Genomewide linkage analysis identifies polymorphism in the human interferon-gamma receptor affecting Helicobacter pylori infection

Helicobacter pylori is considered the most prevalent infectious agent among humans, and it causes gastric inflammation, gastroduodenal ulcers, and a risk of gastric cancer. We performed a genomewide linkage analysis among Senegalese siblings phenotyped for H. pylori-reactive serum immunoglobulin G. A multipoint LOD score of 3.1 was obtained at IFNGR1, the gene that encodes chain 1 of the interferon-gamma (IFN-gamma) receptor. Sequencing of IFNGR1 revealed -56C-->T, H318P, and L450P variants, which were found to be associated with high antibody concentrations. The inclusion of these in the linkage analysis raised the LOD score to 4.2. The variants were more prevalent in Africans than in whites. Our findings indicate that IFN-gamma signaling plays an essential role in human H. pylori infection, and they contribute to an explanation of the observations of high prevalences and relatively low pathogenicity of H. pylori in Africa. Moreover, they provide further support for the value of genomewide linkage studies in the analysis of susceptibility to infection and other complex genetic traits.     

Structural characterization of the peroxodiiron(III) intermediate generated during oxygen activation by the W48A/D84E variant of ribonucleotide reductase protein R2 from Escherichia coli

The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a mu-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by M?ssbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 A was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A). Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster.     

2,3,7,8-tetrachlorodibenzo-p-dioxin induces lecithin: retinol acyltransferase transcription in the rat kidney

Vitamin A (retinoids) has an essential role in development and throughout life of humans and animals. Consequently, effects of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on retinoid metabolism may be contributory to its toxicity. This study was performed to clarify the mechanism behind dioxin-induced retinyl ester formation in the rat kidney. In addition we investigated the possible role of CYP1A1 in dioxin-induced all-trans-retinoic acid (atRA) formation. Male Sprague-Dawley rats were exposed to a single oral dose of TCDD in a combined dose-response and time-course study, with doses ranging from 0.1 to 100 microg/kg bw and time points from 1 to 28 days. Levels of atRA and the expression of two potentially retinoic acid (RA)-controlled proteins critically involved in retinoid storage regulation, lecithin: retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP I), were analyzed in liver and kidney. The expression and activity of cytochrome P4501A1 (assayed as ethoxyresorufin-O-deethylase activity) was assessed to gain insight into its potential role in RA synthesis. There was a significant increase in LRAT mRNA expression in the kidney, whereas no such increase could be observed in the liver, despite significantly increased atRA levels in both tissues. This suggests a tissue-specific regulation of LRAT by TCDD that may be dependent on other factors than atRA. Neither CRBP I mRNA nor protein levels were altered by TCDD. The time-course relationship between CYP1A1 activity and atRA levels in liver and kidney does not exclude a role of CYP1A1 in TCDD-induced RA synthesis. The observed altered regulation of the retinoid-metabolizing enzyme LRAT, together with the low doses and short time required by TCDD to change tissue RA levels, suggest that enzymes involved in retinoid metabolism are specific and/or direct targets of TCDD.     

Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme

Heterodisulfide reductase (Hdr) from methanogenic Archaea catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Upon reaction of the oxidized enzyme with CoM-SH a unique paramagnetic species is formed, which has been shown to be due to a novel type of [4Fe-4S](3+) cluster. In this work, it was addressed whether CoM-SH is directly attached to this [4Fe-4S] cluster using CoM-(33)SH as substrate and purified Hdr from Methanothermobacter marburgensis and Methanosarcina barkeri. With both enzymes treatment with CoM-(33)SH in the presence of duroquinone as an oxidant resulted in a significant broadening of the electron paramagnetic resonance spectrum as compared to CoM-SH as substrate. The signal broadening resulted from an unresolved anisotropic hyperfine coupling between the (33)S nucleus and the paramagnetic center. The results provide compelling evidence for a direct binding of CoM-SH to the [4Fe-4S] cluster in the active site of the enzyme.     

Genomewide comparison of DNA sequences between humans and chimpanzees

A total of 8,859 DNA sequences encompassing ~1.9 million base pairs of the chimpanzee genome were sequenced and compared to corresponding human DNA sequences. Although the average sequence difference is low (1.24%), the extent of changes is markedly different among sites and types of substitutions. Whereas ~15% of all CpG sites have experienced changes between humans and chimpanzees, owing to a 23-fold excess of transitions and a 7-fold excess of transversions, substitutions at other sites vary in frequency, between 0.1% and 0.5%. If the nucleotide diversity in the common ancestral species of humans and chimpanzees is assumed to have been about fourfold higher than in contemporary humans, all possible comparisons between autosomes and X and Y chromosomes result in estimates of the ratio between male and female mutation rates of ~3. Thus, the relative time spent in the male and female germlines may be a major determinant of the overall accumulation of nucleotide substitutions. However, since the extent of divergence differs significantly among autosomes, additional unknown factors must also influence the accumulation of substitutions in the human genome.     

Ala31-Aib32: identification of the key motif for high affinity and selectivity of neuropeptide Y at the Y5-receptor

The turn-inducing sequence Ala-Aib introduced into positions 31 and 32 of neuropeptide Y (NPY) and its analogues has been identified as the key structure for Y(5)-receptor selectivity. Analogues of NPY and PP/NPY chimera containing the motif Ala-Aib were prepared; these peptides turned out to be selective for the Y(5)-receptor. The affinity of the NPY-based peptides was in the range of 6-150 nM, while the affinity of three (Ala-Aib)-containing PP/NPY chimera was in the range of 0.2-0.9 nM. The circular dichroism spectra of the Aib analogues in aqueous solution were all characteristic of an alpha helix; however, they had different intensities of the two negative bands at 220 and 208 nm. Affinity and selectivity for the Y(5)-receptor were correlated with the ratio of the ellipticity at 220 nm versus the one at 208 nm (R), which indicates the presence of a pronounced helix (R > 1) versus a less stabile one (R < 1). When R was in the range 0.74-0.96, the affinity at the Y(5)-receptor was in the range >5 nM, while there was complete loss of affinity at the Y(4)-receptor. R > 1.15 was associated with very high affinity at the Y(5)-receptor and weak affinity at the Y(4)-receptor. These results suggest that the selectivity of the Ala(31)-Aib(32) motif for the Y(5)-receptor derives from a specific conformation that must be correlated with the bioactive conformation of NPY at this subtype.