Biological activity of a new recombinant human coagulation factor VIII and its efficacy in a small animal model.

Deficiency in human coagulation factor VIII (FVIII) causes hemophilia A (HA). Patients with HA may suffer from spontaneous bleeding, which can be life-threatening. Recombinant FVIII (rFVIII) is an established treatment and prevention agent for bleeding in patients with HA. Human plasma-derived FVIII (pdFVIII), commonly used in clinical practice, is relatively difficult to prepare. In this study, we developed a novel B-domain-deleted rFVIII, produced and formulated without the use of animal or human serum-derived components. rFVIII promoted the generation of activated factor X and downstream thrombin, and, similar to that of other available FVIII preparations, its activity was inhibited by FVIII inhibitors. In addition, rFVIII has ideal binding affinity to human von Willebrand factor. Activated FVIII (FVIIIa) could be degraded by activated protein C and lose its procoagulant activity. In vitro, commercially available recombinant FVIII (Xyntha) and pdFVIII were used as controls, and there were no statistical differences between rFVIII and commercial FVIII preparations, which demonstrates the satisfactory efficacy and potency of rFVIII. In vivo, HA mice showed that infusion of rFVIII rapidly corrected activated partial thromboplastin time, similar to Xyntha. Moreover, different batches of rFVIII were comparable. Overall, our results demonstrate the potential of rFVIII as an effective strategy for the treatment of FVIII deficiency.

CARK1 mediates ABA signaling by phosphorylation of ABA receptors.

The function of abscisic acid (ABA) is mediated by its receptors termed RCARs/PYR1/PYLs. Modulation of ABA signaling is vital for plant growth and development. The RCAR-PP2C-SnRK2 regulatory modules have been defined as the core components in ABA signaling. However, it is still not clear whether and how the ABA receptors could be modified at the initial post-translational stage to fine-tune ABA transduction pathway. Here we identify and characterize the putative receptor-like cytoplasmic kinase (RLCK) in Arabidopsis named CARK1, which interacts with RCAR3 (PYL8) and RCAR11 (PYR1) in the manner of phosphorylation. Structural studies of CARK1 revealed the critical active site, N204, which accounts for the kinase activity and the direct interaction with RCAR3/RCAR11. CARK1 phosphorylates RCAR3/RCAR11 at one conserved threonine site, T77/T78. Our genetic analyses further demonstrated that CARK1 positively regulates ABA-mediated physiological responses and overexpression of CARK1 in Arabidopsis distinctly promotes the drought resistance. Moreover, the phosphor-mimic form of RCAR11 in the cark1 mutant is able to functionally complement the ABA sensitivity. CARK1 positively regulates ABA-responsive gene expression and enhances RCAR3/RCAR11's inhibition to Clade A PP2C. Taken together, our studies strongly support the functional significance of CARK1 in positively regulating ABA signaling via phosphorylation on RCAR3/RCAR11 in Arabidopsis.

AtRAE1 is involved in degradation of ABA receptor RCAR1 and negatively regulates ABA signalling in Arabidopsis.

The phytohormone abscisic acid (ABA) plays an important role in regulating plant growth, development, and adaption to various environmental stresses. Regulatory components of ABA receptors (RCARs, also known as PYR/PYLs) sense ABA and initiate ABA signalling through inhibiting the activities of protein phosphatase 2C in Arabidopsis. However, the way in which ABA receptors are regulated is not well known. A DWD protein AtRAE1 (for RNA export factor 1 in Arabidopsis), which may act as a substrate receptor of CUL4-DDB1 E3 ligase, is an interacting partner of RCAR1/PYL9. The physical interaction between RCAR1 and AtRAE1 is confirmed in vitro and in vivo. Overexpression of AtRAE1 in Arabidopsis causes reduced sensitivity of plants to ABA, whereas suppression of AtRAE1 causes increased sensitivity to ABA. Analysis of protein stability demonstrates that RCAR1 is ubiquitinated and degraded in plant cells and AtRAE1 regulates the degradation speed of RCAR1. Our findings indicate that AtRAE1 likely participates in ABA signalling through regulating the degradation of ABA receptor RCAR1.

The Arabidopsis F-box E3 ligase RIFP1 plays a negative role in abscisic acid signalling by facilitating ABA receptor RCAR3 degradation.

