechanisms have been postulated. Ethanol and iso-propanol were oxidized to acetaldehyde and acetone respectively with a potential for useful chemical process. The oxidation of methanol could be developed as a basis for an industrial methanol disposal process.

The use of organotellurium heterocycles as precursors for novel organometallic compounds

The reactions of group 16 heterocycles with organometallic reagents are described. Thiophenes have been used as models for organic sulfur in coal and their reactivity towards triiron dodecacarbonyl has been investigated. Reaction of unsubstituted thiophene with Fe3(CO)12 results in desulfurisation of the heterocycle, with the organic fragment being recovered in the form of the ferrole, C4H4.Fe2(CO)6. In addition a novel organometallic compound of iron is isolated, the formula of which is shown to be C4H4.Fe3(CO)8. Bezothiophene reacts with Fe3(CO)12 to yield benzothiaferrole, C8H6S.Fe2(CO)6, in which the sulfur is retained in the heterocycle. Dibenzothiophene, a more accurate model for organic sulfur in coal, displays no reactivity towards the iron carbonyl, suggesting that the more condensed systems will desulfurise less readily. Microwave methodology has been successful in accelerating the reactions of thiophenes with Fe3(CO)12. However, reaction of benzothiophene does not proceed to the desulfurisation stage while dibenzothiophene is unreactive even under microwave conditions. Tellurophenes (Te analogues of thiophenes) are shown to mimic the behaviour of thiophenes towards certain organometallic reagents with the advantage that their greater reactivity enables recovery of products in higher yields. Hence, reaction of tellurophene with Fe3(CO)12 again affords the ferrole but with an almost ten-fold increase in yield over thiophene. More significantly, dibenzotellurophene is also detellurated by the iron carbonyl affording the previously inaccessible dibenzoferrole, C12H8.Fe2(CO)6, thereby demonstrating the mechanistic feasibility of dechalcogenation of the more condensed aromatic molecules. The potential of tellurium heterocycles to act as precursors for novel organometallics is also recognised owing to the relatively facile elimination of the heteroatom from these systems. Thus, 2-telluraindane reacts with Fe3(CO)12 to yield a novel organometallic compound of formula C16H16.Fe(CO)3, arising from the unsymmetric dimerisation of two organic fragments.

Soft [beta]-adrenoceptor agonists for topical use in psoriasis

Psoriasis is characterised by epidermal proliferation and inflammation resulting in the appearance of elevated erythematous plaques. The ratio of c~AMP/c~GMP is decreased in psoriatic skin and when the epidermal cell surface receptors are stimulated by β-adrenergic agonists, intracellular ATP is transformed into c-AMP, thus restoring the c~AMP/c~GMP levels. This thesis describes a series of β-adrenoceptor agonists for topical delivery based upon the soft-drug approach. Soft drugs are defined as biologically active, therapeutically useful chemical compounds (drugs) characterised by a predictable and controllable In vivo destruction (metabolism) to non-toxic moieties. after they achieve their therapeutic role, The N-substituent can accommodate a broad range of structures and here the alkoxycarbonylethyl group has been used to provide metabolic susceptability. The increased polarity of the dihydroxy acid, expected after metabolic conversion of the soft~drug, ethyl N-[2'-(3',4'-dihydroxyphenyl)-2'-hydroxyethyl]-3- aminopropionate, should eliminate agonist activity. Further. to prevent oxidation and enhance topical delivery, the catechol hydroxyl groups have been esterified to produce a pro-soft-drug which generates the soft-drug in enzymic systems. The chemical hydrolysis of the pro-soft-drug proceeded via the formation of the dlpivaloyloxy acid and it failed to generate the active dihydroxy ester soft-drug. In contrast, in the presence of porcine liver carboxyesterase, the hydrolysis of the pro-soft drug proceeded via the formation of the required active soft-drug. This compound, thus, has the appropnate kinetic features to enable it to be evaluated further as a drug for the treatment of psoriasis. The pH rate-profile for the hydrolysis of soft-drug indicated a maximum stability at pH ∼ 4.0. The individual rate constants for the degradation and the pKa were analysed by nonlinear regression. The pKa of 7.40 is in excellent agreement with that determined by direct titration (7.43) and indicates that satisfactory convergence was achieved. The soft-drug was poorly transported across a silicone membrane; it was also air-sensitive due to oxidation of the catechol group. The transport of the pro-soft-drug was more efficient and, over the donor pH range 3-8, increased with pH. At lower values, the largely protonated species was not transported. However, above pH 7. chemical degradation was rapid so that a donor pH of 5-6 was optimum. The β-adrenergic agonist activity of these compounds was tested in vitro by measuring chronotropic and inotropic responses in the guinea pig atria and relaxation of guinea pig trachea precontracted with acetylcholine (10-3 M). The soft~drug was a full agonist on the tracheal preparation but was less potent than isoprenaline. Responses of the soft~drug were competitively antagonised by propranolol (10-6 M). The soft~drug produced an increase in force and rate of the isolated atrial preparatIon. The propyl analogue was equally potent with ED50 of 6.52 x 10-7 M. In contrast, at equivalent doses, the dihydroxy acid showed no activity; only a marginal effect was observed on the tracheal preparation. For the pro~soft-drug, responses were of slow onset, in both preparations, with a slowly developing relaxatlon of the tracheal preparatlon at high concentrations (10-5 M). This is consistent with in vitro results where the dipivaloyl groups are hydrolysed more readily than the ethyl ester to gIve the active soft-drug. These results confirm the validity tif the pro-soft-drug approach to the deUvery of β-adrenoceptor agonists.

Use of peptide nucleic acids as therapeutic agents

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The use of computational QSAR analysis in the toxicological evaluation of a series of 2-pyridylcarboxamidrazone candidate anti-tuberculosis compounds

A series of N1-benzylidene pyridine-2-carboxamidrazone anti-tuberculosis compounds has been evaluated for their cytotoxicity using human mononuclear leucocytes (MNL) as target cells. All eight compounds were significantly more toxic than dimethyl sulphoxide control and isoniazid (INH) with the exception of a 4-methoxy-3-(2-phenylethyloxy) derivative, which was not significantly different in toxicity compared with INH. The most toxic agent was an ethoxy derivative, followed by 3-nitro, 4-methoxy, dimethylpropyl, 4-methylbenzyloxy, 3-methoxy-4-(-2-phenylethyloxy) and 4-benzyloxy in rank order. In comparison with the effect of selected carboxamidrazone agents on cells alone, the presence of either N-acetyl cysteine (NAC) or glutathione caused a significant reduction in the toxicity of INH, as well as on the 4-benzyloxy derivative, although both increased the toxicity of a 4-N,N-dimethylamino-1-naphthylidene and a 2-t-butylthio derivative. The derivatives from this and three previous studies were subjected to computational analysis in order to derive equations designed to establish quantitative structure activity relationships for these agents. Twenty-five compounds were thus resolved into two groups (1 and 2), which on analysis yielded equations with r2 values in the range 0.65-0.92. Group 1 shares a common mode of toxicity related to hydrophobicity, where cytotoxicity peaked at logP of 3.2, while Group 2 toxicity was strongly related to ionisation potential. The presence of thiols such as NAC and GSH both promoted and attenuated toxicity in selected compounds from Group 1, suggesting that secondary mechanisms of toxicity were operating. These studies will facilitate the design of future low toxicity high activity anti-tubercular carboxamidrazone agents. © 2003 Elsevier Science B.V. All rights reserved.