Core exploration in optimization of chemokine receptor CCR4 antagonists

The design, synthesis, and SAR studies of 'core' variations led to identification of novel, selective, and potent small molecule antagonist (22) of the CC chemokine receptor-4 (CCR4) with improved in vitro activity and liability profile. Compound 22 was efficacious in a murine allergic inflammation model (ED50 approximately 10 mg/kg).     

Synthesis and biological evaluation of 4-amino derivatives of benzimidazoquinoxaline, benzimidazoquinoline, and benzopyrazoloquinazoline as potent IKK inhibitors

We have recently identified BMS-345541 (1) as a highly selective and potent inhibitor of IKK-2 (IC50 = 0.30 microM), which however was considerably less potent against IKK-1 (IC50 = 4.0 microM). In order to further explore the SAR around the imidazoquinoxaline tricyclic structure of 1, we prepared a series of tetracyclic analogues (7, 13, and 18). The synthesis and biological activities of these potent IKK inhibitors are described.     

Construction of a genetic linkage map for Saccharum officinarum incorporating both simplex and duplex markers to increase genome coverage.

Saccharum officinarum L. is an octoploid with 80 chromosomes and a basic chromosome number of x = 10. It has high stem sucrose and contributes 80% of the chromosomes to the interspecific sugarcane cultivars that are grown commercially for sucrose. A genetic linkage map was developed for S. officinarum (clone IJ76-514) using a segregating population generated from a cross between Q165 (a commercial sugarcane cultivar) and IJ76-514. In total, 40 AFLP and 72 SSR primer pairs were screened across the population, revealing 595 polymorphic bands inherited from IJ76-514. These 595 markers displayed a frequency distribution different from all other sugarcane genetic maps produced, with only 40% being simplex markers (segregated 1:1). Of these 240 simplex markers, 178 were distributed on 47 linkage groups (LGs) and 62 remained unlinked. With the addition of 234 duplex markers and 80 biparental simplex markers (segregating 3:1), 534 markers formed 123 LGs. Using the multi-allelic SSR markers, repulsion phase linkage, and alignment with the Q165 linkage map, 105 of the 123 LGs could be grouped into 10 homology groups (HGs). These 10 HGs were further assigned to the 8 HGs observed in cultivated sugarcane and S. spontaneum. Analysis of repulsion phase linkage indicated that IJ76-514 is neither a complete autopolyploid nor an allopolyploid. Detection of 28 repulsion linkages that occurred between 6 pairs of LGs located in 4 HGs suggested the occurrence of limited preferential chromosome pairing in this species.     

Hox patterning of the vertebrate rib cage

Unlike the rest of the axial skeleton, which develops solely from somitic mesoderm, patterning of the rib cage is complicated by its derivation from two distinct tissues. The thoracic skeleton is derived from both somitic mesoderm, which forms the vertebral bodies and ribs, and from lateral plate mesoderm, which forms the sternum. By generating mouse mutants in Hox5, Hox6 and Hox9 paralogous group genes, along with a dissection of the Hox10 and Hox11 group mutants, several important conclusions regarding the nature of the ;Hox code' in rib cage and axial skeleton development are revealed. First, axial patterning is consistently coded by the unique and redundant functions of Hox paralogous groups throughout the axial skeleton. Loss of paralogous function leads to anterior homeotic transformations of colinear regions throughout the somite-derived axial skeleton. In the thoracic region, Hox genes pattern the lateral plate-derived sternum in a non-colinear manner, independent from the patterning of the somite-derived vertebrae and vertebral ribs. Finally, between adjacent sets of paralogous mutants, the regions of vertebral phenotypes overlap considerably; however, each paralogous group imparts unique morphologies within these regions. In all cases examined, the next-most posterior Hox paralogous group does not prevent the function of the more-anterior Hox group in axial patterning. Thus, the ;Hox code' in somitic mesoderm is the result of the distinct, graded effects of two or more Hox paralogous groups functioning in any anteroposterior location.     

