REDD1 Is Essential for Optimal T Cell Proliferation and Survival

REDD1 is a highly conserved stress response protein that is upregulated following many types of cellular stress, including hypoxia, DNA damage, energy stress, ER stress, and nutrient deprivation. Recently, REDD1 was shown to be involved in dexamethasone induced autophagy in murine thymocytes. However, we know little of REDD1’s function in mature T cells. Here we show for the first time that REDD1 is upregulated following T cell stimulation with PHA or CD3/CD28 beads. REDD1 knockout T cells exhibit a defect in proliferation and cell survival, although markers of activation appear normal. These findings demonstrate a previously unappreciated role for REDD1 in T cell function.     

Glycans Flanking the Hypervariable Connecting Peptide between the A and B Strands of the V1/V2 Domain of HIV-1 gp120 Confer Resistance to Antibodies That Neutralize CRF01_AE Viruses

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.     

Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts.

Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5-2.5 mM), anti-inflammatory (0.5-5.0 mM) or antiplatelet (0.18-0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0-10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575+/-3319 vs. 1437+/-348 ng ml(-1) min(-1), mean+/-SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo model.     

Structural,biochemical and biophysical characterization of four oxygen-evolving Photosystem II preparations from spinach

Four procedures utilizing different detergent and salt conditions were used to isolate oxygen-evolving Photosystem II (PS II) preparations from spinach thylakoid membranes. These PS II preparations have been characterized by freeze-fracture electron microscopy, SDS-polyacrylamide gel electrophoresis, steady-state and pulsed oxygen evolution, 77 K fluorescence, and room-temperature electron paramagnetic resonance. All of the O₂-evolving PS II samples were found to be highly purified grana membrane fractions composed of paired, appressed membrane fragments. The lumenal surfaces of the membranes and thus the O₂-evolving enzyme complex, are directly exposed to the external environment. Biochemical and biophysical analyses indicated that all four preparations are enriched in the chlorophyll $\text{a}\text{b-}\text{light-harvesting}$ complex and Photosystem II, and depleted to varying degrees in the stroma-associated components, Photosystem I and the CF₁-ATPase. The four PS II samples also varied in their cytochrome f content. All preparations showed enhanced stability of oxygen production and oxygen-rate electrode activity compared to control thylakoids, apparently promoted by low concentrations of residual detergent in the PS II preparations. A model is presented which summarizes the effects of the salt and detergent treatments on thylakoid structure and, consequently, on the configuration and composition of the oxygen-evolving PS II samples.     

Characterization of the growth and auxin physiology of roots of the tomato mutant,diageotropica

Roots of the tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([³H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [³H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.Abbreviations BCA bicinchoninic acid - IAA indole 3-acetic acid - dgt diageotropica - IC₅₀ concentration for 50% inhibition of growth - NPA N-1-naphthylphthalamic acid - SCB-1 semicarbazone 1 This research was supported by grants from Sandoz Agro, Inc. (G.K.M), the National Aeronautics and Space Administration (NASA) and the National Science Foundation (T.L.L), and NASA (D.L.R.).     

Body composition by air-displacement plethysmography by using predicted and measured thoracic gas volumes

The BOD POD, anew air-displacement plethysmograph for measuring human bodycomposition, utilizes the inverse relationship between pressure andvolume (Boyle's law) to measure body volume directly. The quantity ofair in the lungs during tidal breathing, the average thoracic gasvolume (Vtg), is also measured by the BOD POD by using a standardplethysmographic technique. Alternatively, the BOD POD provides the useof a predicted Vtg (Vtg_pred).The validity of using Vtg_pred inplace of measured Vtg (Vtg_meas)to determine the percentage of body fat (%BF) was evaluated in 50 subjects (36 women, 14 men; ages 18-56 yr). There wasno significant difference betweenVtg_meas andVtg_pred (mean difference ± SE, 53.5 ± 63.3 ml) nor in %BF by usingVtg_meas vs.Vtg_pred (0.2 ± 0.2 %BF). Onan individual basis, %BF measured by usingVtg_meas vs.Vtg_pred differed within ±2.0%BF for 82% of the subjects; maximum differences were 2.9 to+3.0% BF. For comparison, data from 24 subjects who had undergonehydrostatic weighing were evaluated for the validity of using predictedvs. measured residual lung volume(VR_pred vs.VR_meas,respectively). Differences between VR_meas andVR_pred andin %BF calculated by usingVR_meas vs.VR_pred weresignificant (187 ± 46 ml and 1.4 ± 0.3% BF, respectively; P < 0.001). On an individual basis,%BF determined by usingVR_meas vs.VR_preddiffered within ±2.0% BF for 46% of the subjects; maximum differences were 2.9 to +3.8% BF. With respect to %BF measured by air displacement, our findings support the use ofVtg_pred for group mean comparisonsand for purposes such as screening in young to middle-aged individuals.This contrasts with the use ofVR_pred inhydrostatic weighing, which leads to significant errors in theestimation of %BF. Furthermore, although the use ofVtg_pred has some application,determining Vtg_meas is relativelysimple in most cases. Therefore, we recommend that the use ofVtg_meas remain as standardexperimental and clinical practice.

     

Mesohabitat-specific macroinvertebrate assemblage responses to water quality variation in mid-continent (North America) great rivers

We compared the responsiveness of macroinvertebrate assemblages to variation in water quality (ions, nutrients, dissolved metals, and suspended sediment) in two mesohabitats within the main channel of three North American great rivers, the Upper Mississippi, Missouri, and Ohio. Based on about 400 paired samples, we examined the responsiveness of benthic assemblages sampled in the littoral zone and assemblages sampled from the surface of woody snags in the main channel. The assemblages in the two mesohabitats were different in all rivers. Taxa richness was much higher in the benthos than on snags. Macroinvertebrate assemblage response to water quality variation was weak on the Mississippi River, but the reasons for this are unknown. Based on analysis of the similarity between the composition of assemblages from groups of sites with high and low concentrations of water quality variables, benthic assemblages were only slightly more sensitive to water chemistry variation than were snag assemblages. Results of two-sample comparisons between groups of sites with high and low concentrations of water quality variables were consistent with rank correlations of assemblage metrics with water quality. In general, there was little difference between habitats in response to variation in water quality on any river. Our simple method of snag sampling in great rivers is usually much easier than littoral benthic sampling because it does not require wading. Snag sampling in large rivers has some limitations (e.g., natural snags are sometimes absent, samples are semi-quantitative), but lack of sensitivity to water quality gradients compared to the benthos is not among them.