Identification of AIP as a GSK-3 binding protein

GSK-3, a well-known serine/threonine kinase is one of the key players controlling numerous cellular and physiological processes such as protein synthesis, cell poliferation, cellular differentiation, apoptosis and microtubule dynamics. Therefore, GSK-3 phosphorylates and regulates the functions of a diverse group of substrates including many transcription factors, components regulating the cell cycles and signaling proteins. However, the mechanisms by which GSK-3 regulates the functions of many substrates specifically and selectively are not known. In order to understand the molecular basis of GSK-3 regulation and specificity, we attempt to search for novel GSK-3 binding proteins using yeast two-hybrid screening. We have identified AIP (Aurora-A Kinase Interacting Protein) as a protein that interacts with GSK-3. AIP has been reported to be a novel negative regulator of Aurora-A kinase where it might down-regulates Aurora-A kinase through proteasome dependent degradation. Our study showed that AIP is able to bind both the homologous forms of GSK-3, GSK-3a and GSK-3b in intact cells. This binding is not affected by SB216763, a specific GSK-3 inhibitor, indicating that the kinase activity of GSK-3 is not required for the interaction. AIP has the consensus motif –S-X-X-X-S- for substrate phosphorylation by GSK-3b and i sphosphorylated by GSK-3b in vitro. Our results suggest that AIPis a novel binding partner of GSK-3

afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptmyces coelicolor A3(2)

AfsR is a pleiotropic, global regulator that controls the production of actinorhodin, undecylprodigiosin and calcium-dependent antibiotic in Streptomyces coelicolor A3(2). AfsR, with 993 amino acids, is phosphorylated on serine and threonine residues by a protein serine/threonine kinase AfsK and contains an OmpR-like DNA-binding fold at its N-terminal portion and A- and B-type nucleotide-binding motifs in the middle of the protein. The DNA-binding domain, in-dependently of the nucleotide-binding domain, contributed the binding of AfsR to the upstream region of afsS that locates immediately 3′ to afsR and encodes a 63-amino-acid protein. No transcription of afsS in the ΔafsR background and restoration of afsS transcription by afsR on a plasmid in the same genetic background indicated that afsR served as a transcriptional activator for afsS. Interestingly, the AfsR binding site overlapped the promoter of afsS, as determined by DNase I protection assay and high-resolution S1 nuclease mapping. The nucleotide-binding domain contributed distinct ATPase and GTPase activity. The phosphorylation of AfsR by AfsK greatly enhanced the DNA-binding activity and modulated the ATPase activity. The DNA-binding ability of AfsR was independent of the ATPase activity. However, the ATPase activity was essential for transcriptional activation ofafsS, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. Thus, AfsR turns out to be a unique transcriptional factor, in that it is modular, in which DNA-binding and ATPase activities are physically separable, and the two functions are modulated by phosphorylation on serine and threonine residues

Cloning and characterization of MADS-box gene in oil palm

Oil palm has emerged as one of the most important source of oils and fats. The mechanism of floral organs development in this plant is still at its infancy. We describe here the cloning and characterization of a MADS-box gene in oil palm (Elaeis guineensis Jacq.) named EMADS1. It belongs to the AGAMOUS-like2 family of MADS-box gene which plays critical role in flower development as defined by the ABCDE model. EMADS1 was ubiquitously expressed in the immature male and female flower buds and its expression pattern was similar to EgAGL2 and EgMADS8 of oil palm. The EMADS1 transcript also accumulated in embryos of developing seeds. These results suggested that EMADS1 is likely to function at the initial stages of flowering in determination of the inflorescence and the identity of the flower whorls and also embryo development in seeds

ALMA observations of the protostellar disk around the VeLLO IRAS 16253-2429

We present ALMA long-baseline observations toward the Class 0 protostar IRAS 16253-2429 (hereafter IRAS 16253) with a resolution down to 0.12" (~15 au). The 1.3 mm dust continuum emission has a deconvolved Gaussian size of 0.16" x 0. 07" (20 au x 8.8 au), likely tracing an inclined dusty disk. Interestingly, the position of the 1.38 mm emission is offset from that of the 0.87 mm emission along the disk minor axis. Such an offset may come from a torus-like disk with very different optical depths between these two wavelengths. Furthermore, through CO (2 - 1) and C18O (2 - 1) observations, we study rotation and infall motions in this disk-envelope system and infer the presence of a Keplerian disk with a radius of 8 - 32 au. This result suggests that the disk could have formed by directly evolving from a first core, because IRAS16253 is too young to gradually grow a disk to such a size considering the low rotation rate of its envelope. In addition, we find a quadruple pattern in the CO emission at low velocity, which may originate from CO freeze out at the disk/envelope midplane. This suggests that the "cold disk" may appear in the early stage, implying a chemical evolution for the disk around this proto-brown dwarf (or very low-mass protostar) different from that of low-mass stars.Comment: accepted for publication in ApJ, 11 pages, 6 figure

