Kinetic and inhibitor specificity of adenosine transport in guinea pig cerebral cortical synaptosomes: evidence for two nucleoside transporters

The transport of [³H]adenosine at 22°C was investigated in guinea pig cerebral cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions adenosine was not significantly metabolized during the incubation period used to determine the initial rates of adenosine transport. The dose response curves for the inhibition of adenosine transport by nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole were biphasic—approx. 50–60% of the transport activity was inhibited with IC₅₀ values of 0.7, 1 and 9 nM respectively, but the remaining activity was insensitive to concentrations as high as 1 μ M. Adenosine influx by both components was saturable (K_m values of 17 ± 3 and 68 ± 8 μ M; V_max values of 2.8 ± 0.3 and 6.1 ± 0.4 pmol/mg protein per s for NBMPR-sensitive and -insensitive components, respectively), and inhibited by other nucleosides and benzodiazepines. The two transport components also differed in their sensitivity to inhibition by other nucleosides and benzodiazepines indicating that the NBMPR-sensitive component of nucleoside transport in guinea pig synaptosomes exhibits a higher affinity than the NBMPR-insensitive component. However, both components have a broad specificity. Inhibition of adenosine transport by NBMPR was associated with high affinity binding of NBMPR to the synaptosomes (K_d 88 ± 6 pM). Binding of NBMPR to these sites was blocked by dilazep and dipyridamole with K₁ values similar to those measured for inhibiting NBMPR-sensitive adenosine influx. These results, together with previous findings using NBMPR and dipyridamole as ligand probes, suggest that there are two components of nucleoside transport in mammalian cerebral cortical synaptosomes that differ in their sensitivity to inhibition by NBMPR and other transport inhibitors.     

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline‐auxotroph yeast

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl‐CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl‐CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine‐tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity.     

Equity in clinical trials for HIV-associated cryptococcal meningitis: A systematic review of global representation and inclusion of patients and researchers

BackgroundIt is essential that clinical trial participants are representative of the population under investigation. Using HIV-associated cryptococcal meningitis (CM) as a case study, we conducted a systematic review of clinical trials to determine how inclusive and representative they were both in terms of the affected population and the involvement of local investigators.MethodsWe searched Medline, EMBASE, Cochrane, Africa-Wide, CINAHL Plus, and Web of Science. Data were extracted for 5 domains: study location and design, screening, participants, researchers, and funders. Data were summarised and compared over 3 time periods: pre-antiretroviral therapy (ART) (pre-2000), early ART (2000 to 2009), and established ART (post-2010) using chi-squared and chi-squared for trend. Comparisons were made with global disease burden estimates and a composite reference derived from observational studies.ResultsThirty-nine trials published between 1990 and 2019 were included. Earlier studies were predominantly conducted in high-income countries (HICs) and recent studies in low- and middle-income countries (LMICs). Most recent studies occurred in high CM incidence countries, but some highly affected countries have not hosted trials. The sex and ART status of participants matched those of the general CM population. Patients with reduced consciousness and those suffering a CM relapse were underrepresented. Authorship had poor representation of women (29% of all authors), particularly as first and final authors. Compared to trials conducted in HICs, trials conducted in LMICs were more likely to include female authors (32% versus 20% p = 0.014) but less likely to have authors resident in (75% versus 100%, p < 0.001) or nationals (61% versus 93%, p < 0.001) of the trial location.ConclusionsThere has been a marked shift in CM trials over the course of the HIV epidemic. Trials are primarily performed in locations and populations that reflect the burden of disease, but severe and relapse cases are underrepresented. Most CM trials now take place in LMICs, but the research is primarily funded and led by individuals and institutions from HICs.     

Molecular and genetic analyses of Tic20 homologues in Arabidopsis thaliana chloroplasts

The Tic20 protein was identified in pea (Pisum sativum) as a component of the chloroplast protein import apparatus. In Arabidopsis, there are four Tic20 homologues, termed atTic20‐I, atTic20‐IV, atTic20‐II and atTic20‐V, all with predicted topological similarity to the pea protein (psTic20). Analysis of Tic20 sequences from many species indicated that they are phylogenetically unrelated to mitochondrial Tim17‐22‐23 proteins, and that they form two evolutionarily conserved subgroups [characterized by psTic20/atTic20‐I/IV (Group 1) and atTic20‐II/V (Group 2)]. Like psTic20, all four Arabidopsis proteins have a predicted transit peptide consistent with targeting to the inner envelope. Envelope localization of each one was confirmed by analysis of YFP fusions. RT‐PCR and microarray data revealed that the four genes are expressed throughout development. To assess the functional significance of the genes, T‐DNA mutants were identified. Homozygous tic20‐I plants had an albino phenotype that correlated with abnormal chloroplast development and reduced levels of chloroplast proteins. However, knockouts for the other three genes were indistinguishable from the wild type. To test for redundancy, double and triple mutants were studied; apart from those involving tic20‐I, none was distinguishable from the wild type. The tic20‐I tic20‐II and tic20‐I tic20‐V double mutants were albino, like the corresponding tic20‐I parent. In contrast, tic20‐I tic20‐IV double homozygotes could not be identified, due to gametophytic and embryonic lethality. Redundancy between atTic20‐I and atTic20‐IV was confirmed by complementation analysis. Thus, atTic20‐I and atTic20‐IV are the major functional Tic20 isoforms in Arabidopsis, with partially overlapping roles. While the Group 2 proteins have been conserved over approximately 1.2 billion (1.2 × 10⁹) years, they are not essential for normal development.     

Structure-based mutational analysis of eIF4E in relation to sbm1 resistance to pea seed-borne mosaic virus in pea

Background

Pea encodes eukaryotic translation initiation factor eIF4E (eIF4E^S), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4E^R) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection.

Methodology/Principal Findings

To study structure-function relationships between pea eIF4E and PSbMV VPg, we obtained an X-ray structure for eIF4E^S bound to m⁷GTP. The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E. To assess indirectly the importance of key residues in binding to VPg and/or m⁷GTP, an extensive range of point mutants in eIF4E was tested for their ability to complement PSbMV multiplication in resistant pea tissues and for complementation of protein translation, and hence growth, in an eIF4E-defective yeast strain conditionally dependent upon ectopic expression of eIF4E. The mutants also dissected individual contributions from polymorphisms present in eIF4E^R and compared the impact of individual residues altered in orthologous resistance alleles from other crop species. The data showed that essential resistance determinants in eIF4E differed for different viruses although the critical region involved (possibly in VPg-binding) was conserved and partially overlapped with the m⁷GTP-binding region. This overlap resulted in coupled inhibition of virus multiplication and translation in the majority of cases, although the existence of a few mutants that uncoupled the two processes supported the view that the specific role of eIF4E in potyvirus infection may not be restricted to translation.

Conclusions/Significance

The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance. In addition to defining functional domains within the eIF4E structure, we identified eIF4E alleles with the potential to convey novel virus resistance phenotypes.