Human variants of O6-alkylguanine-DNA alkyltransferase

This article summarizes the current understanding of known variant forms of the MGMT gene that encode an altered protein. Epidemiological studies have been carried out to test whether these alterations are associated with altered cancer risk. Laboratory studies using recombinant proteins and cells expressing the known variants have investigated the possible effects of these sequence alterations on the ability of the encoded O(6)-alkylguanine-DNA alkyltransferase protein to protect cells from alkylation damage and to respond to therapeutic inactivators currently undergoing trials for cancer chemotherapy.     

Disruption of the thrombospondin-2 gene alters the lamellar morphology but does not permit vascularization of the adult mouse lumbar disc

Introduction

The biological basis for the avascular state of the intervertebral disc is not well understood. Previous work has suggested that the presence of thrombospondin-1 (TSP-1), a matricellular protein, in the outer annulus reflects a role for this protein in conferring an avascular status to the disc. In the present study we have examined thrombospondin-2 (TSP-2), a matricellular protein with recognized anti-angiogenic activity in vivo and in vitro.

Methods

We examined both the location and expression of TSP-2 in the human disc, and its location in the disc and bordering soft tissues of 5-month-old normal wild-type (WT) mice and of mice with a targeted disruption of the TSP-2 gene. Immunohistochemistry and quantitative histology were utilized in this study.

Results

TSP-2 was found to be present in some, but not all, annulus cells of the human annulus and the mouse annulus. Although there was no difference in the number of disc cells in the annulus of TSP-2-null mice compared with that of WT animals, polarized light microscopy revealed a more irregular lamellar collagen structure in null mouse discs compared with WT mouse discs. Additionally, vascular beds at the margins of discs of TSP-2-null mice were substantially more irregular than those of WT animals. Counts of platelet endothelial cell adhesion molecule-1-positive blood vessels in the tissue margin bordering the ventral annulus showed a significantly larger vascular bed in the tissue bordering the disc of TSP-2-null mice compared with that of WT mice (P = 0.0002). There was, however, no vascular ingrowth into discs of the TSP-2-null mice.

Conclusion

These data confirm a role for TSP-2 in the morphology of the disc and suggest the presence of other inhibitors of angiogenesis in the disc. We have shown that although an increase in vasculature was present in the TSP-2-null tissue in the margin of the disc, vascular ingrowth into the body of the disc did not occur. Our results point to the need for future research to understand the transition from the well-vascularized status of the fetal and young discs to the avascular state of the adult human disc or the small mammalian disc.     

L protease from foot and mouth disease virus confers eIF2-independent translation for mRNAs bearing picornavirus IRES

The leader protease (L^pro) from foot-and-mouth disease virus (FMDV) has the ability to cleave eIF4G, leading to a blockade of cellular protein synthesis. In contrast to previous reports, our present findings demonstrate that FMDV L^pro is able to increase translation driven by FMDV IRES. Additionally, inactivation of eIF2 subsequent to phosphorylation induced by arsenite or thapsigargin in BHK cells blocks protein synthesis directed by FMDV IRES, whereas in the presence of L^pro, significant translation is found under these conditions. This phenomenon was also observed in cell-free systems after induction of eIF2 phosphorylation by addition of poly(I:C).     

Low genetic diversity and fragmentation effects in a wind-pollinated tree, Polylepis multijuga Plige (Rosaceae) in the high Andes

Fragmentation is predicted to increase inbreeding depression and lower the evolutionary potential of organisms by disrupting dispersal. Trees may be more resilient to fragmentation effects due to potential long-distance dispersal mechanisms that genetically connect fragments. Polylepis woodlands in the high Andes are highly fragmented and are currently the focus of reforestation and conservation efforts. Polylepis multijuga Plige (Rosaceae) is a threatened, endemic tree species in the northern Andes of Peru. Samples were collected from 371 adult trees in nine forest fragments separated by 0.5–80 km and genotyped at amplified fragment length polymorphism loci (AFLP) and chloroplast intergenic regions to determine the connectedness of fragments and their suitability for collecting seed for restoration efforts. P. multijuga is wind-pollinated and dispersed; however, genetic diversity in P. multijuga was about half that reported for other wind-pollinated species. Genetic spatial autocorrelation and patterns of chloroplast and AFLP diversity suggest seed dispersal is very limited and that wind dispersed pollen does not effectively connect all fragments. Conservation of this species will require reforestation efforts and possibly augmentation of some fragments to increase their genetic diversity. Collecting seed from multiple large fragments and from individuals separated by at least 25 m within fragments would maximize the genetic diversity of seed collections for reforestation or augmentation. Future studies of this and other Polylepis species should determine how complex topography may affect wind mediated dispersal between fragments and patterns of genetic diversity.     

Targeting conserved water molecules: Design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.     

Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes

The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.     

No increase in radiation-induced chromosome aberration complexity detected by m-FISH after culture in the presence of 5'-bromodeoxyuridine

The thymidine analogue, 5'-bromodeoxyuridine (BrdU), is a known mutagen that is routinely introduced into culture media for subsequent Harlequin stain analysis and determination of cell cycle status. Previously, we examined the induction of chromosome aberrations in human peripheral blood lymphocytes (PBL) known to be in their 1st cell division following exposure to a low dose (0.5 Gy, average one alpha-particle per cell) of high-LET alpha-particles. We found complex chromosome aberrations to be characteristic of exposure to high-LET radiation and suggested the features of complex exchange to reflect qualitatively the spatial deposition of this densely ionising radiation. To exclude the possibility that BrdU addition post-irradiation influenced the complexity of chromosomal damage observed by m-FISH, the effect of increasing BrdU concentration on aberration complexity was investigated. Comparisons between BrdU concentration (0, 10 and 40 microM) and between sham- and alpha-particle-irradiated PBL, were made both independently and in combination to enable discrimination between BrdU and high-LET radiation effects. Aberration type, size, complexity and completeness were assessed by m-FISH, and the relative progression through cell division was evaluated. We found no evidence of any qualitative difference in the complexity of damage as visualised by m-FISH but did observe an increase in the frequency of complex exchanges with increasing BrdU concentration indicative of altered cell cycle kinetics. The parameters measured here are consistent with findings from previous in vitro and in vivo work, indicating that each complex aberration visualised by m-FISH is characteristic of the structure of the high-LET alpha-particle track and the geometry of cell irradiated.