Mutation patterns and structural correlates in human immunodeficiency virus type 1 protease following different protease inhibitor treatments

Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. Mutations at 45 of the 99 amino acid positions in the protease-including 22 not previously associated with drug resistance-were significantly associated with protease inhibitor treatment. Mutations at 17 of the remaining 99 positions were polymorphic but not associated with drug treatment. Pairs and clusters of correlated (covarying) mutations were significantly more likely to occur in treated than in untreated persons: 115 versus 23 pairs and 30 versus 2 clusters, respectively. Of the 115 statistically significant pairs of covarying residues in the treated isolates, 59 were within 8 A of each other-many more than would be expected by chance. In summary, nearly one-half of HIV-1 protease positions are under selective drug pressure, including many residues not previously associated with drug resistance. Structural factors appear to be responsible for the high frequency of covariation among many of the protease residues. The presence of mutational clusters provides insight into the complex mutational patterns required for HIV-1 protease inhibitor resistance.     

DHEA inhibits cell growth and induces apoptosis in BV-2 cells and the effects are inversely associated with glucose concentration in the medium

Dehydroepiandrosterone (DHEA), a major steroid secreted by the adrenal gland which decreases with age after adolescence, is available as a nutritional supplement. DHEA is known to have antiproliferative effects but the mechanism is unclear. In this study using BV-2 cells, a murine microglial cell line, we investigated the effect of DHEA on cell viability and the interaction between DHEA and glucose concentrations in the medium. We showed that DHEA inhibited cell viability and G6PD activity in a dose-dependent manner and that the effect of DHEA on cell viability was inversely associated with glucose concentrations in the medium, i.e. lowered glucose strongly enhanced the inhibition of cell viability by DHEA. DHEA inhibited cell growth by causing cell cycle arrest primarily in the G0--G1 phase, and the effect was more pronounced at zero glucose (no glucose added, G0) than high glucose (4.5 mg/ml of the medium, G4.5). Glucose deprivation also enhanced apoptosis induced by DHEA. At G4.5, DHEA did not induce formation of DNA ladder until it reached 200 microM. However, at G0, 100 microM DHEA was able to induce apoptosis, as evidenced by the formation of DNA ladder, elevation of histone-associated DNA fragmentation and increase in cells positively stained with annexin V-FITC and annexin V-FITC/propidium iodide. The interactions between DHEA and glucose support the contention that DHEA exerts its antiproliferative effects through alteration of glucose metabolism, possibly by inhibition of G6PD activity leading to decreased supply of ribose-5-phosphate for synthesis of DNA and RNA. Although DHEA is only antiproliferative at pharmacological levels, our results indicate that its antiproliferative effect can be enhanced by limiting the supply of glucose such as by energy restriction. In addition, the present study shows that glucose concentration is an important factor to consider when studying the antiproliferative and toxicological effects of DHEA.     

The Impact of Prior Tonsillitis and Treatment Modality on the Recurrence of Peritonsillar Abscess: A Nationwide Cohort Study

Background

Studies suggest an increased risk of peritonsillar abscess (PTA) recurrence in patients with prior tonsillitis. However, this association is inconsistent and could be confounded by different treatment modalities. This study aimed to assess the risk of recurrence among PTA patients with different degrees of prior tonsillitis and treatment modalities, and the role of tonsillectomy in current practice.

Methods

All in-patients with peritonsillar abscess between January 2001 and December 2009 were identified in a nationwide, retrospective cohort study. Recurrence was defined as the first occurrence of PTA ≧30 days from the initial PTA. Factors independently associated with recurrence were analyzed using Cox proportional hazard model after adjusting for demographic and clinical data.

