Cytokinin affects circadian-clock oscillation in a phytochrome B- and Arabidopsis response regulator 4-dependent manner

In higher plants, many developmental processes, such as photomorphogenesis and flowering, are coregulated by light and the phytohormone cytokinin. Interactions between light and cytokinin pathways are presumably mediated by common signaling intermediates. However, the molecular mechanism of these interactions remains unclear. Here, we report that cytokinin specifically induces the expression of the Arabidopsis circadian oscillator genes LATE ELONGATED HYPOCOTYL ( LHY ) and CIRCADIAN CLOCK-ASSOCIATED 1 ( CCA1 ) but represses the expression of TIMING OF CAB EXPRESSION 1 in a light-dependent manner. Consistent with these observations, cytokinin causes a shifted phase of the circadian clock. Mutant studies showed that the altered clock oscillation modulated by cytokinin is dependent on phytochrome B ( PHYB ) and Arabidopsis RESPONSE REGULATOR 4 ( ARR4 ). Whereas overexpression of LHY or CCA1 renders plants slightly more sensitive to cytokinin, phyB and a lhy/cca1 double mutant are less sensitive to the hormone. These results suggest that cytokinin affects the circadian clock oscillation in a PHYB - and ARR4 -dependent manner and that cytokinin signaling is also regulated by light-signaling components, including PHYB , LHY and CCA1 . Therefore, phyB, ARR4 and the circadian oscillator may function as signaling intermediates to integrate light and cytokinin pathways.     

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.Yiwen Deng and Xudong Zhu contributed equally to this work.     

An improvement of cucumber cotyledon greening bioassay for cytokinins

The cucumber cotyledon greening bioassay for cytokinins was modified by using 95% acetone-ethanol instead of 80% acetone as extraction solvent. The cotyledons were extracted directly with a 2:1 (v/v) acetone-ethanol solution in dark for 24 hours, omitting the homogenization and centrifugation operations of the previous bioassay. The modified bioassay is more convenient and especially useful in screening cytokinin-active substance from a large number of samples.     

Nitration of tyrosine 92 mediates the activation of rat microsomal glutathione s-transferase by peroxynitrite

There is increasing evidence that protein function can be modified by nitration of tyrosine residue(s), a reaction catalyzed by proteins with peroxidase activity, or that occurs by interaction with peroxynitrite, a highly reactive oxidant formed by the reaction of nitric oxide with superoxide. Although there are numerous reports describing loss of function after treatment of proteins with peroxynitrite, we recently demonstrated that the microsomal glutathione S-transferase 1 is activated rather than inactivated by peroxynitrite and suggested that this could be attributed to nitration of tyrosine residues rather than to other effects of peroxynitrite. In this report, the nitrated tyrosine residues of peroxynitrite-treated microsomal glutathione S-transferase 1 were characterized by mass spectrometry and their functional significance determined. Of the seven tyrosine residues present in the protein, only those at positions 92 and 153 were nitrated after treatment with peroxynitrite. Three mutants (Y92F, Y153F, and Y92F, Y153F) were created using site-directed mutagenesis and expressed in LLC-PK1 cells. Treatment of the microsomal fractions of these cells with peroxynitrite resulted in an approximately 2-fold increase in enzyme activity in cells expressing the wild type microsomal glutathione S-transferase 1 or the Y153F mutant, whereas the enzyme activity of Y92F and double site mutant was unaffected. These results indicate that activation of microsomal glutathione S-transferase 1 by peroxynitrite is mediated by nitration of tyrosine residue 92 and represents one of the few examples in which a gain in function has been associated with nitration of a specific tyrosine residue.     

Synthesis and characterization of the biodegradable polycaprolactone-graft-chitosan amphiphilic copolymers

A novel synthetic approach to biodegradable amphiphilic copolymers based on poly (epsilon-caprolactone) (PCL) and chitosan was presented, and the prepared copolymers were used to prepare nanoparticles successfully. The PCL-graft-chitosan copolymers were synthesized by coupling the hydroxyl end-groups on preformed PCL chains and the amino groups present on 6-O-triphenylmethyl chitosan and by removing the protective 6-O-triphenylmethyl groups in acidic aqueous solution. The PCL content in the copolymers can be controlled in the range of 10-90 wt %. The graft copolymers were thoroughly characterized by 1H NMR, 13C NMR, FT-IR and DSC. The nanoparticles made from the graft copolymers were investigated by 1H NMR, DLS, AFM and SEM measurements. It was found that the copolymers could form spherical or elliptic nanoparticles in water. The amount of available primary amines on the surface of the prepared nanoparticles was evaluated by ninhydrin assay, and it can be controlled by the grafting degree of PCL.     