An AfsK/AfsR system involved in the response of aerial mycelium formation to glucose in Streptomyces griseus

In Streptomyces coelicolor A3(2), a protein serine/threonine kinase (AfsK) and its target protein (AfsR) control secondary metabolism. AfsK and AfsR homologues (AfsK-g and AfsR-g) from Streptomyces griseus showed high end-to-end similarity in amino acid sequence with the respective S. coelicolor A3(2) proteins, as determined by cloning and nucleotide sequencing. AfsK-g and a fusion protein between AfsK-g and thioredoxin (TRX–AfsK-g) produced in high yield as inclusion bodies in Escherichia coli were solubilized with urea, purified by column chromatography and then refolded to an active form by dialysis to gradually remove the urea. AfsR-g was also fused to glutathione S-transferase (GST–AfsR-g); the fusion product in the soluble fraction in E. coli was purified. Incubation of AfsK-g or TRX–AfsK-g in the presence of [γ-32P]ATP yielded autophosphorylated products containing phosphoserine and phosphothreonine residues. In addition, TRX–AfsK-g phosphorylated serine and threonine residues of GST–AfsR-g in the presence of [γ-32P]ATP. Disruption of chromosomal afsK-g had no effect on A-factor or streptomycin production, irrespective of the culture conditions. The afsK-g disruptants did not form aerial mycelium or spores on media containing glucose at concentrations higher than 1%, but did form spores on mannitol- and glycerol-containing media; this suggests that afsK-g is essential for morphogenesis in the presence of glucose. Introduction of afsK-g restored aerial mycelium formation in the disruptants. The phenotype of afsR-g disruptants was similar to that of afsK-g disruptants; introduction of afsR-g restored the defect in aerial mycelium formation on glucose-containing medium. Thus the AfsK/AfsR system in S. griseus is conditionally needed for morphological differentiation, whereas in S. coelicolor A3(2) it is conditionally involved in secondary metabolism

Solubility of red palm oil in supercritical carbon dioxide: measurement and modelling

The solubility of red palm oil (RPO) in supercritical carbon dioxide (scCO2) was determined using a dynamic method at 8.5–25 MPa and, 313.15–333.15 K and at a fixed scCO2 flow rate of 2.9 g·min− 1 using a full factorial design. The solubility was determined under low pressures and temperatures as a preliminary study for RPO particle formation using scCO2. The solubility of RPO was 0.5–11.3 mg·(g CO2)− 1 and was significantly affected by the pressure and temperature. RPO solubility increased with pressure and decreased with temperature. The Adachi–Lu model showed the best-fit for RPO solubility data with an average relative deviation of 14% with a high coefficient of determination, R2 of 0.9667, whereas the Peng–Robinson equation of state thermodynamic model recorded deviations of 17%–30%

Production of clavulanic acid and cehphamycin C by Stretpmyces clavuligerus in palm oil medium

Palm and palm-kernel oils and their olein and stearin fractions were suitable as the main carbon sources for growth and production of clavulanic acid by Streptomyces clavuligerus. However, oleic and lauric acids were not utilized for growth. A spontaneous mutant, which was selected for higher cephamycin C production, also produced more clavulanic acid with these oils in the medium

Production of tylosin by Streptomyces fradiae in palm oil medium

Streptomyces fradiae (NRRL 2702) produced tylosin when cultured on a synthetic defined medium M3. Palm oil, palm kernel oil and their fractions, as well as fatty acids and glycerol were investigated to serve as the major carbon source in shake flask culture. The lipids, glycerol and fatty acids, particularly palmitic acid but not oleic or lauric acid, were suitable for growth and tylosin production. For palmitic acid, at 168 h, the dry cell yield and tylosin production were 8.9 mg/ml and 0.84 mg/g cell mass respectively