Results

There were 28,837 patients, with a 5.15% recurrence rate and 4.74 years of follow-up. The recurrence rates were significantly higher among subjects with more than five prior tonsillitis or 1–4 prior tonsillitis compared to those without prior tonsillitis (adjusted hazard ratio, 2.82 [95% confidence interval, 2.39–3.33] and 1.59 [95% CI: 1.38–1.82]). The adjusted HR in patients treated with needle aspiration was 1.08 compared to those treated with incision & drainage (95% CI: 0.85–1.38). After age stratification, the adjusted HRs of more than five prior tonsillitis increased to 2.92 and 3.50 in patients aged ≦18 and 19–29 years respectively. The adjusted HR ofneedle aspiration only increased in patients ≦18 years old (aHR: 1.98 [95% CI: 0.99–3.97]). The overall tonsillectomy rate was 1.48% during our study period.

Conclusions

The risk of PTA recurrence increases with higher degrees of prior tonsillitis in all age groups and management by needle aspiration only in the pediatric population. Patients younger than 30 years old with PTA and more than five prior tonsillitis have the greatest risk of recurrence.     

IL-23 p19 Knockout Mice Exhibit Minimal Defects in Responses to Primary and Secondary Infection with Francisella tularensis LVS

Our laboratory’s investigations into mechanisms of protective immunity against Francisella tularensis Live Vaccine Strain (LVS) have uncovered mediators important in host defense against primary infection, as well as those correlated with successful vaccination. One such potential correlate was IL-12p40, a pleiotropic cytokine that promotes Th1 T cell function as part of IL-12p70. LVS-infected IL-12p40 deficient knockout (KO) mice maintain a chronic infection, but IL-12p35 KO mice clear LVS infection; thus the role that IL-12p40 plays in immunity to LVS is independent of the IL-12p70 heterodimer. IL-12p40 can also partner with IL-23p19 to create the heterodimeric cytokine IL-23. Here, we directly tested the role of IL-23 in LVS resistance, and found IL-23 to be largely dispensable for immunity to LVS following intradermal or intranasal infection. IL-23p19 KO splenocytes were fully competent in controlling intramacrophage LVS replication in an in vitro overlay assay. Further, antibody responses in IL-23p19 KO mice were similar to those of normal wild type mice after LVS infection. IL-23p19 KO mice or normal wild type mice that survived primary LVS infection survived maximal doses of LVS secondary challenge. Thus p40 has a novel role in clearance of LVS infection that is unrelated to either IL-12 or IL-23.     

Involvement of the residues of GSKIP, AxinGID, and FRATtide in their binding with GSK3β to unravel a novel C-terminal scaffold-binding region

The specificity and regulation of GSK3β are thought to involve in the docking interactions at core kinase domain because of the particular amino acid residues. Recent X-ray diffraction studies illuminated the relative binding residues on AxinGID and FRATtide for GSK3β docking and appeared that GSK3β Val267Gly (V267G) and Tyr288Phe (Y288F) could distinguish the direct interaction between AxinGID and FRATtide. In order to explore the mode that involved the binding of GSKIP to GSK3β and compare it with that of AxinGID and FRATtide, we pinpointed the binding sites of GSKIP to GSK3β through the single-point mutation of four corresponding sites within GSK3β (residues 260–300) as scaffold-binding region I (designated SBR-I_260–300). Our data showed that these three binding proteins shared similar binding sites on GSK3β. We also found that the binding of GSK3β V267G mutant to GSKIP and AxinGID, but not that of Y288F mutant (effect on FRATtide), was affected. Further, based on the simulation data, the electron-density map of GSKIPtide bore closer similarity to the map AxinGID than to that of FRATtide. Interestingly, many C-terminal helix region point-mutants of GSK3β L359P, F362A, E366K, and L367P were able to eliminate the binding with FRATtide, but not AxinGID or GSKIP. In addition, CABYR exhibited a unique mode in binding to C-terminal helix region of GSK3β. Taken together, our data revealed that in addition to the core kinase domain, SBR-I_260–300, another novel C-terminus helix region, designated SBR-II_339–383, also appeared to participate in the recognition and specificity of GSK3β in binding to other specific proteins.     

Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.     

ADI1, a methionine salvage pathway enzyme,is required for Drosophila fecundity

Background

Methionine, an essential amino acid, is required for protein synthesis and normal cell metabolism. The transmethylation pathway and methionine salvage pathway (MTA cycle) are two major pathways regulating methionine metabolism. Recently, methionine has been reported to play a key role in Drosophila fecundity.

