Monomeric APOBEC3G is catalytically active and has antiviral activity

APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.     

Adherent monomer-misfolded SOD1

Background

Multiple cellular functions are compromised in amyotrophic lateral sclerosis (ALS). In familial ALS (FALS) with Cu/Zn superoxide dismutase (SOD1) mutations, the mechanisms by which the mutation in SOD1 leads to such a wide range of abnormalities remains elusive.

Methodology/Principal Findings

To investigate underlying cellular conditions caused by the SOD1 mutation, we explored mutant SOD1-interacting proteins in the spinal cord of symptomatic transgenic mice expressing a mutant SOD1, SOD1^Leu126delTT with a FLAG sequence (DF mice). This gene product is structurally unable to form a functional homodimer. Tissues were obtained from both DF mice and disease-free mice expressing wild-type with FLAG SOD1 (WF mice). Both FLAG-tagged SOD1 and cross-linking proteins were enriched and subjected to a shotgun proteomic analysis. We identified 34 proteins (or protein subunits) in DF preparations, while in WF preparations, interactions were detected with only 4 proteins.

Conclusions/Significance

These results indicate that disease-causing mutant SOD1 likely leads to inadequate protein-protein interactions. This could be an early and crucial process in the pathogenesis of FALS.     

Electron-microscopic immunocytochemistry of neuropeptide Y immunoreactive innervation of vasopressin neurons in the paraventricular nucleus of the rat hypothalamus

The neuropeptide Y (NPY) immunoreactive synaptic input to neurons containing neurophysin II (NP II), the carrier protein of vasopressin (VP), was observed in the paraventricular nucleus (PVN) of the rat hypothalamus by double-labeling immunocytochemistry combining the preembedding peroxidase-antiperoxidase (PAP) method with the postembedding immunogold staining method at the electron-microscopic level. NPY-like immunoreactivities were detected by the PAP method in the dense granular vesicles (70-100 nm in diameter) in the immunoreactive presynaptic axon terminals. NP II-like immunoreactive large neurosecretory granules labeled with gold particles were found in the neurons receiving synaptic input of the NPY-like immunoreactive terminals. This suggests that NPY may be a neurotransmitter or neuromodulator and that NPY neurons may, through synaptic contacts, regulate the secretion of VP neurons.     

Performance bonuses and the quality of primary health care delivered by family health teams in Brazil: A difference-in-differences analysis

BackgroundPay-for-performance (P4P) programmes to incentivise health providers to improve quality of care have been widely implemented globally. Despite intuitive appeal, evidence on the effectiveness of P4P is mixed, potentially due to differences in how schemes are designed. We exploited municipality variation in the design features of Brazil’s National Programme for Improving Primary Care Access and Quality (PMAQ) to examine whether performance bonuses given to family health team workers were associated with changes in the quality of care and whether the size of bonus mattered.Methods and findingsFor this quasi-experimental study, we used a difference-in-differences approach combined with matching. We compared changes over time in the quality of care delivered by family health teams between (bonus) municipalities that chose to use some or all of the PMAQ money to provide performance-related bonuses to team workers with (nonbonus) municipalities that invested the funds using traditional input-based budgets. The primary outcome was the PMAQ score, a quality of care index on a scale of 0 to 100, based on several hundred indicators (ranging from 598 to 660) of health care delivery. We did one-to-one matching of bonus municipalities to nonbonus municipalities based on baseline demographic and economic characteristics. On the matched sample, we used ordinary least squares regression to estimate the association of any bonus and size of bonus with the prepost change over time (between November 2011 and October 2015) in the PMAQ score. We performed subgroup analyses with respect to the local area income of the family health team. The matched analytical sample comprised 2,346 municipalities (1,173 nonbonus municipalities; 1,173 bonus municipalities), containing 10,275 family health teams that participated in PMAQ from the outset. Bonus municipalities were associated with a 4.6 (95% CI: 2.7 to 6.4; p < 0.001) percentage point increase in the PMAQ score compared with nonbonus municipalities. The association with quality of care increased with the size of bonus: the largest bonus group saw an improvement of 8.2 percentage points (95% CI: 6.2 to 10.2; p < 0.001) compared with the control. The subgroup analysis showed that the observed improvement in performance was most pronounced in the poorest two-fifths of localities. The limitations of the study include the potential for bias from unmeasured time-varying confounding and the fact that the PMAQ score has not been validated as a measure of quality of care.ConclusionsPerformance bonuses to family health team workers compared with traditional input-based budgets were associated with an improvement in the quality of care.

Nasser Fardousi and colleagues investigate the association between performance bonuses and the quality of primary health care delivered by family health teams in Brazil.     

Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase1;1

Rice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1): these are OsGS1;1, OsGS1;2, and OsGS1;3. OsGS1;1 was expressed in all organs tested with higher expression in leaf blades, while OsGS1;2, and OsGS1;3 were expressed mainly in roots and spikelets, respectively. We characterized knockout mutants caused by insertion of endogenous retrotransposon Tos17 into the exon-8 (lines ND8037 and ND9801) or the exon-10 (line NC2327) of OsGS1;1. Mendelian segregation occurred in each progeny. Homozygously inserted mutants showed severe retardation in growth rate and grain filling when grown at normal nitrogen concentrations. Abnormal mRNA for GS1;1 was transcribed, and the GS1 protein and its activity in the leaf blades were barely detectable in these mutants. The glutamine pool in the roots and leaf blades of the mutants was lower than that of the wild type. Re-introduction of OsGS1;1 cDNA under the control of its own promoter into the mutants successfully complemented these phenotypes. Progeny where Tos17 was heterozygously inserted or deleted during segregation showed normal phenotypes. The results indicate that GS1;1 is important for normal growth and grain filling in rice; GS1;2 and GS1;3 were not able to compensate for GS1;1 function.     

Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

Regulation of the expression of the rat angiotensin II receptor mRNA.

Regulation of the expression levels of the rat angiotensin II receptor mRNA in the adrenal, aorta, kidney, and brain was assessed by the competitive polymerase chain reaction method. The bilateral nephrectomy or the administration of Dup753 markedly reduced the expression levels of this receptor mRNA in the adrenal and brain stem, but not in the kidney nor aorta. A continuous infusion of angiotensin II increased the expression level of this receptor mRNA in the adrenal but not in the other tissues. It is suggested that the expression level of this receptor mRNA in the adrenal is dependent on the renin angiotensin aldosterone system.