From plant neighbourhood to landscape scales: how grazing modifies native and exotic plant species richness in grassland

The interactive effect of grazing and soil resources on plant species richness and coexistence has been predicted to vary across spatial scales. When resources are not limiting, grazing should reduce competitive effects and increase colonisation and richness at fine scales. However, at broad scales richness is predicted to decline due to loss of grazing intolerant species. We examined these hypotheses in grasslands of southern Australia that varied in resources and ungulate grazing intensity since farming commenced 170 years ago. Fine-scale species richness was slightly greater in more intensively grazed upper slope sites with high nutrients but low water supply compared to those that were moderately grazed, largely due to a greater abundance of exotic species. At broader scales, exotic species richness declined with increasing grazing intensity whether nutrients or water supply were low or high. Native species richness declined at all scales in response to increasing grazing intensity and greater resource supply. Grazing also reduced fine-scale heterogeneity in native species richness and although exotics were also characterised by greater heterogeneity at fine scales, grazing effects varied across scales. In these grasslands patterns of plant species richness did not match predictions at all scales and this is likely to be due to differing responses of native and exotic species and their relative abundance in the regional species pool. Over the past 170 years intolerant native species have been eliminated from areas that are continually and heavily grazed, whereas transient, light grazing increases richness of both exotics and natives. The results support the observation that the processes and scales at which they operate differ between coevolved ungulate—grassland systems and those in transition due to recent invasion of herbivores and associated plant species.     

Remarkable morphological stasis in an extant vertebrate despite tens of millions of years of divergence

The relationship between genotypic and phenotypic divergence over evolutionary time varies widely, and cases of rapid phenotypic differentiation despite genetic similarity have attracted much attention. Here, we report an extreme case of the reverse pattern--morphological stasis in a tropical fish despite massive genetic divergence. We studied the enigmatic African freshwater butterfly fish (Pantodon buchholzi), whose distinctive morphology earns it recognition as a monotypic family. We sequenced the mitochondrial genome of Pantodon from the Congo basin and nine other osteoglossomorph taxa for comparison with previous mitogenomic profiles of Pantodon from the Niger basin and other related taxa. Pantodon populations form a monophyletic group, yet their mitochondrial coding sequences differ by 15.2 per cent between the Niger and Congo basins. The mitogenomic divergence time between these populations is estimated to be greater than 50 Myr, and deep genetic divergence was confirmed by nuclear sequence data. Among six sister-group comparisons of osteoglossomorphs, Pantodon exhibits the slowest rate of morphological divergence despite a level of genetic differentiation comparable to both species-rich (e.g. Mormyridae) and species-poor (e.g. Osteoglossidae) families. Morphological stasis in these two allopatric lineages of Pantodon offers a living vertebrate model for investigating phenotypic stability over millions of generations in the face of profound fluctuations in environmental conditions.     

Increased diacylglycerols characterize hepatic lipid changes in progression of human nonalcoholic fatty liver disease; comparison to a murine model

Background and Aims

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD.

Methods

Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts.

Results

Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts.

Conclusions

Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD.     

A ‘Chinese Spring’ wheat (Triticum aestivum L.) bacterial artificial chromosome library and its use in the isolation of SSR markers for targeted genome regions

A bacterial artificial chromosome (BAC) library was constructed from the bread wheat (Triticum aestivum L.) genotype ‘Chinese Spring’ (‘CS’). The library consists of 395,136 clones with an estimated average insert size of 157 kb. This library provides an estimated 3.4-fold genome coverage for this hexaploid species. The genome coverage was confirmed by RFLP analysis of single-copy RFLP clones. The CS BAC library was used to develop simple sequence repeat (SSR) markers for targeted genome regions using five sequence-tagged-site (STS) markers designed from the chromosome arm of 3BS. The SSR markers for the targeted genome region were successfully obtained. However, similar numbers of new SSR markers were also generated for the other two homoeologous group 3 chromosomes. This data suggests that BAC clones belonging to all three chromosomes of homoeologous group 3 were isolated using the five STS primers. The potential impacts of these results on marker isolation in wheat and on library screening in general are discussed.