Results

Here, we revealed that the MTA cycle plays a crucial role in Drosophila fecundity using the mutant of aci-reductone dioxygenase 1 (DADI1), an enzyme in the MTA cycle. In dietary restriction condition, the egg production of adi1 mutant flies was reduced compared to that of control flies. This fecundity defect in mutant flies was rescued by reintroduction of Dadi1 gene. Moreover, a functional homolog of human ADI1 also recovered the reproduction defect, in which the enzymatic activity of human ADI1 is required for normal fecundity. Importantly, methionine supply rescued the fecundity defect in Dadi1 mutant flies. The detailed analysis of Dadi1 mutant ovaries revealed a dramatic change in the levels of methionine metabolism. In addition, we found that three compounds namely, methionine, SAM and Methionine sulfoxide, respectively, may be required for normal fecundity.

Conclusions

In summary, these results suggest that ADI1, an MTA cycle enzyme, affects fly fecundity through the regulation of methionine metabolism.     

Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Balpha subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Balpha subunit binding without greatly affecting methylation, indicating that Balpha subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Balpha subunit but not the amount that could be coimmunoprecipitated with Aalpha subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Balpha subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Balpha subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.     

Determination of electric current distributions in animals and humans exposed to a uniform 60-Hz high-intensity electric field

The thermographic method for determining specific absorption rate (SAR) in animals and models of tissues or bodies exposed to electromagnetic fields was applied to the problem of quantifying the current distribution in homogeneous bodies of arbitrary shape exposed to 60-Hz electric fields. The 60-Hz field exposures were simulated by exposing scale models of high electrical conductivity to 57.3-MHz VHF fields of high strength in a large 3.66 × 3.66 × 2.44-m TE₁₀₁ mode resonant cavity. After exposure periods of 2–30 s, the models were quickly disassembled so that the temperature distribution (maximum value up to 7 °C) along internal cross-sectional planes of the model could be recorded thermographically. The SAR, W′, calculated from the temperature changes at any point in the scale model was used to determine the SAR, W, for a full-scale model exposed to a 60-Hz electric field of the same strength by the relation W = (60/ f² · (σ′/σ) · W′ where f′ is the model exposure frequency, σ′ is the conductivity of the scale model at the VHF exposure frequency, and σ is the conductivity of the full-scale subject at 60 Hz. The SAR was used to calculate either the electric field strength or the current density for the full-scale subject. The models were used to simulate the exposure of the full-scale subject located either in free space or in contact with a conducting ground plane. Measurements made on a number of spheroidal models with axial ratios from 1 to 10 and conductivity from 1 to 10 s/m agreed well with theoretical predictions. Maximum current densities of 200 nA/cm² predicted in the ankles of man models and 50 nA/cm² predicted in the legs of pig models exposed to 60-Hz fields at 1kV/m agreed well with independent measurements on full-scale models.     

Theoretical and experimental determination of SAR patterns for spherical tissue models in a rectangular resonant cavity

Specific absorption rates (SARs) were determined theoretically and experimentally for several spherical models of tissue exposed to electrical fields of TE101 mode in a rectangular cavity of 57.3 MHz resonant frequency. The approximate theoretical SAR can be calculated according to the Mie theory by superposition of four plane waves representing the fields excited in the cavity. The theoretical and thermographically determined SAR patterns in spheres with radii of 5, 7.5, and 10 cm and with conductivities of 0.1, 1, and 10 S/m were compared. For a sphere with radius less than 7.5 cm and conductivity less than 1 S/m, the SAR was quite uniform. When conductivity was increased to 10 S/m, the SAR patterns showed higher absorption in the periphery of the largest sphere (10-cm radius). These characteristics are important in evaluating the scaling technique of exposing a model of a human to very-high-frequency fields to obtain power absorption data in humans exposed to high-frequency or very-low-frequency fields.