Discovery and synthesis of 6,7,8,9-tetrahydro-5H-pyrimido-[4,5-d]azepines as novel TRPV1 antagonists

Utilization of a tetrahydro-pyrimdoazepine core as a bioisosteric replacement for a piperazine-urea resulted in the discovery a novel series of potent antagonists of TRPV1. The tetrahydro-pyrimdoazepines have been identified as having good in vitro and in vivo potency and acceptable physical properties.     

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.     

Can long-distance dispersal shape the local and regional species pool?

Long-distance dispersal events are irregular and their role in shaping plant diversity is often discussed and modeled but rarely studied experimentally. We mimicked long-distance dispersal experimentally by sowing eleven exotic and fourteen native species into a calcareous grassland community in Estonia. Exotic species were randomly chosen from the collection of 500 herbaceous species in the Botanical Garden of the Tartu University. All exotic species were able to complete their life-cycles under the climatic and edaphic conditions in the garden. Native species originated from open dry calcareous habitats in the surroundings of the study site, but did not occur in the experimental grassland. Seven exotic species and seven native species established during the first year. In the third year, there were still three exotic species with five premature individuals, and three sown native species with sixteen individuals in the plots. These results show that long-distance dispersal both within and between regions may have an impact on species composition in target plant communities. If relatively the best established exotic speciesPhyteuma scheuchzeri would be classified as casual, one may conclude that transition among introduction and casual stages corresponds to ten’ rule. The species richness of seedlings, taking both local and sown species into account, was higher in plots with higher native established plant species richness.     

Amyotrophic lateral sclerosis: an emerging era of collaborative gene discovery

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND). It is currently incurable and treatment is largely limited to supportive care. Family history is associated with an increased risk of ALS, and many Mendelian causes have been discovered. However, most forms of the disease are not obviously familial. Recent advances in human genetics have enabled genome-wide analyses of single nucleotide polymorphisms (SNPs) that make it possible to study complex genetic contributions to human disease. Genome-wide SNP analyses require a large sample size and thus depend upon collaborative efforts to collect and manage the biological samples and corresponding data. Public availability of biological samples (such as DNA), phenotypic and genotypic data further enhances research endeavors. Here we discuss a large collaboration among academic investigators, government, and non-government organizations which has created a public repository of human DNA, immortalized cell lines, and clinical data to further gene discovery in ALS. This resource currently maintains samples and associated phenotypic data from 2332 MND subjects and 4692 controls. This resource should facilitate genetic discoveries which we anticipate will ultimately provide a better understanding of the biological mechanisms of neurodegeneration in ALS.     

Association of Pericardial Fat With Liver Fat and Insulin Sensitivity After Diet‐Induced Weight Loss in Overweight Women

Pericardial adipose tissue (PAT) is positively associated with fatty liver and obesity‐related insulin resistance. Because PAT is a well‐known marker of visceral adiposity, we investigated the impact of weight loss on PAT and its relationship with liver fat and insulin sensitivity independently of body fat distribution. Thirty overweight nondiabetic women (BMI 28.2–46.8 kg/m², 22–41 years) followed a 14.2 ± 4‐weeks low‐calorie diet. PAT, abdominal subcutaneous (SAT), and visceral fat volumes (VAT) were measured by magnetic resonance imaging (MRI), total fat mass, trunk, and leg fat by dual‐energy X‐ray absorptiometry and intrahepatocellular lipids (IHCL) by (¹)H‐magnetic resonance spectroscopy. Euglycemic hyperinsulinemic clamp (M) and homeostasis model assessment of insulin resistance (HOMA_IR) were used to assess insulin sensitivity or insulin resistance. At baseline, PAT correlated with VAT (r = 0.82; P < 0.001), IHCL (r = 0.46), HOMA_IR (r = 0.46), and M value (r = ?0.40; all P < 0.05). During intervention, body weight decreased by ?8.5%, accompanied by decreases of ?12% PAT, ?13% VAT, ?44% IHCL, ?10% HOMA2‐%B, and +24% as well as +15% increases in HOMA2‐%S and M, respectively. Decreases in PAT were only correlated with baseline PAT and the loss in VAT (r = ?0.56; P < 0.01; r = 0.42; P < 0.05) but no associations with liver fat or indexes of insulin sensitivity were observed. Improvements in HOMA_IR and HOMA2‐%B were only related to the decrease in IHCL (r = 0.62, P < 0.01; r = 0.65, P = 0.002) and decreases in IHCL only correlated with the decrease in VAT (r = 0.61, P = 0.004). In conclusion, cross‐sectionally PAT is correlated with VAT, liver fat, and insulin resistance. Longitudinally, the association between PAT and insulin resistance was lost suggesting no causal relationship between the two.     

