Biomedical subjects
David Wheeler
Publications and source records attributed to David Wheeler.
Analysis of regions within the bacteriophage T4 AsiA protein involved in its binding to the sigma70 subunit of E. coli RNA polymerase and its role as a transcriptional inhibitor and co-activator.
Bacteriophage T4 AsiA, a protein of 90 amino acid residues, binds to the sigma(70) subunit of Escherichia coli RNA polymerase and inhibits host or T4 early transcription or, together with the T4 MotA protein, activates T4 middle transcription. To investigate which regions within AsiA are involved in forming a complex with sigma(70) and in providing transcriptional functions we generated random mutations throughout AsiA and targeted mutations within the C-terminal region. We tested mutant proteins for their ability to complement the growth of T4 asiA am phage under non-suppressing conditions, to inhibit E. coli growth, to interact with sigma(70) region 4 in a two-hybrid assay, to bind to sigma(70) in a native protein gel, and to inhibit or activate transcription in vitro using a T4 middle promoter that is active with RNA polymerase alone, is inhibited by AsiA, and is activated by MotA/AsiA. We find that substitutions within the N-terminal half of AsiA, at amino acid residues V14, L18, and I40, rendered the protein defective for binding to sigma(70). These residues reside at the monomer-monomer interface in recent NMR structures of the AsiA dimer. In contrast, AsiA missing the C-terminal 44 amino acid residues interacted well with sigma(70) region 4 in the two-hybrid assay, and AsiA missing the C-terminal 17 amino acid residues (Delta74-90) bound to sigma(70) and was fully competent in standard in vitro transcription assays. However, the presence of the C-terminal region delayed formation of transcriptionally competent species when the AsiA/polymerase complex was pre-incubated with the promoter in the absence of MotA. Our results suggest that amino acid residues within the N-terminal half of AsiA are involved in forming or maintaining the AsiA/sigma(70) complex. The C-terminal region of AsiA, while not absolutely required for inhibition or co-activation, aids inhibition by slowing the formation of transcription complexes between a promoter and the AsiA/polymerase complex.
Protein intake is positively associated with body cell mass in weight-stable HIV-infected men.
Depletion of body cell mass (BCM) in human immunodeficiency virus (HIV)-infected patients is strongly associated with disease progression and death. Although whole-body protein turnover is increased in HIV infection, it is not known whether protein intake is independently associated with BCM. The purpose of this study was to determine the associations, if any, between protein intake and several body composition variables in 467 weight-stable, HIV-infected men with CD4 <200 cells/mm(3) enrolled in a multicenter nutritional supplementation trial. Baseline BCM, total body fat and extracellular mass as measured by bioelectrical impedance analysis, dietary intake (24 h food recall) and muscle building activity assessed by structured interview were analyzed to determine association(s) between body composition variables and macronutrient intake. Multiple regression analysis showed that BCM was positively associated with body weight (P = 0.001), height (P < 0.001), protein intake (P < 0.001), muscle-building activity (P < 0.001) and African-American ethnicity (P < 0.05) and negatively associated with carbohydrate intake (P < 0.05), age (P < 0.001) and number of prior AIDS-related diagnoses (P < 0.001). We conclude that protein intake is associated with increased BCM, whereas carbohydrate intake is negatively associated with BCM in HIV-infected men, independently of muscle building activity.
Safety and antiviral activity at 48 weeks of lopinavir/ritonavir plus nevirapine and 2 nucleoside reverse-transcriptase inhibitors in human immunodeficiency virus type 1-infected protease inhibitor-experienced patients.
The safety and antiviral activity of lopinavir (Lpv), a protease inhibitor (PI) coformulated with ritonavir (Rtv) to enhance its pharmacokinetic properties, were evaluated in 70 patients with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels of 1000-100,000 copies/mL on a first PI-containing regimen. Patients were randomized to substitute only the PI with Lpv/Rtv, 400/100 mg or 400/200 mg twice daily. On day 15, nevirapine (200 mg 2x/day) was added, and nucleoside reverse-transcriptase inhibitors were changed. Despite a >4-fold reduction in phenotypic susceptibility to the preentry PI in 63% of patients, mean plasma HIV-1 RNA levels declined by 1.14 log(10) copies/mL after 2 weeks of Lpv/Rtv. At week 48, 86% of subjects receiving treatment had plasma HIV-1 RNA levels of <400 copies/mL; 76% had levels <50 HIV-1 RNA copies/mL (intent-to-treat: 70% and 60%, respectively). Mean CD4 cell counts increased by 125 cells/muL. Three patients discontinued therapy for drug-related adverse events.