Intrinsic enoyl-CoA isomerase activity of rat acyl-CoA oxidase I

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. It was found that rat acyl-CoA oxidase I has intrinsic enoyl-CoA isomerase activity, which was confirmed using incubation followed with HPLC analysis in this study. Various 3-enoyl-CoA substrates with cis or trans configuration were synthesized and used in the study of enzyme substrate specificity. The isomerase activity of the enzyme was characterized through studies of kinetics, pH dependence, and enzyme inhibition. Most k(cat)/K(M) values of rat peroxisomal acyl-CoA oxidase I for isomerization reaction are comparable with those of authentic rat liver peroxisomal Delta(3)-Delta(2)-enoyl-CoA isomerase and rat liver peroxisomal multifunctional enzyme 1 when hexenoyl-CoA and octenoyl-CoA with cis- or trans-configuration were used as substrate. Glu421 was found to be the catalytic residue for both oxidase and isomerase activities of the enzyme. The isomerase activity of rat peroxisomal acyl-CoA oxidase I is probably due to a spontaneous process driven by thermodynamic equilibrium with formation of a conjugated structure after deprotonation of substrate alpha-proton. The energy level of transition state may be lowered by a stable dienolate intermediate, which gain further stabilization via charge transfer with electron-deficient FAD cofactor of the enzyme.     

A parallel coiled-coil tetramer with offset helices

Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between alpha helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of alpha-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valine-containing mutant forms a discrete alpha-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 A resolution crystal structure of the tetramer reveals a parallel four-stranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.     

Core structure of S2 from the human coronavirus NL63 spike glycoprotein

Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an alpha-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 degrees C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.     

Preparation of submicron unilamellar liposomes by freeze-drying double emulsions

A novel method is described for the preparation of sterile submicron unilamellar liposomes. The method is based on the lyophilization of double emulsions containing disaccharides as lyoprotectants in both the inner and outer aqueous phase. Using various phospholipids or mixtures of lipids as emulsifiers, the double emulsions can be prepared by a two-step emulsification, including hydrophilic agents in the inner aqueous phase or lipophilic agents in the oil phase. Then, the double emulsions are lyophilized after sterilization by passing them through a 0.22-microm pore filter. Rehydration of the lyophilized products results in liposomes with a relatively high encapsulation efficiency (for calcein, 87%; 5-fluorouracil, 19%; flurbiprofen, 93%) and a size below 200 nm measured by the dynamic light scattering technique (DLS) and the atomic force microscopy (AFM). The liposomes were found to be unilamellar from freeze-fracture electron micrographs and X-ray diffraction patterns. In addition, the liposomes can be reconstituted just before use by rehydration of the lyophilized products which are relatively stable. Thus, this reproducible and simple technique can be used to prepare sterilized, submicron unilamellar liposomes with a relatively high encapsulation efficiency, and excellent stability during long-term storage.     

Single-point mutations of hepatitis C virus NS3 that impair p53 interaction and anti-apoptotic activity of NS3

The N-terminal domain of NS3 of hepatitis C virus (HCV) possesses serine protease activity, which is essential for virus replication. This portion is also implicated in malignant transformation of hepatocytes. We previously demonstrated that an N-terminal portion of NS3 formed a complex with the tumor suppressor p53 and suppressed actinomycin D-induced apoptosis. We report here that single-point mutations of NS3 at position 106 from Leu to Ala (L106A), and position 43 from Phe to Ala (F43A) to a lesser extent, significantly impaired complex formation with p53. Moreover, the L106A mutation impaired an otherwise more distinct anti-apoptotic activity of NS3. F43A and L106A mutations also inhibited serine protease activity of NS3. These results collectively suggest the possibility that Leu106 and Phe43 are involved in p53 interaction and serine protease activity, and therefore, can be a good target for certain low-molecular-weight compound(s) to inhibit both oncogenic and replicative abilities of HCV.