A high-density admixture scan in 1,670 African Americans with hypertension

Hypertension (HTN) is a devastating disease with a higher incidence in African Americans than European Americans, inspiring searches for genetic variants that contribute to this difference. We report the results of a large-scale admixture scan for genes contributing HTN risk, in which we screened 1,670 African Americans with HTN and 387 control individuals for regions of the genome with elevated proportion of African or European ancestry. No loci were identified that were significantly associated with HTN. We also searched for evidence of an admixture signal at 40 candidate genes and eight previously reported linkage peaks, but none appears to contribute substantially to the differential HTN risk between African and European Americans. Finally, we observed nominal association at one of the loci detected in the admixture scan of Zhu et al. 2005 (p = 0.016 at 6q24.3 correcting for four hypotheses tested), although we caution that the significance is marginal and the estimated odds ratio of 1.19 per African allele is less than what would be expected from the original report; thus, further work is needed to follow up this locus.     

In vitro metabolism of [14C]4-chlorobiphenyl and [14C]2,2',5,5'-tetrachlorobiphenyl by hepatic microsomes from rats and pigeons. Evidence against an obligatory arene oxide in aromatic hydroxylation reactions.

1. The catalytic activities of cytochromes P-450IA1 and P-450IIB1 in control and Aroclor 1254 treated rats and pigeons (1 mmol/kg) were assessed using [14C]4-chloro- and [14C]2,2',5,5'-tetrachlorobiphenyl as substrates. Treatment of rats resulted in increases of the total amount of chloroform-extractable metabolites of [14C]4-chlorobiphenyl from 37.2 (control) to 199.4 and 221.6 nmol/hr per mg microsomal protein at 48 and 120 hr post treatment. The portion of [14C]4-chloro-3',4'-dihydroxybiphenyl (M4) and of a second unidentified dihydroxylated metabolite (M3) increased during these incubations from 13.7% for controls to 53.5% at 48 hr and 69.12% at 120 hr post treatment. 2. [14C]4-chloro-3'-hydroxybiphenyl (M1) and [14C]4-chloro-4'-hydroxybiphenyl (M2) were the major metabolites formed by pigeon hepatic microsomes; however, the amounts formed were 38.7- and 29.3-fold less, respectively, than in untreated rats. Treatment of pigeons with Aroclor 1254 increased the metabolite formation from 1.0 (control) to 13.6 and 22.4 nmol/hr per mg microsomal protein at 48 hr and 120 hr post treatment respectively; however, only small amounts of metabolites M3 (0.5 nmol/hr per mg protein) and M4 (2.0 nmol/hr per mg protein) were detected. 3. Treatment of rats with Aroclor 1254 resulted in an approximately two-fold increase in the rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl, and the ratio of 3- to 4-hydroxylation increased from 0.45 (control) to 0.6 and 0.8 at 48 hr and 120 hr post treatment respectively. The rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl by control and Aroclor 1254 treated pigeons was up to 23-fold lower than in rats and there was no evidence for the formation of the diol metabolite M3. However, as with rats, the ratio of meta- to para-carbon atom hydroxylation increased from 0.58 (controls) to 0.72 at 120 hr post treatment. 4. From the evidence presented, it is suggested that cytochromes P-450IA1 and P-450IIB1 may not metabolize PCB-congeneric substrates via an obligatory arene oxide intermediate.     

