Genetic variants predict plasma exposure to efavirenz and nelfinavir, and they may predict virologic failure and/or emergence of drug-resistant virus. These associations with treatment responses must be validated in other studies.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.

SummaryHelicobacter pylori strains demonstrate substantial variability in the efficiency of transformation by plasmids from Escherichia coli, and many strains are completely resistant to transformation. Among the barriers to transformation are numerous strainspecific restriction-modification systems in H. pylori. We have developed a method to protect plasmid DNA from restriction by in vitro site-specific methylation using cell-free extracts of H. pylori before transformation. In two cases, plasmid DNA treated with cell-free extracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain. Among three strains examined in detail, the transformation frequency by treated plasmid shuttle and suicide vectors was significantly increased compared with mock-treated plasmid DNA. The results indicate that the restriction barrier in H. pylori can be largely overcome by specific DNA methylation in vitro. The approach described should significantly enhance the ability to manipulate gene function in H. pylori and other organisms that have substantial restriction barriers to transformation.

The pyrBI operon of Escherichia coli K-12 encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase,-EC 2.1.3.2). Expression of this operon apparently is negatively regulated by the intracellular levels of UTP. To elucidate the regulatory mechanism in which UTP functions, the nucleotide sequence of the promoter-regulatory region of the pyrBI operon was determined and DNA fragments containing this region were transcribed in vitroQ These experiments revealed a p-independent transcriptional terminator (attenuator) located only 23 base pairs before the promoter-proximal end of the structural genes. Transcription, initiated upstream at either of two potential pyrBI promoters was efficiently ("-98%) terminated at this site, indicating that the regulation of pyrBI expression involves atten-. uation control. Additional features identified suggest a model for regulation in which the relative-rates of UTP-dependent transcription within the pyrBI leader region and coupled translation of the leader transcript control transcriptional termination at the attenuator.In Escherichia coli K-12 and closely related bacteria, de novo synthesis of UMP is catalyzed by six enzymes encoded by six unlinked pyrimidine genes and operons (1-3). The expression of these genes and operons appears to be noncoordinately regulated by pyrimidine nucleotides (4), but little is known about the regulatory mechanisms involved. The pyrBI operon encodes the catalytic (pyrB) and regulatory (pyrl) subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (ATCase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) (3). Previous in vivo studies have indicated that pyrBI expression is negatively regulated over a several hundredfold range by the levels of a uridine nucleotide (4). Recent experiments using an in vitro coupled transcription-translation system have identified UTP as the principal pyrimidine regulatory effector of this operon (5). This result and the observation that ATCase synthesis is preferentially stimulated by sublethal concentrations of inhibitors of transcriptional elongation in Salmonella typhimurium (unpublished data) suggest that the rate of UTP-dependent transcription is involved in the regulation of pyr'BI expression. In addition, ATCase synthesis was shown to be selectively inhibited in. a hisT strain of S. typhimunium (6), in which the rate of translational elongation is' slowed (ref. 7; D. Palmer and S. Artz, personal communication). This result suggests that the rates of both transcription and translation, perhaps of a leader sequence preceding the pyrBI structural genes, are involved in regulation. The regulatory mechanism could be similar to the attenuation control mechanisms of amino acid biosynthetic operons (8).In this study we determined the nucleotide sequence of the promoter-regulatory region of the pyrBI operon of E. coli K-12 and characterized in vitro transcription of DNA fragments containing this reg...

The discovery of methods for generating proteins with inherent cell membrane-translocating activity will expand our ability to study and manipulate various intracellular processes in living systems. We report a method to engineer proteins with cell-membrane permeability. After a 12-amino acid residue membrane-translocating sequence (MTS) was fused to the C-terminus of glutathione S-transferase (GST), the resultant GST-MTS fusion proteins were efficiently imported into NIH 3T3 fibroblasts and other cells. To explore the applicability of this nondestructive import method to the study of intracellular processes, a 41-kDa GST-Grb2SH2-MTS fusion protein containing the Grb2 SH2 domain was tested for its effect on the epidermal growth factor (EGF)-stimulated signaling pathway. This fusion protein entered cells, formed a complex with phosphorylated EGF receptor (EGFR), and inhibited EGF-induced EGFR-Grb2 association and mitogen-activated protein kinase activation.

