Rapid localization of Gag/GagPol complexes to detergent-resistant membrane during the assembly of human immunodeficiency virus type 1

During human immunodeficiency virus type 1 (HIV-1) assembly in HIV-1-transfected COS7 cells, almost all steady-state Gag/Gag and Gag/GagPol complexes are membrane bound. However, exposure to 1% Triton X-100 gives results indicating that while all Gag/GagPol complexes remain associated with the detergent-resistant membrane (DRM), only 30% of Gag/Gag complexes are associated with the DRM. Analysis of the localization of newly synthesized Gag/Gag and Gag/GagPol to the membrane indicates that after a 10-min pulse with radioactive [(35)S]Cys-[(35)S]Met, all newly synthesized Gag/GagPol is found at the DRM. Only 30% of newly synthesized Gag/Gag moves to the membrane, and at 0 min of chase, only 38% of this membrane-bound Gag/Gag is associated with the DRM. During the first 30 min of chase, most membrane-bound Gag/Gag moves to the DRM, while between 30 and 60 min of chase, there is a significant decrease in membrane-bound Gag/Gag and Gag/GagPol. Since the localization of newly synthesized Gag/Gag to the DRM and the interaction of GagPol with Gag both depend upon Gag multimerization, the rapid localization of GagPol to the DRM probably reflects the interaction of all newly synthesized GagPol with the first newly synthesized polymeric Gag to associate with the DRM.     

The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly

Human lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNALys isoacceptors. Evidence exists that Gag alone is sufficient for the incorporation of LysRS into virions. Herein, using both in vitro and in vivo methods, we begin to map regions in Gag and LysRS that are required for this interaction. In vitro reactions between wild-type and truncated HIV-1 Gag and human LysRS were monitored using GST-tagged molecules and glutathione-agarose chromatography. Gag/LysRS interaction in vivo was detected in 293FT cells cotransfected with plasmids coding for wild-type or mutant HIV-1 Gag and LysRS, either by monitoring Gag.LysRS complexes immunoprecipitated from cell lysate with anti-LysRS or by measuring the ability of LysRS to be packaged into budded Gag viral-like particles. Based on these studies, we conclude that the Gag/LysRS interaction depends upon Gag sequences within the C-terminal domain of capsid (the last 54 amino acids) and amino acids 208-259 of LysRS. The latter domain includes the class II aminoacyl-tRNA synthetase consensus sequence known as motif 1. Both regions have been implicated in homodimerization of capsid and LysRS, respectively. Sequences falling outside these amino acid stretches can be deleted from either molecule without affecting the Gag/LysRS interaction, further supporting the observation that LysRS is incorporated into Gag viral-like particles independent of its ability to bind tRNALys.     

Analysis of skeletal muscle metabolome: Evaluation of extraction methods for targeted metabolite quantification using liquid chromatography tandem mass spectrometry

Functional metabolomics of skeletal muscle involves the simultaneous identification and quantification of a large number of metabolites. For this purpose, the extraction of metabolites from animal tissues is a crucial technical step that needs to be optimized. In this work, five extraction methods for skeletal muscle metabolome analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) were tested. Bird skeletal muscles sampled postmortem and quenched in liquid nitrogen were used. Three replicates of the same sample were extracted using the following solvent systems of varying polarity: boiling water (BW, +100 °C), cold pure methanol (CPM, −80 °C), methanol/chloroform/water (MCW, −20 °C), boiling ethanol (BE, +80 °C), and perchloric acid (PCA, −20 °C). Three injections by extraction were performed. The BW extraction showed the highest recovery of metabolites with the lowest variability (<10%) except for creatine-phosphate (creatine-P). Considering yield (area of the peaks), reproducibility, and ease, the current experiment drew a scale for the muscle metabolome extraction starting from the best to the least convenient: BW > MCW > CPM > PCA ? BE. In addition, the semiquantification of metabolites in two muscles showing different metabolic and contractile properties was carried out after BW extraction and showed expected differences in metabolite contents, thereby validating the technique for biological investigations. In conclusion, the BW extraction is recommended for analysis of skeletal muscle metabolome except for creatine-P, which was poorly recovered with this technique.     