Biodegradation efficiency of functionally important populations selected for bioaugmentation in phenol- and oil-polluted area

Denaturing gradient gel electrophoresis of amplified fragments of genes coding for 16S rRNA and for the largest subunit of multicomponent phenol hydroxylase (LmPH) was used to monitor the behaviour and relative abundance of mixed phenol-degrading bacterial populations (Pseudomonas mendocina PC1, P. fluorescens strains PC18, PC20 and PC24) during degradation of phenolic compounds in phenolic leachate- and oil-amended microcosms. The analysis indicated that specific bacterial populations were selected in each microcosm. The naphthalene-degrading strain PC20 was the dominant degrader in oil-amended microcosms and strain PC1 in phenolic leachate microcosms. Strain PC20 was not detectable after cultivation in phenolic leachate microcosms. Mixed bacterial populations in oil-amended microcosms aggregated and formed clumps, whereas the same bacteria had a planktonic mode of growth in phenolic leachate microcosms. Colony hybridisation data with catabolic gene specific probes indicated that, in leachate microcosms, the relative proportions of bacteria having meta (PC1) and ortho (PC24) pathways for degradation of phenol and p-cresol changed alternately. The shifts in the composition of mixed population indicated that different pathways of metabolism of aromatic compounds dominated and that this process is an optimised response to the contaminants present in microcosms.     

Diverse compounds mimic Alzheimer disease-causing mutations by augmenting Abeta42 production

Increased Abeta42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Abeta42. Among the more potent Abeta42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2-selective NSAID. Many COX-2-selective NSAIDs tested raised Abeta42, including multiple COX-2-selective derivatives of two Abeta42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Abeta42. These compounds seem to target the gamma-secretase complex, increasing gamma-secretase-catalyzed production of Abeta42 in vitro. Short-term in vivo studies show that two Abeta42-raising compounds increase Abeta42 levels in the brains of mice. The elevations in Abeta42 by these compounds are comparable to the increases in Abeta42 induced by Alzheimer disease-causing mutations in the genes encoding amyloid beta protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Abeta42 production in humans.     

The effects of dexpramipexole (KNS-760704) in individuals with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by upper and lower motor neuron dysfunction and loss, rapidly progressive muscle weakness, wasting and death. Many factors, including mitochondrial dysfunction, may contribute to ALS pathogenesis. Riluzole, which has shown only modest benefits in a measure of survival time without demonstrated effects on muscle strength or function, is the only approved treatment for ALS. We tested the putative mitochondrial modulator dexpramipexole (KNS-760704; (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine) in subjects with ALS in a two-part, double-blind safety and tolerability study, with a preliminary assessment of its effects on functional decline and mortality. In part 1, the effects of dexpramipexole (50, 150 or 300 mg d(-1)) versus placebo were assessed over 12 weeks. In part 2, after a 4-week, single-blind placebo washout, continuing subjects were re-randomized to dexpramipexole at 50 mg d(-1) or 300 mg d(-1) as double-blind active treatment for 24 weeks. Dexpramipexole was safe and well tolerated. Trends showing a dose-dependent attenuation of the slope of decline of the ALS Functional Rating Scale-Revised (ALSFRS-R) in part 1 and a statistically significant (P = 0.046) difference between groups in a joint rank test of change from baseline in ALSFRS-R and mortality in part 2 strongly support further testing of dexpramipexole in ALS.     

Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly

The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1^liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1^liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1^liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles.     

Use of balance methods for assessment of short-term changes in body composition

Balance methods reveal changes in body energy, nitrogen, macro‐ and micronutrients as well as fluid in response to different feeding regimens. Under metabolic ward conditions, where physical activity is restricted and activity and food intake are controlled, the errors of estimates of energy intake, energy expenditure, and energy losses are about 2, 4, and 2%, respectively. Balance techniques can be used to validate techniques of in vivo body composition analysis (BCA). This is necessary since immediate and transient changes in body composition in response to a change in diet adversely affect the validity of techniques by violating the assumptions underlying standard methods (i.e., a constant composition or hydration of lean mass). Using two compartment reference methods, like densitometry, dual X‐ray absorptiometry (DXA) or deuterium dilution, changes in fat mass with caloric restriction and overfeeding can be measured with a minimal detectable change (MDC) of 1.0–2.0 kg. However, when compared against balance data, the validity of these techniques to measure short‐term changes in body composition is poor. The noninvasive and rapid new quantitative magnetic resonance (QMR) technique has a high precision with a MDC of 0.18 kg of fat mass. The validity of QMR to assess short‐term changes in fat mass is challenged by comparison to balance data. Today, techniques used for in vivo BCA should be related to steady state conditions only, while in the nonsteady state, the use of balance methods is recommended to assess short‐term changes in body composition.