Vibrio fischeri genes hvnA and hvnB encode secreted NAD(+)-glycohydrolases

HvnA and HvnB are proteins secreted by Vibrio fischeri ES114, an extracellular light organ symbiont of the squid Euprymna scolopes, that catalyze the transfer of ADP-ribose from NAD(+) to polyarginine. Based on this activity, HvnA and HvnB were presumptively designated mono-ADP-ribosyltransferases (ARTases), and it was hypothesized that they mediate bacterium-host signaling. We have cloned hvnA and hvnB from strain ES114. hvnA appears to be expressed as part of a four-gene operon, whereas hvnB is monocistronic. The predicted HvnA and HvnB amino acid sequences are 46% identical to one another and share 44% and 34% identity, respectively, with an open reading frame present in the Pseudomonas aeruginosa genome. Four lines of evidence indicate that HvnA and HvnB mediate polyarginine ADP-ribosylation not by ARTase activity, but indirectly through an NAD(+)-glycohydrolase (NADase) activity that releases free, reactive, ADP-ribose: (i) like other NADases, and in contrast to the ARTase cholera toxin, HvnA and HvnB catalyzed ribosylation of not only polyarginine but also polylysine and polyhistidine, and ribosylation was inhibited by hydroxylamine; (ii) HvnA and HvnB cleaved 1, N(6)-etheno-NAD(+) and NAD(+); (iii) incubation of HvnA and HvnB with [(32)P]NAD(+) resulted in the production of ADP-ribose; and (iv) purified HvnA displayed an NADase V(max) of 400 mol min(-1) mol(-1), which is within the range reported for other NADases and 10(2)- to 10(4)-fold higher than the minor NADase activity reported in bacterial ARTase toxins. Construction and analysis of an hvnA hvnB mutant revealed no other NADase activity in culture supernatants of V. fischeri, and this mutant initiated the light organ symbiosis and triggered regression of the light organ ciliated epithelium in a manner similar to that for the wild type.     

Mechanism of Tet repressor induction by tetracyclines: length compensates for sequence in the alpha8-alpha9 loop

Natural Tet repressor (TetR) variants are alpha-helical proteins bearing a large loop between helices 8 and 9, which is variable in sequence and length. We have deleted this loop consisting of 14 amino acid residues in TetR(D) and rebuilt it stepwise with up to 42 alanine residues. All except the mutant with the longest alanine loop show wild-type repression, but none is inducible with tetracycline. This demonstrates the importance of the alpha8-alpha9 loop and its amino acid sequence for induction. The induction efficiencies increase with loop length, when the more tightly binding inducer anhydrotetracycline is used. The largest increase of inducibility was observed for TetR mutants with loop lengths between eight and 17 alanine residues. Since loop residues Asp/Glu157 and Arg158 are conserved in the natural TetR sequence variants, we constructed a mutant in which all other residues of the loop were replaced by alanine. This mutant exhibits increased anhydrotetracycline induction compared to the corresponding alanine variant. Thus, these residues are important for induction. Binding constants for the anhydrotetracycline-TetR interaction are below the detection level of 10(5) M(-1) for the mutant with a loop of two alanine residues and increase sharply until a loop size of ten residues is reached. TetR variants with longer loops have similar anhydrotetracycline-binding constants, ranging between 2.6 x 10(9) M(-1) and 8.0 x 10(9) M(-1), about 500-fold lower than wild-type TetR. The increase of the affinity occurs at shorter loop lengths than that of inducibility. We conclude that the induction defect of the polyalanine variants arises from two increments: (i) the loop must have a minimal length-to allow efficient inducer binding; (ii) the loop must structurally participate in the conformational change associated with induction.     

DNA-(N4-cytosine)-methyltransferase from Bacillus amyloliquefaciens: kinetic and substrate binding properties

Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer oligonucleotide duplex containing the palindrome recognition site GGATCC was studied by methods of steady-state and presteady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaric aldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward a 20-mer oligonucleotide substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower then the concentrations of substrates. The kcat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s-1. The Km values for the duplex and SAM are 0.35 and 1.6 microM, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer oligonucleotide duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s-1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in the course of the enzyme single turnover, which coincides with the earlier data on EcoRI MTase. Regardless of the order of the enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme-substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding of DNA. In gel retardation experiments, there was no stoichiometrically simple complexes with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.     

