A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation.

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry).

Improving biosynthesis of AuPd core-shell nanoparticles through Escherichia coli with the assistance of phytochelatin for catalytic enhanced chemiluminescence and benzyl alcohol oxidation.

In this work, AuPd core-shell nanoparticles (NPs) biosynthesized through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (Au-Pd/AtPCS1-E. coli) with catalytic enhanced chemiluminescence (CL) and benzyl alcohol oxidation (BAO) was investigated. Such biosynthesis of AuPd core-shell NPs was obviously enhanced due to insertion of the gene sequence of Arabidopsis thaliana phytochelatin synthase (AtPCS1) to a plasmid vector (pET-28b) of Escherichia coli (E.

Enhanced biosynthesis of CdS nanoparticles through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli with fluorescence effect in detection of pyrogallol and gallic acid.

In this work, CdS nanoparticles (CdS NPs) biosynthesized through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (CdS/AtPCS1-E. coli) with fluorescence (FL) performance for detection of pyrogallol and gallic acid was investigated. Through expression of the AtPCS1 gene inside E.

Decreased aluminium tolerance in the growth of Saccharomyces cerevisiae with SSO2 gene disruption.

Aluminium ions inhibit growth of the budding yeast Saccharomyces cerevisiae. Disruption of the SSO2 gene increased the susceptibility to aluminium. Sso2p belongs to the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family.

The Mode of Action of Cyclo(l-Ala-l-Pro) in Inhibiting Aflatoxin Production of Aspergillus flavus.

Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of was investigated. A glutathione -transferase (GST) of the fungus, designated AfGST, was identified as a binding protein of cyclo(l-Ala-l-Pro) in an experiment performed using cyclo(l-Ala-l-Pro)-immobilized Sepharose beads.

Inhibitory Activities of Alkyl Syringates and Related Compounds on Aflatoxin Production.

Inhibitors of aflatoxin production of aflatoxigenic fungi are useful for preventing aflatoxin contamination in crops. As methyl syringate weakly inhibits aflatoxin production, aflatoxin production inhibitory activities of additional alkyl syringates with alkyl chains from ethyl to octyl were examined. Inhibitory activity toward aflatoxin production of Aspergillus flavus became stronger as the length of the alkyl chains on the esters became longer.

Search for aflatoxin and trichothecene production inhibitors and analysis of their modes of action.

Mycotoxin contamination of crops is a serious problem throughout the world because of its impact on human and animal health as well as economy. Inhibitors of mycotoxin production are useful not only for developing effective methods to prevent mycotoxin contamination, but also for investigating the molecular mechanisms of secondary metabolite production by fungi. We have been searching for mycotoxin production inhibitors among natural products and investigating their modes of action.

Epidermal growth factor receptor phosphorylates protein kinase C {delta} at Tyr332 to form a trimeric complex with p66Shc in the H2O2-stimulated cells.

Protein kinase C (PKC) delta is phosphorylated at Tyr311 and Tyr332 and its catalytic activity is enhanced in the H(2)O(2)-stimulated cells, but the enzymes that recognize these tyrosine residues, especially Tyr332, have been remained to be clarified. The analysis of the endogenous proteins in COS-7 cells revealed that PKCdelta binds to p66Shc, an adaptor protein containing two phosphotyrosine-binding domains, in a manner dependent on its tyrosine phosphorylation upon H(2)O(2) stimulation. The studies using the mutated PKCdelta clarified that PKCdelta associates with p66Shc through the phosphorylated Tyr332 residue.

Biochemical assays for multiple activation states of protein kinase C.

This protocol describes biochemical procedures to monitor the activation of the protein kinase C (PKC) family using PKCdelta as the representative. The PKC family is composed of ten isoforms divided into cPKC, nPKC and aPKC groups, and their catalytic activity is regulated by multiple mechanisms. For example, PKCdelta in the nPKC group is activated by diacylglycerol as a second messenger in the receptor-coupled manner, through tyrosine phosphorylation and protein complex formation in stress-stimulated cells, and by the caspase-catalyzed cleavage during apoptosis.

Protein kinase Cdelta binds TIRAP/Mal to participate in TLR signaling.

Toll-like receptor (TLR) family members recognize specific molecular patterns within pathogens. Signaling through TLRs results in a proximal event that involves direct binding of adaptor proteins to the receptors. We observed that TIRAP/Mal, an adaptor protein for TLR2 and TLR4, binds protein kinase Cdelta (PKCdelta).

The complex formation of PKCdelta through its C1- and C2-like regions in H2O2-stimulated cells.

PKCdelta was revealed to make a homologous protein complex that shows a high protein kinase activity upon H(2)O(2) stimulation by expressing the enzymes having different epitope tags in COS-7 cells. The association of the endogenous PKCdelta in the cells was observed by sucrose density gradients. Analysis using the mutant replacing the tyrosine phosphorylation sites showed that PKCdelta is activated without tyrosine phosphorylation in the stimulated cells, and the time course of the activation was parallel with that of the complex formation.

Regulation of intracellular localization and transcriptional activity of FOXO4 by protein kinase B through phosphorylation at the motif sites conserved among the FOXO family.

FOXO4 transcription factor, also referred to AFX, contains three putative phosphorylation motif sites for protein kinase B (PKB), Thr32, Ser197, and Ser262, and it is proposed that phosphorylated FOXO4 stays in the cytosol and is imported to the nucleus through dephosphorylation to induce target gene expression. These three sites were revealed to be phosphorylated by PKB in vitro on phosphopeptide analysis, and in cultured cells on immunoblotting with phosphorylation-site specific antibodies. The mutants with either Thr32 or Ser197 replaced by Ala were found mostly in the nuclear but not the cytosol fraction, and treatment with platelet-derived growth factor did not change their distributions in the cells.

Distinct activation mechanisms of protein kinase B by growth-factor stimulation and heat-shock treatment.

Protein kinase B (PKB) alpha, having the pleckstrin homology (PH) and catalytic domains in its amino- and carboxyl-terminal regions, respectively, is activated in the signaling pathway of growth factors as a downstream target of phosphatidylinositol 3-kinase and becomes an active form in heat-shocked cells in a manner independent of the lipid kinase. Therefore, the activation mechanisms of PKBalpha were compared in platelet-derived growth factor (PDGF)-stimulated and heat-shocked cells by monitoring the protein kinase activity and phosphorylation of the mutant molecules expressed in COS-7 cells. In heat-shocked cells, PKBalpha was activated to a certain level without phosphorylation on Thr-308 in the activation loop and on Thr-450 and Ser-473 in the carboxyl-terminal end region, which is critical for growth-factor-induced activation of PKBalpha.

Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cdelta in the Golgi complex.

Protein kinase C (PKC), a Ca(2+)/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCdelta in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events.

Protein kinase C delta (PKC delta): activation mechanisms and functions.

Protein kinase C (PKC)delta was the first new/novel PKC isoform to be identified by the screening of mammalian cDNA libraries, based on the structural homology of its nucleotide sequences with those of classical/conventional PKC isoforms. PKC delta is expressed ubiquitously among cells and tissues. It is activated by diacylglycerol produced by receptor-mediated hydrolysis of membrane inositol phospholipids as well as by tumor-promoting phorbol ester through the binding of these compounds to the C1 region in its regulatory domain.