Differential Sensitivity of “Old” versus “New” APOBEC3G to Human Immunodeficiency Virus Type 1 Vif

HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inhibiting its encapsidation into virions. Here, we compared the relative sensitivity to Vif of APOBEC3G in stable HeLa cells containing APOBEC3G (HeLa-A3G cells) versus that of newly synthesized APOBEC3G. We observed that newly synthesized APOBEC3G was more sensitive to degradation than preexisting APOBEC3G. Nevertheless, preexisting and transiently expressed APOBEC3G were packaged with similar efficiencies into vif-deficient human immunodeficiency virus type 1 (HIV-1) virions, and Vif inhibited the encapsidation of both forms of APOBEC3G into HIV particles equally well. Our results suggest that HIV-1 Vif preferentially induces degradation of newly synthesized APOBEC3G but indiscriminately inhibits encapsidation of “old” and “new” APOBEC3G.     

Engineering of a wheat germ expression system to provide compatibility with a high throughput pET-based cloning platform

Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT^® SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354–359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two ¹⁵N-, ¹³C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline.     

Genetic and morphologic features for melanoma classification

Melanoma is comprised of biologically distinct subtypes. The defining clinical, histomorphologic, and molecular features are not fully established. This study sought to validate the association between genetic and histomorphologic features previously described and to determine their reproducibility and association with important clinical variables. Detailed clinical and histomorphologic features of 365 primary cutaneous melanomas were assessed by 11 pathologists and correlated with mutation status of BRAF and NRAS. There was substantial agreement in the quantitative assessment of histomorphologic features showing similar or better interobserver reproducibility than the established World Health Organization classification scheme. We confirmed that melanomas with BRAF mutations showed characteristic morphologic features (P < 0.0001) and metastasized more frequently to regional lymph nodes (P = 0.046). Importantly, melanomas without mutations were a heterogeneous group, with a subset having very similar clinical and morphological features as those with BRAF mutation raising the possibility that they are biologically related. Our study confirms an association between histomorphologic features, mutation status, and pattern of metastasis, providing criteria for a refined melanoma classification aimed at defining biologically homogeneous disease subgroups.     

Distinct signature of altered homeostasis in aging rod photoreceptors: implications for retinal diseases

Background

Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown.

Methodology/Principal Findings

To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging (1.5, 5, and 12 month old mice). We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types (oxidative phosphorylation, stress and immune response).

Conclusions/Significance

Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases.     

Direct somatic embryogenesis and plant regeneration from mature sugarbeet (Beta vulgaris L.) zygotic cotyledons

An in vitro protocol has been developed for direct somatic embryogenesis of zygotic cotyledons from mature sugarbeet (Beta vulgaris L.) embryos. Explants were sequentially cultured on modified Murashige and Skoog (MS) medium supplemented with different combinations of 2,4-D, NAA, BAP and TIBA. Somatic embryogenesis was induced within 4 weeks of culture on embryogenesis induction medium which contained MS medium supplemented with BAP and TIBA. Proliferation of somatic embryos was observed on embryo proliferation medium, which contained MS medium supplemented with BAP and NAA within 4 weeks of culture. Plants were regenerated on hormone free half; strength MS medium containing a low sucrose concentration. With some sugarbeet lines, high frequencies of plant regeneration in excess of 90percnt; were observed. The incorporation of TIBA in the media was essential for successful regeneration.     

Properties of ATP-dependent Phosphofructokinase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains

ATP-dependent phosphofructokinase (ATP:D-fructose S-phosphate, 1-phosphotransferase, EC 2.7.1.11, PFK) from endosperm of developing wheat grains was purified to apparent homogeneity with about 45% recovery using ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sepharose CL-SB and Sephadex G-200. The purified enzyme with a molecular weight of about 182 kD, was a heterotetramer with subunit molecular weights ranging between 20 and 80 kD. The enzyme exhibited maximum activity at pH 7.9 and was highly specific for its substrates. The enzyme had absolute requirement for Mg2+. At pH 7.9, the Km values as determined by Lineweaver-Burk plots were 1.43 and 0.70 mM, respectively for fru-S-P and ATP. Fru-2, S-P2 had no effect on the activity of the enzyme. The enzyme was inhibited strongly by citrate, ADP, 3-PGA and PEP with Ki values of 2.40, 1.75, 2.10 and 0.80 mM, respectively. Citrate and PEP inhibited the enzyme competitively with respect to both fru-S-P and ATP. ADP and 3-PGA inhibited the enzyme non-competitively and competitively, respectively with respect to fru-S-P and in a mixed manner with respect to ATP. Hill plot values indicated co-operative interaction of citrate, 3-PGA and PEP with the enzyme.     

A modified pedigree method of selection

Summary A dismal 81% failure of newly bulked lines against checks was observed in Indian wheat material evolved by the pedigree method. This is considered to be the consequence of selection in space planting in early generations followed by yield tests in a competitive environment which did not allow any scope of selection for competitive ability. Thus, most of the homozygous lines, which were until now raised and carried forward under space planting, failed to compete with the checks for yield in close planting. It is, therefore, necessary to modify the method to combine both the competitive ability and identity of selected plants. The proposed modified procedure involves raising the population under spaced planting as well as under drilling from F₂ onwards. Evaluation for tiller index marks an important step. On the basis of tiller index, single plants are isolated from spaced populations. Steps of the modified procedure are outlined in detail. The modified method, which economises on breeders' workload and other physical inputs, besides having other advantages, can also be used for certain other grain crops. Preliminary results based on the modified method during the past two seasons have indicated its utility.