Maize ATR safeguards genome stability during kernel development to prevent early endosperm endocycle onset and cell death

The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.

Differently from Arabidopsis, ATR activity in maize plays an essential role under control growth conditions, ensuring genome stability during kernel Development.     

Patients lacking classical poor prognostic markers might also benefit from a step-down glucocorticoid bridging scheme in early rheumatoid arthritis: week 16 results from the randomized multicenter CareRA trial

IntroductionConsidering a lack of efficacy data in patients with early rheumatoid arthritis (eRA) presenting without classical markers of poor prognosis, we compared methotrexate (MTX) with or without step-down glucocorticoids in the CareRA trial.MethodsDisease-modifying antirheumatic drug–naïve patients with eRA were stratified into a low-risk group based on prognostic markers that included non-erosiveness, anti–citrullinated protein antibodies and rheumatoid factor negativity and low disease activity (Disease Activity Score in 28 joints based on C-reactive protein (DAS28(CRP)) ≤3.2). Patients were randomized to 15 mg of MTX weekly (MTX with tight step-up (MTX-TSU)) or 15 mg of MTX weekly with prednisone bridging, starting at 30 mg and tapered to 5 mg daily from week 6 (COmbinatie therapie bij Reumatoïde Artritis (COBRA Slim)). A TSU approach was applied. Outcomes assessed were DAS28(CRP)-determined remission, cumulative disease activity, Health Assessment Questionnaire (HAQ) scores and adverse events (AEs) after 16 treatment weeks.ResultsWe analyzed 43 COBRA Slim and 47 MTX-TSU patients and found that 65.1% in the COBRA Slim group and 46.8% in the MTX-TSU group reached remission (P = 0.081). Mean ± standard deviation area under the curve values of DAS28(CRP) were 13.84 ± 4.58 and 11.18 ± 4.25 for the MTX-TSU and COBRA Slim patients, respectively (P = 0.006). More COBRA Slim patients had an HAQ score of 0 (51.2% versus 23.4%, P = 0.006) at week 16. Therapy-related AEs between groups did not differ.ConclusionIn patients with low-risk eRA, MTX with step-down glucocorticoid bridging seems more efficacious than MTX step-up monotherapy, with a comparable number of AEs observed over the first 16 treatment weeks.

Trial registration

EU Clinical Trials Register Identifier: EudraCT number 2008-007225-39. Registered 5 November 2008.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0611-8) contains supplementary material, which is available to authorized users.     

Resistance of the dopamine D4 receptor to agonist-induced internalization and degradation

Dopamine receptors are G-protein-coupled receptors involved in the control of motivation, learning, and fine-tuning of motor movement, as well as modulation of neuroendocrine signalling. Stimulation of G-protein-coupled receptors normally results in attenuation of signalling through desensitization, followed by internalization and down-regulation of the receptor. These processes allow the cell to regain homeostasis after exposure to extracellular stimuli and offer protection against excessive signalling.Here, we have investigated the agonist-mediated attenuation properties of the dopamine D4 receptor.We found that several hallmarks of signal attenuation such as receptor phosphorylation, internalization and degradation showed a blunted response to agonist treatment. Moreover, we did not observe recruitment of β-arrestins upon D4 receptor stimulation. We also provide evidence for the constitutive phosphorylation of two serine residues in the third intracellular loop of the D4 receptor.These data demonstrate that, when expressed in CHO, HeLa and HEK293 cells, the human D4 receptor shows resistance to agonist-mediated internalization and down-regulation. Data from neuronal cell lines, which have been reported to show low endogenous D4 receptor expression, such as the hippocampal cell line HT22 and primary rat hippocampal cells, further support these observations.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC–MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO₂)AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mangrove forests in a peri-urban setting: the case of Mombasa (Kenya)

The structure and regeneration patterns of the peri-urban mangrove vegetation of Mombasa at Tudor creek were studied along belt transects at two forest sites of Kombeni and Tsalu. Based on the species importance values, the dominant mangrove species were Rhizophora mucronata Lam. (Rhizophoraceae) and Avicennia marina (Forssk.) Vierh. (Avicenniaceae). Lumnitzera racemosa Willd., reported in an earlier floristic survey, was not encountered. Tree density varied from 1,264 trees ha^–1 at Kombeni to 1,301 trees ha^–1 at Tsalu and mean tree height was higher at the former site compared to the latter. The size-class structure at both localities showed the numerical dominance of small trees over larger trees. The spatial distribution pattern of adults and juveniles varied greatly between sites and showed a close to uniform pattern (Morisita’s Index I _δ ≪ 1) for adult trees, but a tendency to clustered distribution (I _δ ≫ 1) for juveniles. The present paper shows that unmanaged but exploited peri-urban mangroves are structurally stressed, having enlarged canopy gaps that are characterised by spatial and temporal site heterogeneity that influences regeneration, implying longer periods for canopy closure. Diversifying uses of mangrove products and establishing reserves as no cut zones with regulated harvesting will minimise canopy gap sizes, and promote conservation practices. The proposed management strategy shall boost the ecosystem resilience to both anthropogenic and natural stressors expected in the peri-urban setting in the long run.     

