In vitro biotransformations of the prostaglandin D2 (DP) antagonist MK-0524 and synthesis of metabolites

Metabolites of the potent DP antagonist, MK-0524, were generated using in vitro systems including hepatic microsomes and hepatocytes. Four metabolites (two hydroxylated diastereomers, a ketone and an acyl glucuronide) were characterized by LC-MS/MS and 1H NMR. Larger quantities of these metabolites were prepared by either organic synthesis or biosynthetically to be used as standards in other studies. The propensity for covalent binding was assessed and was found to be acceptable (<50 pmol-equiv/mg protein).     

Assessing the Bioavailability and Risk from Metal-Contaminated Soils and Dusts

Exposure to contaminated soil and dust is an important pathway in human health risk assessment. Physical and chemical characteristics and biological factors determine the bioaccessibility/bioavailability of soil and dust contaminants. Within a single sample, contamination may arise from multiple sources of toxic elements that may exist as different species that impact bioavailability. In turn, the bioaccessibility/bioavailability of soil and dust contaminants directly impacts human health risk. Research efforts focusing on development and application of in vitro and in vivo methods to measure the bioaccessibility/bioavailability of metal-contaminated soils have advanced in recent years. The objective of this workshop was to focus on developments in assessing the bioaccessibility/bioavailability of arsenic-contaminated soils, metals’ contamination in urban Canadian residences and potential children's exposures to toxic elements in house dust, an urban community-based study (i.e., West Oakland Residential Lead Assessment), bioavailability studies of soil cadmium, chromium, nickel, and mercury and human exposures to contaminated Brownfield soils. These presentations covered issues related to human health and bioavailability along with the most recent studies on community participation in assessing metals’ contamination, studies of exposures to residential contamination, and in vitro and in vivo methods development for assessing the bioaccessibility/bioavailability of metals in soils and dusts.     

Fibroblasts derived from long‐lived insulin receptor substrate 1 null mice are not resistant to multiple forms of stress

Reduced signalling through the insulin/insulin-like growth factor-1 signalling (IIS) pathway is a highly conserved lifespan determinant in model organisms. The precise mechanism underlying the effects of the IIS on lifespan and health is currently unclear, although cellular stress resistance may be important. We have previously demonstrated that mice globally lacking insulin receptor substrate 1 (Irs1^−/−) are long-lived and enjoy a greater period of their life free from age-related pathology compared with wild-type (WT) controls. In this study, we show that primary dermal fibroblasts and primary myoblasts derived from Irs1^−/− mice are no more resistant to a range of oxidant and nonoxidant chemical stressors than cells derived from WT mice.     

Estimating the Hidden Burden of Bovine Tuberculosis in Great Britain

The number of cattle herds placed under movement restrictions in Great Britain (GB) due to the suspected presence of bovine tuberculosis (bTB) has progressively increased over the past 25 years despite an intensive and costly test-and-slaughter control program. Around 38% of herds that clear movement restrictions experience a recurrent incident (breakdown) within 24 months, suggesting that infection may be persisting within herds. Reactivity to tuberculin, the basis of diagnostic testing, is dependent on the time from infection. Thus, testing efficiency varies between outbreaks, depending on weight of transmission and cannot be directly estimated. In this paper, we use Approximate Bayesian Computation (ABC) to parameterize two within-herd transmission models within a rigorous inferential framework. Previous within-herd models of bTB have relied on ad-hoc methods of parameterization and used a single model structure (SORI) where animals are assumed to become detectable by testing before they become infectious. We study such a conventional within-herd model of bTB and an alternative model, motivated by recent animal challenge studies, where there is no period of epidemiological latency before animals become infectious (SOR). Under both models we estimate that cattle-to-cattle transmission rates are non-linearly density dependent. The basic reproductive ratio for our conventional within-herd model, estimated for scenarios with no statutory controls, increases from 1.5 (0.26–4.9; 95% CI) in a herd of 30 cattle up to 4.9 (0.99–14.0) in a herd of 400. Under this model we estimate that 50% (33–67) of recurrent breakdowns in Britain can be attributed to infection missed by tuberculin testing. However this figure falls to 24% (11–42) of recurrent breakdowns under our alternative model. Under both models the estimated extrinsic force of infection increases with the burden of missed infection. Hence, improved herd-level testing is unlikely to reduce recurrence unless this extrinsic infectious pressure is simultaneously addressed.     

Carotid artery stenting: update

"Extracranial carotid artery stenosis is responsible for approximately 20-30% of ischemic strokes. Traditionally, carotid artery stenosis has been treated with carotid endarterectomy. However, the low periprocedural complication rate and the mid term durability of carotid artery stenting has made it a competitive alternative treatment strategy. This update reviews the technical aspects of carotid artery stenting, clinical data supporting carotid artery stenting-particularly in high risk patients, and the complications associated with carotid artery stenting."     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

Parasite aquaporins: Current developments in drug facilitation and resistance

Background

Although being situated in a niche, research on parasite aquaporins is a lively field that has provided new insight into basic aquaporin structure–function relationships and physiological roles of water and solute transport. Moreover, it bears the potential to find novel approaches to antiparasitic chemotherapy.

Scope of review

Here, we summarize the current knowledge about the structure and substrate selectivity of aquaporins from protozoan and helminth parasites, review the current views on their physiological roles, and discuss their potency for chemotherapy.

Major conclusions

Parasite aquaporins fulfill highly diverse tasks in the physiology of the various organisms, yet their general protein structure is well conserved. Aquaporins are directly (antimonials) and indirectly (melarsoprol, pentamidine) linked to the uptake of antiparasitic drugs. Unfortunately, drug-like aquaporin inhibitors are still missing.

General significance

Aquaporins expression levels determine the degree of parasite resistance against certain drugs. Further studies on parasite aquaporins may provide data about overcoming drug resistance mechanisms or even spark novel treatments. This article is part of a Special Issue entitled Aquaporins.     

Characterization of Glyoxalase I (E. coli)—Inhibitor Interactions by Electrospray Time-of-Flight Mass Spectrometry and Enzyme Kinetic Analysis

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-{2-[3-hexyloxybenzoyl]-vinyl}glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme.     

Aspects of amino acid requirements for in vitro evagination of imaginal leg discs inDrosophila melanogaster

Summary A method of staging late third instar larvae on the basis of salivary gland morphology is described. Using this technique, we investigated stage related amino acid requirements forDrosophila leg disc evagination in vitro. It was found that the requirement for glutamine lasted longer than that of proline. The staging technique should help in the detailed exploration of the late 3rd instar time period in order to bridge the gap between biochemistry and morphogenesis in the initiation of disc evagination.