Expanded CAG repeats activate the DNA damage checkpoint pathway

Trinucleotide repeats (TNRs) are sequences whose expansion causes several genetic diseases and chromosome breakage. We report a novel finding that expanded CAG repeats activate the DNA damage response. Mutations in yeast MEC1, RAD9, or RAD53 genes result in increased rates of fragility of a CAG repeat tract while single or double deletions of RAD17 or RAD24 have only a modest effect on TNR fragility, indicating that signaling down the Rad9 pathway and not the Rad17-Rad24 pathway plays a major role in sensing and repairing CAG-tract breaks. Deletion of CHK1 had no effect on CAG fragility, suggesting that a Chk1-mediated G2 arrest is not required for TNR repair. Absence of Mec1, Ddc2, Rad17, Rad24, or Rad53 also gives rise to increased frequency of CAG repeat contractions, indicating that components of the checkpoint machinery play an active role in the maintenance of both chromosomal integrity and repeat stability at expanded CAG sequences.     

The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system

GPR54 is a G-protein-coupled receptor that displays a high percentage of identity in the transmembrane domains with the galanin receptors. The ligand for GPR54 has been identified as a peptide derived from the KiSS-1 gene. KiSS-1 has been shown to have anti-metastatic effects, suggesting that KiSS-1 or its receptor represents a potential therapeutic target. To further our understanding of the physiological function of this receptor, we have generated a mutant mouse line with a targeted disruption of the GPR54 receptor (GPR54 -/-). The analysis of the GPR54 mutant mice revealed developmental abnormalities of both male and female genitalia and histopathological changes in tissues which normally contain sexually dimorphic features. These data suggest a role for GPR54/KiSS-1 in normal sexual development, and indicate that study of the GPR54 mutant mice may provide valuable insights into human reproductive syndromes.     

A dominant-negative mutant of human DNA helicase B blocks the onset of chromosomal DNA replication

A cDNA encoding a human ortholog of mouse DNA helicase B, which may play a role in DNA replication, has been cloned and expressed as a recombinant protein. The predicted human DNA helicase B (HDHB) protein contains conserved helicase motifs (superfamily 1) that are strikingly similar to those of bacterial recD and T4 dda proteins. The HDHB gene is expressed at low levels in liver, spleen, kidney, and brain and at higher levels in testis and thymus. Purified recombinant HDHB hydrolyzed ATP and dATP in the presence of single-stranded DNA, displayed robust 5'-3' DNA helicase activity, and interacted physically and functionally with DNA polymerase alpha-primase. HDHB proteins with mutations in the Walker A or B motif lacked ATPase and helicase activity but retained the ability to interact with DNA polymerase alpha-primase, suggesting that the mutants might be dominant over endogenous HDHB in human cells. When purified HDHB protein was microinjected into the nucleus of cells in early G(1), the mutant proteins inhibited DNA synthesis, whereas the wild type protein had no effect. Injection of wild type or mutant protein into cells at G(1)/S did not prevent DNA synthesis. The results suggest that HDHB function is required for S phase entry.     

Field-based evaluation of biopesticide impacts on native biodiversity: malagasy coleoptera and anti-locust entomopathogenic fungi

A community of 225 species of Coleoptera was used as a surrogate to evaluate nontarget effects of entomopathogenic fungi under development as biopesticides for use against the Malagasy migratory locust Locusta migratoria capito Saussure (Orthoptera: Acrididae). Evaluation of a standard chemical treatment of fenitrothion + esfenvalerate, two indigenous isolates of Metarhiziumflavoviride Gams & Roszsypol (SP3 and SP9), and an indigenous isolate of Beauveria bassiana (Balsamo) Vuillemin (SP16) against an untreated control in a replicated field trial in southern Madagascar showed that one of the isolates of M. flavoviride (SP3) and fenitrothion + esfenvalerate had distinct effects on nontarget beetle communities that were similar to each other. The other two isolates had no detectable effects compared with the untreated control. Based on an evaluation of the species affected, the similar effects of SP3 and the chemical pesticide are hypothesized to be the result of a perturbation of predator-prey relationships, with a distinct tendency to be manifested via predators. The data indicate that use of SP9 and SP16 would have minimal detrimental effects on the biodiversity of nontarget beetles, but that SP3 needs further testing.     

