Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115

Per os infectivity factors PIF1 (Ac119) and PIF2 (Ac022), like P74, are essential for oral infection of lepidopteran larval hosts of Autographa californica M nucleopolyhedrovirus (AcMNPV). Here we show that Ac115 also is a PIF (PIF3) and that, unlike PIF1 and PIF2, it does not mediate specific binding of AcMNPV occlusion-derived virus (ODV) to midgut target cells. We used an improved in vivo fluorescence dequenching assay to compare binding, fusion, and competition among control AcMNPV ODV and the ODVs of AcMNPV PIF1, PIF2, and PIF3 deletion mutants. Our results showed that binding and fusion of PIF1 and PIF2 mutants, but not the PIF3 mutant, were both qualitatively and quantitatively different from those of control ODV. Unlike control and PIF3-deficient ODV, an excess of PIF1- or PIF2-deficient ODV failed to compete effectively with control ODV's binding to specific receptors on midgut epithelial cells. Moreover, the levels of PIF1- and PIF2-deficient ODV binding were depressed threefold compared to control levels. Binding, fusion, and competition by PIF3-deficient ODV, however, were all indistinguishable from those of control ODV. These results implicated PIF1 and PIF2 as ODV envelope attachment proteins that mediate specific binding to primary target cells within the midgut. In contrast, PIF3 mediates another unidentified, but critical, early event during primary infection.     

Multiple myosin II heavy chain kinases: roles in filament assembly control and proper cytokinesis in Dictyostelium

Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A(-)/B(-)/C(-) triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified.     

The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology

Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.     

Microbial production of L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone by Ketogulonicigenium vulgare DSM 4025

Ketogulonicigenium vulgare DSM 4025, known as a 2-keto-L-gulonic acid producing strain from L-sorbose via L-sorbosone, surprisingly produced L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone as the substrate under a growing or resting condition. As the best result, K. vulgare DSM 4025 produced 1.37 g per liter of L-AA from 5.00 g per liter of L-sorbosone during 4 h incubation time at 30 degrees C under the resting cell condition having 5.70 g per liter of wet cells. The precursor of L-AA formation from D-sorbitol and L-sorbose, except for L-gulose, was thought to be the putative furanose form of L-sorbosone. This is the first time it is reported that bacteria can produce vitamin C via L-sorbosone.     

Novel functional biodegradable polymer II: fibroblast growth factor-2 activities of poly(gamma-glutamic acid)-sulfonate

Basic fibroblast growth factor (FGF-2) mitogenic activities of sulfonated poly(gamma-glutamic acid) (gamma-PGA-S) were investigated with chlorate-treated L929 fibroblast culture tests. When 72% of the carboxyl groups in gamma-PGA were sulfonated (gamma-PGA-S72), cell numbers reached a maximum. The activity of gamma-PGA-S72 was higher than that of gamma-PGA and synthetic heparinoids and was almost comparable to that of heparin. Cytotoxicity of gamma-PGA-S72 was not observed, regardless of the degree of sulfonation. FGF-2-protective effects of gamma-PGA-S72 against acid and thermal inactivation were also evaluated, and gamma-PGA-S72 showed higher FGF-2-protective effects in comparison to nonsulfonated gamma-PGA. The steric structures of various sulfonated gamma-PGA-Ss were analyzed by molecular modeling (molecular orbital method (MOPAC)) and indicated that gamma-PGA-Ss are helical in vacuo. Results from MOPAC and the molecular mechanics method (MM2) demonstrated that electrostatic interactions can take place between sulfonic and carboxyl groups of gamma-PGA-S and basic amino acid residues in FGF-2. gamma-PGA-S72 can interact with FGF-2 strongly.     

Crystallization and preliminary X-ray crystallographic analysis of extracellular giant hemoglobin from pogonophoran Oligobrachia mashikoi

An extracellular giant hemoglobin of Oligobrachia mashikoi, composed of 24 globins with the molecular mass of approximately 400 kDa was crystallized in its intact form. Two crystal forms were obtained by the vapor-diffusion method. Form I crystals obtained using sodium acetate as a precipitant belong to the space group P6(1)22 or P6(5)22, with unit-cell parameters a=112.41, c=621.25 A, and diffracted X-rays beyond 3.0 A resolution. Form II crystals obtained using PEG 10000 as a precipitant belong to the space group R32, with unit-cell parameters a=111.50, c=276.84 A, and diffracted X-rays beyond 2.9 A resolution. The crystals are suitable for X-ray crystallography to determine the supramacromolecular assembly of this giant hemoglobin.     

Valproic acid induces apoptosis dependent of Yca1p at concentrations that mildly affect the proliferation of yeast

Valproic acid (VPA) inhibited the growth of yeast in a dose-dependent manner with complete inhibition attained at 100 mM. When cells were exposed to 25 mM VPA, the wild-type died showing apoptotic markers, while yca1Delta deleted of YCA1 encoding yeast caspase 1 survived. On the other hand, when cells were exposed to 50 mM VPA, both the wild-type and yca1Delta died showing morphological features similar to those of the autophagic death of cdc28 which was also independent of YCA1. Thus, these results suggested that yeast cells die via YCA1-dependent apoptosis when their proliferative activity is mildly impaired.     

Electro-transfer of small interfering RNA ameliorated arthritis in rats

RNA interference provides the powerful means of sequence-specific gene silencing. Particularly, small interfering RNA (siRNA) duplexes may be potentially useful for therapeutic molecular targeting of human diseases, although novel delivery systems should be devised to achieve efficient and organ-specific transduction of siRNA. In the present study, we demonstrated that electro-transfer of a siRNA-polyamine complex made efficient and specific gene knockdown possible in the articular synovium. Targeted suppression of the tumor necrosis factor-alpha gene through this procedure significantly ameliorated collagen-induced arthritis in rats. Our results suggest the potential feasibility of therapeutic intervention with RNA medicines for treatment of rheumatoid and other locomotor diseases.