Nascent pectin formed in Golgi apparatus of pea epicotyls by addition of uronic acids has different properties from nascent pectin at the stage of galactan elongation

Microsomal membranes were prepared from etiolated pea (Pisum sativum L.) epicotyls and used to form nascent [Uronic acid-14C]pectin. The enzyme products were characterized by selective enzymic degradation, gel permeation chromatography and analysis of cellulose binding properties. The product obtained had a molecular weight of around 40 kDa, which was significantly lower than that of nascent [Gal-14C]pectin prepared from the same tissues. It is composed mainly of polygalacturonan and perhaps also rhamnogalacturonan (RG-I). Evidence was obtained for the presence of a protein attached to the nascent [Uronic acid-14C]pectin, but it was unaffected by endoglucanase and did not bind to cellulose. Hence, no xyloglucan appeared to be attached to the nascent [Uronic acid-14C]pectin. A model is proposed in which xyloglucan is attached to nascent pectin after formation of homogalacturonan, but before the pectin leaves the Golgi apparatus.     

A protein processing filter method for bacterial identification by mass spectrometry-based proteomics

A "one-pot" alternative method for processing proteins and isolating peptide mixtures from bacterial samples is presented for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and data reduction. The conventional in-solution digestion of the protein contents of bacteria is compared to a small disposable filter unit placed inside a centrifuge vial for processing and digestion of bacterial proteins. Each processing stage allows filtration of excess reactants and unwanted byproduct while retaining the proteins. Upon addition of trypsin, the peptide mixture solution is passed through the filter while retaining the trypsin enzyme. The peptide mixture is then analyzed by LC-MS/MS with an in-house BACid algorithm for a comparison of the experimental unique peptides to a constructed proteome database of bacterial genus, specie, and strain entries. The concentration of bacteria was varied from 10 × 10(7) to 3.3 × 10(3) cfu/mL for analysis of the effect of concentration on the ability of the sample processing, LC-MS/MS, and data analysis methods to identify bacteria. The protein processing method and dilution procedure result in reliable identification of pure suspensions and mixtures at high and low bacterial concentrations.     

Gap junctions mediate STAT5-independent β-casein expression in CID-9 mammary epithelial cells

Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent β-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed β-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed β-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced β-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed β-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.     

Therapeutic vaccination with simian immunodeficiency virus (SIV)-DNA + IL-12 or IL-15 induces distinct CD8 memory subsets in SIV-infected macaques

DNA vaccination is an invaluable approach for immune therapy in that it lacks vector interference and thus permits repeated vaccination boosts. However, by themselves, DNA-based vaccines are typically poor inducers of Ag-specific immunity in humans and non-human primates. Cytokines, such as IL-12 and IL-15, have been shown to be potent adjuvants for the induction and maintenance of cellular immune responses, in particular during HIV infection. In this study, we examined the ability of therapeutic vaccination with SIV-DNA+IL-12 or IL-15 as molecular adjuvants to improve DNA vaccine potency and to enhance memory immune responses in SIV-infected macaques. Our results demonstrate that incorporating IL-12 into the vaccine induces SIV-specific CD8 effector memory T cell (T(EM)) functional responses and enhances the capacity of IFN-gamma-producing CD8 T(EM) cells to produce TNF. Lower levels of PD-1 were expressed on T cells acquiring dual function upon vaccination as compared with mono-functional CD8 T(EM) cells. Finally, a boost with SIV-DNA+IL-15 triggered most T cell memory subsets in macaques primed with either DNA-SIV or placebo but only CD8 T(EM) in macaques primed with SIV-DNA+IL-12. These results indicate that plasmid IL-12 and IL-15 cytokines represent a significant addition to enhance the ability of therapeutic DNA vaccines to induce better immunity.