Discovery of potent azaindazole leucine-rich repeat kinase 2 (LRRK2) inhibitors possessing a key intramolecular hydrogen bond – Part 2

The discovery of disease-modifying therapies for Parkinson’s Disease (PD) represents a critical need in neurodegenerative medicine. Genetic mutations in LRRK2 are risk factors for the development of PD, and some of these mutations have been linked to increased LRRK2 kinase activity and neuronal toxicity in cellular and animal models. As such, research towards brain-permeable kinase inhibitors of LRRK2 has received much attention. In the course of a program to identify structurally diverse inhibitors of LRRK2 kinase activity, a 5-azaindazole series was optimized for potency, metabolic stability and brain penetration. A key design element involved the incorporation of an intramolecular hydrogen bond to increase permeability and potency against LRRK2. This communication will outline the structure-activity relationships of this matched pair series including the challenge of obtaining a desirable balance between metabolic stability and brain penetration.     

Statistical Aspects of the Use of Biomarkers in Nutritional Epidemiology Research

Few strong and consistent associations have arisen from observational studies of dietary consumption in relation to chronic disease risk. Measurement error in self-reported dietary assessment may be obscuring many such associations. Attempts to correct for measurement error have mostly used a second self-reported assessment in a subset of a study cohort to calibrate the self-reported assessment used throughout the cohort, under the dubious assumption of uncorrelated measurement errors between the two assessments. The use, instead, of objective biomarkers of nutrient consumption to produce calibrated consumption estimates provides a promising approach to enhance study reliability. As summarized here, we have recently applied this nutrient biomarker approach to examine energy, protein, and percent of energy from protein, in relation to disease incidence in Women’s Health Initiative cohorts, and find strong associations that are not evident without biomarker calibration. A major bottleneck for the broader use of a biomarker-calibration approach is the rather few nutrients for which a suitable biomarker has been developed. Some methodologic approaches to the development of additional pertinent biomarkers, including the possible use of a respiratory quotient from indirect calorimetry for macronutrient biomarker development, and the potential of human feeding studies for the evaluation of a range of urine- and blood-based potential biomarkers, will briefly be described.     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3 extended DNAs, with the following sequence: 5-GTTAGGCTT-3 3-CAATCCGAA-5. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3 extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5 extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.     

A model of radioiodine transfer to goat milk incorporating the influence of stable iodine

Previously reported models for radioiodine in ruminants cannot account for the effect of variations in stable iodine intake including large countermeasure doses of stable iodine on the transfer of radioiodine to goat milk. A metabolically based model of radioiodine transfer in goats has been parameterised using new experimental data on the effect of countermeasure doses of stable iodine on radioiodine transfer to milk. To account for the effect of dietary stable iodine levels, the model represents the transfer of iodine from the extracellular fluid to milk with Michaelis-Menten kinetics. The model shows good agreement with the experimental data, and the estimated parameters compare favourably with values which can be estimated from the literature. The parameterised model accounts for 95% of the variation in the observed data for milk, faeces, urine and thyroid (n=199). The model has been used to predict the effects of variation in stable iodine intake and the extent of consequent chemical contamination of milk by stable iodine. The time taken for radio-iodine to reach peak concentrations in milk following a deposition event is predicted to vary significantly (ca. 2 days) over a range of expected stable iodine intakes. Doses of stable iodine sufficient to reduce the radioiodine transfer to milk will result in stable iodine concentrations in milk greatly in excess of internationally advised limits. Therefore, we recommend that stable iodine supplementation not be used as a countermeasure to reduce radioiodine transfer to milk. Indeed, model predictions suggest that reductions in stable iodine intake would be a more effective countermeasure. However, this is unlikely to be feasible since the short physical half-life of ¹³¹I may not allow adequate time to implement changes in feed manufacture. The model described in this paper is freely available in ModelMaker 3.0 format (http://www.notingham.ac.uk/environmental-modelling/). Received: 16 August 1999 / Accepted in revised form: 15 November 1999     

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.     

Evaluation of tebufenozide carry-over and residual effects on spruce budworm (Lepidoptera: Tortricidae)

Laboratory and field studies investigated carry-over effects of tebufenozide on spruce budworm, Choristoneura fumiferana (Clemens). In the laboratory, third and fourth instars were fed either sublethal doses of tebufenozide (10 ppm) or water onAbies balsamea (L.) Mill. needles, reared to adulthood and allowed to oviposit on laying surfaces 1 or 14 d after being sprayed with water or tebufenozide concentrations of 17.5, 35.0, and 70.0 g/liter. Percentage adult emergence and sex ratio were not affected by larval ingestion of the tebufenozide. Also, the mean number of eggs laid on untreated wax paper by moths reared from tebufenozide-treated larvae was similar to the controls. Hence, tebufenozide did not inhibit carry-over effects on treated larvae. Oviposition on tebufenozide-treated wax paper by moths reared from untreated larvae was affected by both the substrate concentration and the age of the treatment residue. When offered treated and untreated laying surfaces simultaneously, C. fumiferana did not show a preference. However, significantly fewer eggs were laid on both laying surfaces by fewer females than when tebufenozide was absent. Residual tebufenozide on wax paper did not affect egg hatch but topical applications were toxic to eggs. Field studies appear to corroborate laboratory results and suggest that although the ingestion of tebufenozide by larval spruce budworm might not impair adult reproduction, the insecticide's presence in the environment could inhibit oviposition. This inhibition was considered to be a primary factor in tebufenozide's multi-year effects against spruce budworm populations.     

Stable fly (Stomoxys calcitrans: Diptera, Muscidae) trap response to changes in effective trap height caused by growing vegetation

Stable flies (Stomoxys calcitrans L.) are blood-feeding pests of cattle, whose populations are often monitored using sticky traps. Trap responses at different heights were compared with: 1) a choice and no-choice test, with sticky cards set at 30 and 121 cm heights (above ground), and 2) tall (120 cm) traps placed in short (3 cm) and tall (40 cm) grass to assess how vegetation height affects trap catches. In the first experiment, the percentage of upper to lower trap catches were similar at choice (16%) and no-choice traps (15%). In the vegetation study, stable fly catch height data were fitted to gamma distributions to determine the most productive trap heights; 20 cm above short grass and 24 cm above tall grass (from lower edge of trap). The results indicate that traps used to monitor stable fly populations should be maintained at a constant distance above surrounding vegetation rather than ground surface, otherwise trap data can be misleading.     

BRCT domains: Easy as one,two, three

BRCA1 C-terminal (BRCT) domains are integral signaling modules in the DNA damage response (DDR). Aside from their established roles as phospho-peptide binding modules, BRCT domains have been implicated in phosphorylation-independent protein interactions, DNA binding and poly(ADP-ribose) (PAR) binding. These numerous functions can be attributed to the diversity in BRCT domain structure and architecture, where domains can exist as isolated single domains or assemble into higher order homo- or hetero-domain complexes. In this review, we incorporate recent structural and biochemical studies to demonstrate how structural features allow single and tandem BRCT domains to attain a high degree of functional diversity.Key words: BRCT domain, DNA repair, phosphorylation, phospho-peptide interaction, protein interaction, DNA binding, DNA damage response