Identification of novel susceptibility loci for inflammatory bowel disease on chromosomes 1p, 3q, and 4q: Evidence for epistasis between 1p and IBD1

The idiopathic inflammatory bowel diseases, Crohn’s disease (CD) and ulcerative colitis (UC), are chronic, frequently disabling diseases of the intestines. Segregation analyses, twin concordance, and ethnic differences in familial risks have established that CD and UC are complex, non-Mendelian, related genetic disorders. We performed a genome-wide screen using 377 autosomal markers, on 297 CD, UC, or mixed relative pairs from 174 families, 37% Ashkenazim. We observed evidence for linkage at 3q for all families (multipoint logarithm of the odds score (MLod) = 2.29, P = 5.7 × 10−4), with greatest significance for non-Ashkenazim Caucasians (MLod = 3.39, P = 3.92 × 10−5), and at chromosome 1p (MLod = 2.65, P = 2.4 × 10−4) for all families. In a limited subset of mixed families (containing one member with CD and another with UC), evidence for linkage was observed on chromosome 4q (MLod = 2.76, P = 1.9 × 10−4), especially among Ashkenazim. There was confirmatory evidence for a CD locus, overlapping IBD1, in the pericentromeric region of chromosome 16 (MLod = 1.69, P = 2.6 × 10−3), particularly among Ashkenazim (MLod = 1.51, P = 7.8 × 10−3); however, positive MLod scores were observed over a very broad region of chromosome 16. Furthermore, evidence for epistasis between IBD1 and chromosome 1p was observed. Thirteen additional loci demonstrated nominal (MLod > 1.0, P < 0.016) evidence for linkage. This screen provides strong evidence that there are several major susceptibility loci contributing to the genetic risk for CD and UC.

NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal α-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal α-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

Pharmacological and immunohistochemical evidence for a functional nitric oxide synthase system in rat peritoneal eosinophils

Eosinophil migration in vivo is markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (l-NAME). In this study, we investigated the existence of a NOS system in eosinophils. Our results demonstrated that rat peritoneal eosinophils strongly express both type II (30.2 ± 11.6% of counted cells) and type III (24.7 ± 7.4% of counted cells) NOS, as detected by immunohistochemistry using affinity purified mouse mAbs. Eosinophil migration in vitro was evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used were N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 × 10−8 M) and leukotriene B4 (LTB4, 10−8 M). l-NAME (but not d-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM; P < 0.05) and LTB4 (61% reduction for 1.0 mM; P < 0.05). In addition, the type II NOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P < 0.05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and LTB4- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration by l-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism.

From trial data to practical knowledge: qualitative study of how general practitioners have accessed and used evidence about statin drugs in their management of hypercholesterolaemia

Objectives To explore how general practitioners have accessed and evaluated evidence from trials on the use of statin lipid lowering drugs and incorporated this evidence into their practice. To draw out the practical implications of this study for strategies to integrate clinical evidence into general medical practice.

The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: Neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the protein huntingtin (htt). Pathogenesis in HD appears to involve the formation of ubiquitinated neuronal intranuclear inclusions containing N-terminal mutated htt, abnormal protein interactions, and the aggregate sequestration of a variety of proteins (noticeably, transcription factors). To identify novel htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library. We found a predicted WW domain protein (ZK1127.9) that interacts with N-terminal fragments of htt in two-hybrid tests. A human homologue of ZK1127.9 is CA150, a transcriptional coactivator with a N-terminal insertion that contains an imperfect (Gln-Ala)38 tract encoded by a polymorphic repeat DNA. CA150 interacted in vitro with full-length htt from lymphoblastoid cells. The expression of CA150, measured immunohistochemically, was markedly increased in human HD brain tissue compared with normal age-matched human brain tissue, and CA150 showed aggregate formation with partial colocalization to ubiquitin-positive aggregates. In 432 HD patients, the CA150 repeat length explains a small, but statistically significant, amount of the variability in the onset age. Our data suggest that abnormal expression of CA150, mediated by interaction with polyglutamine-expanded htt, may alter transcription and have a role in HD pathogenesis.

Paleoclimate and Amerindians: Evidence from stable isotopes and atmospheric circulation

Two Amerindian demographic shifts are attributed to climate change in the northwest plains of North America: at ≈11,000 calendar years before present (yr BP), Amerindian culture apparently split into foothills–mountains vs. plains biomes; and from 8,000–5,000 yr BP, scarce archaeological sites on the open plains suggest emigration during xeric “Altithermal” conditions. We reconstructed paleoclimates from stable isotopes in prehistoric bison bone and relations between weather and fractions of C4 plants in forage. Further, we developed a climate-change model that synthesized stable isotope, existing qualitative evidence (e.g., palynological, erosional), and global climate mechanisms affecting this midlatitude region. Our isotope data indicate significant warming from ≈12,400 to 11,900 yr BP, supporting climate-driven cultural separation. However, isotope evidence of apparently wet, warm conditions at 7,300 yr BP refutes emigration to avoid xeric conditions. Scarcity of archaeological sites is best explained by rapid climate fluctuations after catastrophic draining of the Laurentide Lakes, which disrupted North Atlantic Deep Water production and subsequently altered monsoonal inputs to the open plains.

Molecular and genealogical evidence for a founder effect in Fanconi anemia families of the Afrikaner population of South Africa

Fanconi anemia (FA) is a rare, genetically heterogeneous autosomal recessive disorder associated with progressive aplastic anemia, congenital abnormalities, and cancer. FA has a very high incidence in the Afrikaner population of South Africa, possibly due to a founder effect. Previously we observed allelic association between polymorphic markers flanking the FA group A gene (FANCA) and disease chromosomes in Afrikaners. We genotyped 26 FA families with microsatellite and single nucleotide polymorphic markers and detected five FANCA haplotypes. Mutation scanning of the FANCA gene revealed association of these haplotypes with four different mutations. The most common was an intragenic deletion of exons 12–31, accounting for 60% of FA chromosomes in 46 unrelated Afrikaner FA patients, while two other mutations accounted for an additional 20%. Screening for these mutations in the European populations ancestral to the Afrikaners detected one patient from the Western Ruhr region of Germany who was heterozygous for the major deletion. The mutation was associated with the same unique FANCA haplotype as in Afrikaner patients. Genealogical investigation of 12 Afrikaner families with FA revealed that all were descended from a French Huguenot couple who arrived at the Cape on June 5, 1688, whereas mutation analysis showed that the carriers of the major mutation were descendants of this same couple. The molecular and genealogical evidence is consistent with transmission of the major mutation to Western Germany and the Cape near the end of the 17th century, confirming the existence of a founder effect for FA in South Africa.

Evidence for DNA phosphate backbone alkylation and cleavage by pyrrolo[1,2-a]benzimidazoles: small molecules capable of causing base-pair-specific phosphodiester bond hydrolysis.

This report presents evidence that a reduced pyrrolo[1,2-a]benzimidazole (PBI) cleaves DNA as a result of phosphate alkylation followed by hydrolysis of the resulting phosphate triester. The base-pair specificity of the phosphate alkylation results from Hoogsteen-type hydrogen bonding of the reduced PBI in the major groove at only A.T and G.C base pairs. Alkylated phosphates were detected by 31P NMR and the cleavage products were detected by 1H NMR and HPLC. Evidence is also presented that a reduced PBI interacts with DNA in the major groove rather than in the minor groove or by intercalation.