Fluorescence-quenching screening of protein kinase C ligands with an environmentally sensitive fluorophore

A novel fluorescence-quenching screening method for protein kinase C (PKC) ligands was developed utilizing solvatochromic fluorophores. Solvatochromic dyes, highly sensitive to the presence or the absence of competitive ligands in their binding to the C1b domain of PKCδ (δC1b), were combined with a known pharmacophoric moiety of 1,2-diacylglycerol (DAG) lactones, PKC ligands. Addition of δC1b to the fluorescent compounds caused a gradual increase in the fluorescent intensity in proportion to the increase of δC1b. As a competitive ligand was added to the complex of δC1b domain and fluorescent compounds, a gradual decrease in the fluorescent intensity was observed. The relative binding affinities of known ligands were successfully determined by this fluorescent method and corresponded well to the K(i) values measured by a radioisotope method. These results indicate that washing, which is a laborious step in binding evaluations, is not required for this environmentally sensitive fluorophore based system. Screening with the system was performed for 2560 preselected library compounds with possible pharmacophores, and some lead compounds were found. This fluorescence-based method could be applied widely to known ligand-receptor combinations.     

Mutations in two non‐canonical Arabidopsis SWI2/SNF2 chromatin remodeling ATPases cause embryogenesis and stem cell maintenance defects

SWI2/SNF2 chromatin remodeling ATPases play important roles in plant and metazoan development. Whereas metazoans generally encode one or two SWI2/SNF2 ATPase genes, Arabidopsis encodes four such chromatin regulators: the well‐studied BRAHMA and SPLAYED ATPases, as well as two closely related non‐canonical SWI2/SNF2 ATPases, CHR12 and CHR23. No developmental role has as yet been described for CHR12 and CHR23. Here, we show that although strong single chr12 or chr23 mutants are morphologically indistinguishable from the wild type, chr12 chr23 double mutants cause embryonic lethality. The double mutant embryos fail to initiate root and shoot meristems, and display few and aberrant cell divisions. Weak double mutant embryos give rise to viable seedlings with dramatic defects in the maintenance of both the shoot and the root stem cell populations. Paradoxically, the stem cell defects are correlated with increased expression of the stem cell markers WUSCHEL and WOX5. During subsequent development, the meristem defects are partially overcome to allow for the formation of very small, bushy adult plants. Based on the observed morphological defects, we named the two chromatin remodelers MINUSCULE 1 and 2. Possible links between minu1 minu2 defects and defects in hormone signaling and replication‐coupled chromatin assembly are discussed.     

Enhanced Production of Virus-Inhibiting Factor (Interferon) in Human Diploid Cells by Ultraviolet Irradiation and Temperature Shift-Down after Stimulation with Newcastle Disease Virus

The production of the virus-inhibiting factor or interferon (IF) was highest in cells incubated at 37 C after inoculation with Newcastle disease (ND) virus and decreased as the incubation temperature was lowered. Shift-down of incubation temperature to 32 C or 34 C after incubation at 37 C for 4–7 hr enhanced IF production in cell cultures stimulated with ND virus, as compared with cultures incubated continuously at 37 C. Shift-down to 32 C after incubation at 37 C for 6 hr. was optimal for this enhancement of IF yield. Enhanced IF production was also observed in cell cultures irradiated by ultraviolet light 4–7 hr after stimulation with ND virus.     

Crystal structural analysis of protein-protein interactions drastically destabilized by a single mutation

The complex of barnase (bn) and barstar (bs), which has been widely studied as a model for quantitative analysis of protein-protein interactions, is significantly destabilized by a single mutation, namely, bs Asp39 --> Ala, which corresponds to a change of 7.7 kcal x mol(-1) in the free energy of binding. However, there has been no structural information available to explain such a drastic destabilization. In the present study, we determined the structure of the mutant complex at 1.58 A resolution by X-ray crystallography. The complex was similar to the wild-type complex in terms of overall and interface structures; however, the hydrogen bond network mediated by water molecules at the interface was significantly different. Several water molecules filled the cavity created by the mutation and consequently caused rearrangement of the hydrated water molecules at the interface. The water molecules were redistributed into a channel-like structure that penetrated into the complex. Furthermore, molecular dynamics simulations showed that the mutation increased the mobility of water molecules at the interface. Since such a drastic change in hydration was not observed in other mutant complexes of bn and bs, the significant destabilization of the interaction may be due to this channel-like structure of hydrated water molecules.     

Efficient injection of bull spermatozoa into oocytes using a Piezo-driven pipette

Recently, mouse and human offspring have been successfully obtained from embryos developed after intracytoplasmic sperm injection(ICSI), using a Piezo micromanipulator. In this study, the Piezo-ICSI procedure was used with in vitro matured bovine oocytes known to be difficult to fertilize microsurgically. The efficacy of Piezo-ICSI versus conventional ICSI was examined after oocytes were activated and fertilized with or without calcium ionophore (A23187) exposure. In conventional ICSI, the rate of fertilization was 19% (11/59) with A23187 and 5% (2/38) without it. However, when the Piezo-ICSI procedure was performed, the fertilization rate was 72% (47/65) with A23187 and 72% (28/39) without it. The rate of oocyte survival after microinjection was nearly similar for both methods. We suggest that the bovine oocyte is successfully activated and fertilized when an immobilized spermatozoon is injected exactly into the ooplasm through the oolemma, perforated easily by the pulsation of the Piezo. Moreover, an activating procedure such as exposure of oocytes to A23187 is not necessary, because the so-called sperm factor (oocyte activating substances) is incorporated into the ooplasm along with a spermatozoon. In this respect, the Piezo-ICSI was more efficient than the conventional ICSI method for fertilizing and thus obtaining more bovine embryos.     

Tomoregulin ectodomain shedding by proinflammatory cytokines

The shedding mechanism for the tomoregulin (TR) ectodomain, which contains two follistatin modules and a single epidermal growth factor (EGF)-like domain, remains unclear. Our study provides the first evidence that proinflammatory cytokines, IL-1beta and TNF-alpha, induce TR-ectodomain shedding in cultured A172 human glioma cells, without affecting TR mRNA expression. In addition, it appears that this shedding process is induced via activation of the NF-kappaB signaling pathway; with consequent increase in the production of metalloproteinases. Furthermore, since due to erbB4 tyrosine phosphorylation TR may have functions similar to EGF/neuregulin (NRG) family growth factors, our results suggest that following inflammation-induced injury, increases in TR shedding may contribute to tissue growth and repair in the central nervous system.     

Structural Analysis of Hen Egg Lysozyme Refolded after Denaturation at Acidic pH

The Protein Journal - Protein structures fluctuate in solution; therefore, proteins have multiple stable structures that are slightly different from each other. In this study, we determined the...