Synthesis and biological evaluation of gem-diamine 1-N-iminosugars related to L-iduronic acid as inhibitors of heparan sulfate 2-O-sulfotransferase

A variety of gem-diamine 1-N-iminosugars related to L-iduronic acid were synthesized and evaluated as inhibitors of heparan sulfate uronyl 2-O-sulfotransferase using an in vitro enzyme assay. Two iminosugars containing guanidino groups acted as potent in vitro inhibitors of the enzyme.     

The Temperature-Sensitive brush Mutant of the Legume Lotus japonicus Reveals a Link between Root Development and Nodule Infection by Rhizobia

The brush mutant of Lotus japonicus exhibits a temperature-dependent impairment in nodule, root, and shoot development. At 26°C, brush formed fewer nodules, most of which were not colonized by rhizobia bacteria. Primary root growth was retarded and the anatomy of the brush root apical meristem revealed distorted cellular organization and reduced cell expansion. Reciprocal grafting of brush with wild-type plants indicated that this genotype only affected the root and that the shoot phenotype was a secondary effect. The root and nodulation phenotype cosegregated as a single Mendelian trait and the BRUSH gene could be mapped to the short arm of chromosome 2. At 18°C, the brush root anatomy was rescued and similar to the wild type, and primary root length, number of infection threads, and nodule formation were partially rescued. Superficially, the brush root phenotype resembled the ethylene-related thick short root syndrome. However, treatment with ethylene inhibitor did not recover the observed phenotypes, although brush primary roots were slightly longer. The defects of brush in root architecture and infection thread development, together with intact nodule architecture and complete absence of symptoms from shoots, suggest that BRUSH affects cellular differentiation in a tissue-dependent way.     

Intermediate host availability masks the strength of experimentally-derived colonisation patterns in echinostome trematodes

A fundamental goal of parasite evolutionary ecology is to elucidate patterns of host use and determine the underlying mechanisms of parasite colonisation. In order to distinguish the relative contributions of host encounter rates and host compatibility to infection outcomes, we compared host use in both field and experimental laboratory settings. Two years of bi-weekly snail sampling at a freshwater pond demonstrated fluctuating availability among three potential second intermediate snail host species and suggested that two trematode species (Echinostoma revolutum and Echinoparyphium sp.) did not colonise the three potential snail host species, Lymnaea elodes, Physa gyrina and Helisoma trivolvis, differentially. However, a series of experimental infections demonstrated that both parasites colonised H. trivolvis more so than the other two host species. Thus, more echinostome parasites utilised snail hosts that cannot serve as their first intermediate host. In experimental infections, host size and vagility were not strong determinants of infection. By utilising field and laboratory approaches, we were able to compare the strength of host compatibility under controlled conditions with patterns of infection in nature. Based on the results from these studies, it appears that host encounter is the primary mechanism dictating infection outcomes in the field.     

Inhibitors of hepatitis C virus NS3/4A: α-Ketoamide based macrocyclic inhibitors

A novel series of hepatitis C virus (HCV) NS3/4A protease inhibitors bearing a P2-P4 macrocycle and a P1-P1′ α-ketoamide serine trap is reported. The NS3 protease, which is essential for viral replication, is considered one of the most attractive targets for developing novel anti-HCV therapies. The optimization of both the macrocycle and the warhead portions led to the discovery of compounds 8b and 8g with a good activity both in the enzyme as well as in the cell based (replicon) assays with favorable PK profile in a preclinical species.     

Quantifying Transmission of Campylobacter jejuni in Commercial Broiler Flocks

Since meat from poultry colonized with Campylobacter spp. is a major cause of bacterial gastroenteritis, human exposure should be reduced by, among other things, prevention of colonization of broiler flocks. To obtain more insight into possible sources of introduction of Campylobacter into broiler flocks, it is essential to estimate the moment that the first bird in a flock is colonized. If the rate of transmission within a flock were known, such an estimate could be determined from the change in the prevalence of colonized birds in a flock over time. The aim of this study was to determine the rate of transmission of Campylobacter using field data gathered for 5 years for Australian broiler flocks. We used unique sampling data for 42 Campylobacter jejuni-colonized flocks and estimated the transmission rate, which is defined as the number of secondary infections caused by one colonized bird per day. The estimate was 2.37 ± 0.295 infections per infectious bird per day, which implies that in our study population colonized flocks consisting of 20,000 broilers would have an increase in within-flock prevalence to 95% within 4.4 to 7.2 days after colonization of the first broiler. Using Bayesian analysis, the moment of colonization of the first bird in a flock was estimated to be from 21 days of age onward in all flocks in the study. This study provides an important quantitative estimate of the rate of transmission of Campylobacter in broiler flocks, which could be helpful in future studies on the epidemiology of Campylobacter in the field.     

Trf1 Is Not Required for Proliferation or Functional Telomere Maintenance in Chicken DT40 Cells

The telomere end-protection complex prevents the ends of linear eukaryotic chromosomes from degradation or inappropriate DNA repair. The homodimeric double-stranded DNA-binding protein, Trf1, is a component of this complex and is essential for mouse embryonic development. To define the requirement for Trf1 in somatic cells, we deleted Trf1 in chicken DT40 cells by gene targeting. Trf1-deficient cells proliferated as rapidly as control cells and showed telomeric localization of Trf2, Rap1, and Pot1. Telomeric G-strand overhang lengths were increased in late-passage Trf1-deficient cells, although telomere lengths were unaffected by Trf1 deficiency, as determined by denaturing Southern and quantitative FISH analysis. Although we observed some clonal variation in terminal telomere fragment lengths, this did not correlate with cellular Trf1 levels. Trf1 was not required for telomere seeding, indicating that de novo telomere formation can proceed without Trf1. The Pin2 isoform and a novel exon 4, 5–deleted isoform localized to telomeres in Trf1-deficient cells. Trf1-deficient cells were sensitive to DNA damage induced by ionizing radiation. Our data demonstrate that chicken DT40 B cells do not require Trf1 for functional telomere structure and suggest that Trf1 may have additional, nontelomeric roles involved in maintaining genome stability.     

Heparan Sulfate 2-O-Sulfotransferase Is Required for Triglyceride-rich Lipoprotein Clearance

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153–164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st^f/f) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1^f/f) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st^f/fAlbCre⁺ mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1^f/fAlbCre⁺ mice. In contrast, Hs6st1^f/fAlbCre⁺ mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.     

Circulating mediators of bone remodeling in psoriatic arthritis: implications for disordered osteoclastogenesis and bone erosion

Introduction  

Diverse bone pathologies are observed in patients with psoriatic arthritis (PsA). Uncoupling of bone remodeling with disordered osteoclastogenesis has been implicated in the pathogenesis of PsA. The aim of this study was to examine the role of soluble mediators of bone remodeling within the circulation of patients with PsA.