Variants of the CD40 ligand gene are not associated with increased susceptibility to tuberculosis in West Africa

Evidence for linkage between tuberculosis and human chromosomal region Xq26 has previously been described. The costimulatory molecule CD40 ligand, encoded by TNFSF5 and located at Xq26.3, is a promising positional candidate. Interactions between CD40 ligand and CD40 are involved in the development of humoral- and cell-mediated immunity, as well as the activation of macrophages, which are the primary host and effector cells for Mycobacterium tuberculosis. We hypothesised that common variation within TNFSF5 might affect susceptibility to tuberculosis disease and, thus, might be responsible for the observed linkage to Xq26. Sequencing 32 chromosomes from a Gambian population identified nine common polymorphisms within the coding, 3 and 5 regulatory sequences of the gene. Six single nucleotide polymorphisms (SNPs) and a 3 microsatellite were genotyped in 121 tuberculosis patients and their available parents. No association with tuberculosis was detected for these variants using a transmission disequilibrium test, although one SNP at –726 showed some evidence of association in males. This finding, however, did not replicate in a separate case control study of over 1,200 West African individuals. We conclude that common genetic variation in TNFSF5 is not likely to affect tuberculosis susceptibility in West Africa and the linkage observed in this region is not due to variation in TNFSF5.Sadly, Professor Steve Bennett passed away in March 2003     

POCUS: mining genomic sequence annotation to predict disease genes

Here we present POCUS (prioritization of candidate genes using statistics), a novel computational approach to prioritize candidate disease genes that is based on over-representation of functional annotation between loci for the same disease. We show that POCUS can provide high (up to 81-fold) enrichment of real disease genes in the candidate-gene shortlists it produces compared with the original large sets of positional candidates. In contrast to existing methods, POCUS can also suggest counterintuitive candidates.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

Multicenter evaluation of the phosphorylcholine-coated biodivYsio stent in short de novo coronary lesions: The SOPHOS study

AIMS: The BiodivYsio trade mark stent (Biocompatibles Ltd, Farnham, UK) is coated with a phosphorylcholine (PC)-containing copolymer to confer biocompatibility. The SOPHOS (Study Of PHosphorylcholine coating On Stents) study was designed to assess the safety and efficacy of this novel coronary stent and by indirect comparison to indicate equivalence with other formal stent studies. METHODS AND RESULTS: Patients with angina and a single short (#x2A7F;12 mm) de novo lesion in a native coronary artery of >/=2.75 mm diameter were included. A total of 425 patients were allocated in 24 centers. Clinical data were collected at one-, six- and nine-month follow-up. Angiography was performed before and after the stent implantation. In addition, in the first 200 patients (SOPHOS A) angiography was routinely performed at six months. The following 225 patients (SOPHOS B) were merely followed up clinically. The primary end-point of the study, the six-month MACE-rate (MACE = Major Adverse Cardiac Events) was 13.4% (two cardiac death; five Q-wave/nine non-Q-wave myocardial infarctions (MI); nine CABG and 32 target lesion revascularization (TLR), which is similar to the calculated 15% MACE-rate in comparable reference studies. Secondary end-points included among others restenosis at six months in the SOPHOS A population. The target vessel diameter was 2.98 +/- 0.48 mm. Minimal lumen diameter pre/post procedure and at follow-up was 1.00 +/- 0.32, 2.69 +/- 0.37, 1.91 +/- 0.71 mm, respectively. The binary restenosis rate (>/=50% diameter stenosis at follow-up) was 17.7%. CONCLUSION: The coronary BiodivYsio stent is safe and effective as a primary device for the treatment of native coronary artery lesions in patients with stable or unstable angina pectoris. Clinical and angiographic results are in the statistical range of equivalence with comparable studies with other current stents.     

Human herpesvirus 8 open reading frame 21 is a thymidine and thymidylate kinase of narrow substrate specificity that efficiently phosphorylates zidovudine but not ganciclovir

Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to encode a protein similar to the thymidine kinase (TK) enzyme of other herpesviruses. Expressed in mammalian cells, ORF 21 was found to have low TK activity, based on poor growth in media containing hypoxanthine-aminopterin-thymidine (HAT) and low incorporation of [(3)H]thymidine into high-molecular-weight DNA. Kinetic analysis using HHV8 TK as a purified glutathione S-transferase (GST) fusion protein showed that the enzyme has a comparatively high K(m) for thymidine (dThd) of approximately 33.2 microM. Nearly 50% of the phosphorylated product of the reaction with dThd was thymidylate. This monophosphate kinase activity was more pronounced with 3'-azido-3'-deoxythymidine (AZT), in which 78% of the reaction product was AZT diphosphate. Thymidine analogs competitively inhibited dThd phosphorylation by HHV8 TK, while 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, and corresponding analogs did not. Further competition experiments revealed that the nucleoside analog ganciclovir (GCV), at up to 1,000-fold molar excess, could not significantly inhibit dThd phosphorylation by the enzyme. In support of these data, 143B TK(-) cells expressing HHV8 TK phosphorylated GCV very poorly and were not susceptible to GCV toxicity compared to parental cells. Phosphorylation of [(3)H]GCV by a purified GST-HHV8 TK fusion protein was not detected by high-pressure liquid chromatography analysis. Structural features of HHV8 TK substrate recognition were investigated. Therapeutic implications of these findings are discussed.