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The HIV-1 virion infectivity factor (Vif) is required during viral replication to inactivate the host cell anti-viral factor, APOBEC3G (A3G). Vif binds A3G and a Cullin5-ElonginBC E3 ubiquitin ligase complex which results in the proteasomal degradation of A3G. The Vif PPLP motif (amino acids 161-164) is essential for normal Vif function because mutations in this motif reduce the infectivity of virions produced in T-cells. In this report, we demonstrate that mutation of the Vif PPLP motif reduces Vif binding to A3G without affecting its interaction with ElonginC and Cullin5. We demonstrate that the failure of the Vif mutant to bind A3G resulted in A3G incorporation into assembling virions with loss of viral infectivity.

The iceA locus of Helicobacter pylori includes one of two mutually exclusive gene families, iceA1 and iceA2. Colonization with iceA1 strains is associated with enhanced acute mucosal inflammation, and adherence to gastric epithelial cells in vitro induces expression of iceA1 but not iceA2 mRNA; however, both transcripts can be detected in vivo. The aim of this study was to determine whether differing levels of iceA transcription in vivo may contribute to disease pathogenesis. RNA from 41 H. pylori-positive gastric biopsy specimens was reverse transcribed to cDNA. Quantitative PCR was performed using biotinylated iceA1, iceA2, and 16S rRNA primers, and binding of biotinylated products to streptavidin-coated plates was detected by hybridization with a fluoresceinlabeled probe. iceA genotypes were determined by PCR and sequence analysis. All 41 samples contained detectable H. pylori 16S rRNA, with similar levels in iceA1-(n ‫؍‬ 10) and iceA2 (n ‫؍‬ 31)-colonized patients (P ‫؍‬ 0.34). Biopsy specimens from four (40%) and 19 (61%) persons colonized with iceA1 or iceA2 strains, respectively, had detectable iceA RNA. Acute inflammatory scores were significantly higher in iceA1 RNA-positive patients than in iceA1 RNA-negative, iceA2 RNA-positive, or iceA2 RNA-negative subjects (P < 0.05 for each). Within the iceA2 RNA-positive group, H. pylori strains with a single 35-amino-acid cassette were associated with significantly higher mucosal iceA2 transcript levels (P ‫؍‬ 0.014 versus strains with two cassettes). These results indicate that the levels of transcription of H. pylori iceA1 and iceA2 and of 16S rRNA are independent and that particular iceA2 gene structures are associated with enhanced transcription. The finding that iceA1 transcription levels are significantly associated with the intensity of neutrophilic infiltration suggests that heterogeneity in inflammatory scores among persons colonized with H. pylori iceA1 strains reflects levels of iceA1 transcription in vivo.Helicobacter pylori induces gastric inflammation in virtually all colonized individuals, and such gastritis increases the risk for peptic ulcer disease and distal gastric adenocarcinoma (8,14,15,21,23,27). However, only a minority of persons carrying H. pylori develop clinical sequelae, suggesting that particular bacterial products may contribute to pathogenesis (4). The first strain-specific gene identified in H. pylori was cagA, a component of the cag pathogenicity island (1, 7, 9, 30); persons colonized with H. pylori cagA-positive strains are at increased risk for developing peptic ulceration and distal gastric cancer compared to persons harboring cagA-negative strains (5, 10-12, 22, 24, 29). The gene vacA represents a second H. pylori locus of heterogeneity, and strains that possess a vacA s1 signal sequence allele are also associated with peptic ulcer disease (3, 32). However, the majority of persons colonized with cagA ϩ vacA s1 strains remain asymptomatic, suggesting that additional H. pylori genes may also be important in disease pathogenes...