The phytohormone abscisic acid (ABA) plays a vital role in plant growth and development. The function of ABA is mediated by a group of newly discovered ABA receptors, named PYRABACTIN RESISTANCE 1/PYR-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORs (PYR1/PYLs/RCARs). Here, we report that an Arabidopsis thaliana F-box protein RCAR3 INTERACTING F-BOX PROTEIN 1 (RIFP1) interacts with ABA receptor (RCAR3) and SCF E3 ligase complex subunits Arabidopsis SKP1-LIKE PROTEINs (ASKs) in vitro and in vivo. The rifp1 mutant plants displayed increased ABA-mediated inhibition of seed germination and water loss of detached leaves, while the overexpression of RIFP1 in Arabidopsis led to plants being insensitive to ABA. Meanwhile, the rifp1 mutant plants showed greater tolerance to water deficit. In addition, the RCAR3 protein level was more stable in the rifp1 mutant plants than in the wild-type plants, indicating that RIFP1 facilitates the proteasome degradation of RCAR3. Accordingly, the loss of RIFP1 increased the transcript levels of several ABA-responsive genes. Taken together, these data indicate that RIFP1 plays a negative role in the RCAR3-mediated ABA signalling pathway and likely functions as an adaptor subunit of the SCF ubiquitin ligase complex to regulate ABA receptor RCAR3 stability.

UDP-glucosyltransferase71c5, a major glucosyltransferase, mediates abscisic acid homeostasis in Arabidopsis.

Abscisic acid (ABA) plays a key role in plant growth and development. The effect of ABA in plants mainly depends on its concentration, which is determined by a balance between biosynthesis and catabolism of ABA. In this study, we characterize a unique UDP-glucosyltransferase (UGT), UGT71C5, which plays an important role in ABA homeostasis by glucosylating ABA to abscisic acid -: glucose ester (GE) in Arabidopsis (Arabidopsis thaliana). Biochemical analyses show that UGT71C5 glucosylates ABA in vitro and in vivo. Mutation of UGT71C5 and down-expression of UGT71C5 in Arabidopsis cause delay in seed germination and enhanced drought tolerance. In contrast, overexpression of UGT71C5 accelerates seed germination and reduces drought tolerance. Determination of the content of ABA and ABA-GE in Arabidopsis revealed that mutation in UGT71C5 and down-expression of UGT71C5 resulted in increased level of ABA and reduced level of ABA-GE, whereas overexpression of UGT71C5 resulted in reduced level of ABA and increased level of ABA-GE. Furthermore, altered levels of ABA in plants lead to changes in transcript abundance of ABA-responsive genes, correlating with the concentration of ABA regulated by UGT71C5 in Arabidopsis. Our work shows that UGT71C5 plays a major role in ABA glucosylation for ABA homeostasis.

Arabidopsis ABA receptor RCAR1/PYL9 interacts with an R2R3-type MYB transcription factor, AtMYB44.

Abscisic acid (ABA) signaling plays important roles in plant growth, development and adaptation to various stresses. RCAR1/PYL9 has been known as a cytoplasm and nuclear ABA receptor in Arabidopsis. To obtain further insight into the regulatory mechanism of RCAR1/PYL9, a yeast two-hybrid approach was performed to screen for RCAR1/PYL9-interacting proteins and an R2R3-type MYB transcription factor, AtMYB44, was identified. The interaction between RCAR1/PYL9 and AtMYB44 was further confirmed by glutathione S-transferase (GST) pull-down and bimolecular fluorescence complementation (BiFC) assays. Gene expression analysis showed that AtMYB44 negatively regulated the expression of ABA-responsive gene RAB18, in contrast to the opposite role reported for RCAR1/PYL9. Competitive GST pull-down assay and analysis of phosphatase activity demonstrated that AtMYB44 and ABI1 competed for binding to RCAR1/PYL9 and thereby reduced the inhibitory effect of RCAR1/PYL9 on ABI1 phosphatase activity in the presence of ABA in vitro. Furthermore, transient activation assay in protoplasts revealed AtMYB44 probably also decreased RCAR1/PYL9-mediated inhibition of ABI1 activity in vivo. Taken together, our work provides a reasonable molecular mechanism of AtMYB44 in ABA signaling.

4-[4-(3-Methoxy-benzamido)phen-oxy]-N-methyl-picolinamide.

In the title compound, C(21)H(19)N(3)O(4), the central benzene ring makes dihedral angles of 78.54 (6) and 75.30 (6)° with the pyridine and 3-methoxy-phenyl rings, respectively. An intra-molecular N-H⋯N interaction occurs, generating an S(?). The crystal packing shows inter-molecular N-H⋯O hydrogen-bonding inter-actions between the N-H groups and the O atoms of the 3-methoxy-phenyl ring and the carbonyl groups of the amide functions. Inter-molecular C-H⋯O inter-actions are also present.