Expression and homology modeling of sterol 14alpha-demethylase from Penicillium digitatum

Green mold of citrus, caused by Penicillium digitatum, is the most serious postharvest disease of citrus. Sterol 14alpha-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in biological kingdoms and is a prime target of antifungal drugs. To exploit novel 14alpha-demethylase inhibitor (DMI) fungicides, DNA and total RNA were isolated from P. digitatum. The CYP51 of P. digitatum was cloned and expressed in Escherichia coli, yielding recombinant protein with a molecular weight of c. 59 kDa. The P. digitatum CYP51 protein (PdCYP51) was purified and polyclonal antibodies were prepared. Compared with the sequence of P. digitatum PD5 in GenBank, there were four mutated nucleotides which resulted in four mutated amino acids. The three-dimensional (3D) model of P. digitatum CYP51 was established based on structure template of 1e9x.pdb and diniconazole was docked into the active site by FlexX. According to spectral data, it is suggested that the purified soluble protein had high affinity with diniconazole, a potent inhibitor of CYP51 reaction in fungi. At the same time, these spectral data suggested that the 3D model and the docking model were reasonable, which we hope can be used to provide a virtual screening of novel DMI drugs.     

Long-term depression of mGluR1 signaling

Glutamate produces both fast excitation through activation of ionotropic receptors and slower actions through metabotropic receptors (mGluRs). To date, ionotropic but not metabotropic neurotransmission has been shown to undergo long-term synaptic potentiation and depression. Burst stimulation of parallel fibers releases glutamate, which activates perisynaptic mGluR1 in the dendritic spines of cerebellar Purkinje cells. Here, we show that the mGluR1-dependent slow EPSC and its coincident Ca transient were selectively and persistently depressed by repeated climbing fiber-evoked depolarization of Purkinje cells in brain slices. LTD(mGluR1) was also observed when slow synaptic current was evoked by exogenous application of a group I mGluR agonist, implying a postsynaptic expression mechanism. Ca imaging further revealed that LTD(mGluR1) was expressed as coincident attenuation of both limbs of mGluR1 signaling: the slow EPSC and PLC/IP3-mediated dendritic Ca mobilization. Thus, different patterns of neural activity can evoke LTD of either fast ionotropic or slow mGluR1-mediated synaptic signaling.     

Changes in components, glycyrrhizin and glycyrrhetinic acid, in raw Glycyrrhiza uralensis Fisch, modify insulin sensitizing and insulinotropic actions

We hypothesized that roasted Glycyrrhizae Radix (Glycyrrhizin Radix Praeparata, GRP) might modify anti-diabetic action due to compositional changes. Then we examined the anti-diabetic effect and mechanism of raw Glycyrrhizae Radix (GR) and GRP extracts and their major respective components, glycyrrhizin and glycyrrhetinic acid. In partial pancreatectomized (Px) diabetic mice, both GR and GRP improved glucose tolerance, but only GRP enhanced glucose-stimulated insulin secretion as much as exendin-4. Both GR and GRP extracts enhanced insulin-stimulated glucose uptake through peroxisome proliferation-activated receptor (PPAR)-gamma activation in 3T3-L1 adipocytes. Consistently with the results of the mice study, only GRP and glycyrrhetinic acid enhanced glucose-stimulated insulin secretion in isolated islets. In addition, they induced mRNA levels of insulin receptor substrate-2, pancreas duodenum homeobox-1, and glucokinase in the islets, which contributed to improving beta-cell viability. In conclusion, GRP extract containing glycyrrhetinic acid improved glucose tolerance better than GR extract by enhancing insulinotropic action. Thus, GRP had better anti-diabetic action than GR.     

A CpG oligodeoxynucleotide inducing anti-coxsackie B3 virus activity in human peripheral blood mononuclear cells

Coxsackie B3 virus (CVB3) is the most significant pathogen causing myocarditis in humans, and antiviral therapy would be most effective in the early stages of the disease. Here we provide evidence that BW001, a C-type CpG oligodeoxynucleotide, induces anti-CVB3 activity in human peripheral blood mononuclear cells (PBMCs). In parallel, we have demonstrated that BW001 induces human PBMCs to express mRNAs of multiple types of interferon (IFN), including IFN-alpha, IFN-beta, IFN-omega and IFN-gamma, and to express mRNAs of at least 11 subtypes of IFN-alpha. The induced IFNs may contribute to the anti-CVB3 activity. The results suggest that BW001 could be developed into a medication with the potential to treat CVB3 infectious diseases by inducing natural mixed IFNs.