Peptides induce persistent signaling from endosomes by a nutrient transceptor

The yeast Gap1 transceptor mediates amino acid activation of the protein kinase A pathway and undergoes endocytic internalization following amino acid transport. We identified three specific γ-glutamyl dipeptides that cause persistent cyclic AMP-independent activation of protein kinase A, prevent Gap1 vacuolar sorting and cause Gap1 accumulation in endosomes. To our knowledge, these are the first examples of persistent agonists of a transceptor. In yeast mutants blocked in multivesicular body sorting, L-citrulline mimicked persistent signaling, further supporting that the internalized Gap1 transceptor keeps signaling. Unexpectedly, these dipeptides were transported by Gap1 and not by the regular dipeptide transporters. Their uptake was unusually sensitive to external pH and caused transient intracellular acidification. High external pH, NHA1 deletion or V-ATPase inhibition overcame the vacuolar sorting defect. Hence, this work has identified specific dipeptides that cause enhanced proton influx through the Gap1 symporter, resulting in its defective vacuolar sorting, and independently transform it into a persistently signaling transceptor.     

Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 agonist, promotes potent systemic and mucosal responses to intranasal immunization with HIVgp140

Successful vaccine development against HIV will likely require the induction of strong, long-lasting humoral and cellular immune responses in both the systemic and mucosal compartments. Based on the known immunological linkage between the upper-respiratory and urogenital tracts, we explored the potential of nasal adjuvants to boost immunization for the induction of vaginal and systemic immune responses to gp140. Mice were immunized intranasally with HIV gp140 together with micellar and emulsion formulations of a synthetic TLR4 agonist, Glucopyranosyl Lipid Adjuvant (GLA) and responses were compared to R848, a TLR7/8 agonist, or chitosan, a non TLR adjuvant. GLA and chitosan but not R848 greatly enhanced serum immunoglobulin levels when compared to antigen alone. Both GLA and chitosan induced high IgG and IgA titers in nasal and vaginal lavage and feces. The high IgA and IgG titers in vaginal lavage were associated with high numbers of gp140-specific antibody secreting cells in the genital tract. Whilst both GLA and chitosan induced T cell responses to immunization, GLA induced a stronger Th17 response and chitosan induced a more Th2 skewed response. Our results show that GLA is a highly potent intranasal adjuvant greatly enhancing humoral and cellular immune responses, both systemically and mucosally.     

In Vivo Phosphorylation of Ser21 and Ser83 during Nutrient-induced Activation of the Yeast Protein Kinase A (PKA) Target Trehalase

The readdition of an essential nutrient to starved, fermenting cells of the yeast Saccharomyces cerevisiae triggers rapid activation of the protein kinase A (PKA) pathway. Trehalase is activated 5–10-fold within minutes and has been used as a convenient reporter for rapid activation of PKA in vivo. Although trehalase can be phosphorylated and activated by PKA in vitro, demonstration of phosphorylation during nutrient activation in vivo has been lacking. We now show, using phosphospecific antibodies, that glucose and nitrogen activation of trehalase in vivo is associated with phosphorylation of Ser²¹ and Ser⁸³. Unexpectedly, mutants with reduced PKA activity show constitutive phosphorylation despite reduced trehalase activation. The same phenotype was observed upon deletion of the catalytic subunits of yeast protein phosphatase 2A, suggesting that lower PKA activity causes reduced trehalase dephosphorylation. Hence, phosphorylation of trehalase in vivo is not sufficient for activation. Deletion of the inhibitor Dcs1 causes constitutive trehalase activation and phosphorylation. It also enhances binding of trehalase to the 14-3-3 proteins Bmh1 and Bmh2, suggesting that Dcs1 inhibits by preventing 14-3-3 binding. Deletion of Bmh1 and Bmh2 eliminates both trehalase activation and phosphorylation. Our results reveal that trehalase activation in vivo is associated with phosphorylation of typical PKA sites and thus establish the enzyme as a reliable read-out for nutrient activation of PKA in vivo.