DNA-(N4-Cytosine)-Methyltransferase from Bacillus amyloliquefaciens: Kinetic and Substrate-binding Properties

Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer duplex containing a palindromic recognition site GGATCC was studied by methods of steady-state and pre-steady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaraldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward the 20-mer substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower than the concentrations of substrates. The k _cat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s^–1. The K _M values for the duplex and SAM are 0.35 and 1.6 M, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s^–1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in a single turnover of the enzyme, which coincides with the earlier data on EcoRI MTase. Regardless of the order of enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme–substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding with DNA. In gel retardation experiments, there was no stoichiometrically simple complex with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.     

The nuclear dot protein sp100, characterization of domains necessary for dimerization, subcellular localization, and modification by small ubiquitin-like modifiers

The Sp100 and promyelocytic leukemia proteins (PML) are constituents of nuclear domains, known as nuclear dots (NDs) or PML bodies, and are both covalently modified by the small ubiquitin-related protein SUMO-1. NDs play a role in autoimmunity, virus infections, and in the etiology of acute promyelocytic leukemia. To date, little is known about the function of the Sp100 protein. Here we analyzed Sp100 domains that determine its subcellular localization, dimerization, and SUMOylation. A functional nuclear localization signal and an ND-targeting region that coincides with an Sp100 homodimerization domain were mapped. Sequences similar to the Sp100 homodimerization/ND-targeting region occur in several other proteins and constitute a novel protein motif, termed HSR domain. The lysine residue of the Sp100 protein, to which SUMO-1 is covalently linked, was mapped within and may therefore modulate the previously described HP1 protein-binding site. A consensus sequence for SUMOylation of proteins in general is suggested. SUMOylation strictly depended on a functional nuclear localization signal but was not necessary for nuclear import or ND targeting. A three-dimensional structure of Sp100, which supports the mapping data and provides additional information on Sp100 structure/function relationships, was generated by computer modeling. Taken together, our studies indicate the existence of well defined Sp100 domains with functions in ND targeting, nuclear import, nuclear SUMOylation, and protein-protein interaction.     

Selective Irreversible Inactivation of Replicating Mengovirus by Nucleoside Analogues: a New Form of Viral Interference

We describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (BrTu) and tubercidin (Tu). At a concentration of 5 microgram/ml, BrTu reversibly blocked the synthesis of cellular mRNA and rRNA but did not inhibit either mengovirus RNA synthesis or multiplication. BrTu is a potent inhibitor of adenosine kinase, and low concentrations of BrTu (e.g., 0.5 microgram/ml), which did not by themselves inhibit cell growth, blocked phosphorylation of Tu and thus protected uninfected cells against irreversible cytotoxicity resulting from Tu incorporation into nucleic acids. In contrast, in mengovirus-infected cells, BrTu did not completely inhibit Tu incorporation into mengovirus RNA, allowing the formation of Tu-containing functionally defective polynucleotides that aborted the virus development cycle. This increased incorporation of Tu coupled to mengovirus infection could be attributed either to a reduction in the inhibitory action of BrTu and/or its nucleotide derivatives at the level of nucleoside and nucleotide kinases and/or, perhaps, to an effect upon the nucleoside transport system. The virus life cycle in nucleoside-treated cells progressed to the point of synthesis of negative strands and probably to the production of a few defective new positive strands. Irreversible virus growth arrest was achieved if the nucleoside mixture of BrTu (0.5 to 10 microgram/ml) and Tu (1 to 20 microgram/ml) was added no later than 30 min after virus infection and maintained for periods of 2 to 8 h. The cultures thus "cured" of mengovirus infection could be maintained and transferred for several weeks, during which they neither produced detectable virus nor showed a visible cytopathic effect; however, the infected and cured cells themselves, while metabolically viable, were permanently impaired in RNA synthesis and unable to divide. Although completely resistant to superinfecting picornaviruses, they retained the ability to support the growth of several other viruses (vaccinia virus, reovirus, and vesicular stomatitis virus), showing that cured cells had, in general, retained the metabolic and structural machinery needed for virus production. The resistance of cured cells to superinfection with picornaviruses seemed attributable neither to interferon action nor to destruction or blockade of virus receptors but more likely to the consumption of some host factor(s) involved in the expression of early viral functions during the original infection.