Synergistic induction of ornithine decarboxylase by diacylglycerol, A23187, and cholera toxin in guinea pig lymphocytes

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetyl-glycerol (OAG), A23187, and cholera toxin, ornithine decarboxylase activity was induced synergistically, peaking at 6 h. Addition of 12-O-tetradecanoyl-phorbol 13-acetate (TPA), A23187, and dibutyryl cAMP caused the same kind of induction. Cholera toxin potentiated the ability of A23187 to induce ornithine decarboxylase, but not that of OAG. Dibutyryl cAMP augmented the induction caused by A23187 but not by TPA. These results suggest that both the activation of Ca++-sensitive, phospholipid-dependent protein kinase (protein kinase C) and the increase in intracellular levels of Ca++ and cAMP are necessary for this induction. cAMP may potentiate the induction by modulating a Ca++ messenger system other than that for protein kinase C activation.     

Peptidomimetic modification improves cell permeation of bivalent farnesyltransferase inhibitors

Bivalent enzyme inhibitors, in which a surface binding module is linked to an active site binding module through a spacer, are a robust approach for site-selectively delivering a minimally-sized agent to a protein surface to regulate its functions, such as protein–protein interactions (PPIs). Previous research revealed that these agents effectively disrupt the interaction between farnesyltransferase (FTase) and the C-terminal region of K-Ras4B protein. However, the whole cell activity of these peptide-based agents is limited due to their low membrane permeability. In this study, we tested a peptidomimetic modification of these bivalent agents using a previously developed inhibitor, FTI-249, and evaluated their cell permeability and biological activity in cells. Confocal cell imaging using fluorescently-labeled agents showed that the peptidomimetic 3-BODIPY penetrated cells, while the peptide-based 1-BODIPY did not. Cell-based evaluation demonstrated that peptidomimetic 3 at a concentration of 100 μM inhibited HDJ-2 processing in cells, indicating that this peptidomimetic modification improves cell permeability, thus leading to enhanced whole cell activity of the bivalent compounds.     

Mutational analysis of the length of the J3/4 domain of Escherichia coli ribonuclease P ribozyme

We prepared a series of length variants of the J3/4 domain of Escherichia coli ribonuclease P (RNase P) ribozyme: the four-base long J3/4 domain (A(62)G(63)G(64)A(65)) was replaced with GGA (denoted DeltaA), GA (DeltaAG), A (DeltaAGG), AAGGA (SigmaA), AAAGGA (SigmaAA), and AAAAGGA (SigmaAAA). The results indicated that truncating and inserting operations of the J3/4 domain drastically reduced ribozyme activity (WT>SigmaAA>SigmaA>SigmaAAA>DeltaAG>DeltaA, DeltaAGG), but did not affect the cleavage site selection of a substrate by the ribozyme. The reduced ribozyme activity of each mutant was rescued to some extent by the addition of a high concentration of magnesium ions. Our data indicate that the conserved AGGA sequence was important for efficient ribozyme reactions, and suggested that the length mutations affected ribozyme activity through metal ion binding steps.     

The effects of aggregation-inducing motifs on amyloid formation of model proteins related to neurodegenerative diseases

To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.     

Effects of sugar concentration on recombinant human alpha(1)-antitrypsin production by genetically engineered rice cell

Productivity of recombinant human alpha(1)-antitrypsin (rAAT) with a genetically engineered rice cell using an inducible promoter has been studied by batch-wise and continuous production. A simple model explained the effect of proteases released from the disrupted cells on the rAAT degradation. Glucose concentration in the medium significantly affected the rAAT productivity in the continuous production, because the rAAT was induced by sugar depletion. When the fresh medium containing 5mM glucose was supplied to the continuous bioreactor, induction time was long and the productivity was low, indicating that the glucose concentration in the cells was high enough as to repress the promoter. When the glucose concentration in the fresh medium was reduced to 0.5mM, total amount of rAAT produced in 70h cultivation reached 6.7-7.6mg/g-dry cell, which was two times larger than the control medium without glucose.     

“Sensory Switching” in Elbow Reconstruction

In the treatment of the soft tissue defect of the elbow, flap reconstruction is necessitated in many cases because of thinness of soft tissue at this region. In addition, reacquirement of tactile sensation is desirable because of the anatomical and specific functions of the elbow. Of three cases treated for elbow defects, one was reconstructed with a pedicled island forearm flap containing the lateral cutaneous nerve of the forearm, another was reconstructed with a venoneuro-accompanying artery fasciocutaneous flap (VNAF flap) containing the basilic vein, and the third with the VNAF flap containing the cephalic vein. The three cases demonstrated a sudden change of sensory territory 4 to 6 months after surgery, which was confirmed by touching the reconstructed region with patients'' eye-closed: from its original territory to the elbow in a “switching”-like action. Here we describe and discuss the concept of “sensory switching.”     

3,3-diphenylpentane skeleton as a steroid skeleton substitute: novel inhibitors of human 5alpha-reductase 1

We designed and synthesized novel type 1 5alpha-reductase inhibitors by using 3,3-diphenylpentane skeleton as a substitute for the usual steroid skeleton. 4-(3-(4-(N-Methylacetamido)phenyl)pentan-3-yl)phenyl dibenzylcarbamate (11k) is a competitive 5alpha-reductase inhibitor with the IC(50) value of 0.84 microM.