Selective Activation of Phospholipase Cγ1 and Distinct Protein Kinase C Subspecies in Intracellular Signaling by Hepatocyte Growth Factor/Scatter Factor in Primary Cultured Rat Neocortical Cells

Abstract: Hepatocyte growth factor/scatter factor (HGF) was recently reported to function as a neurotrophic factor in the CNS. To investigate the intracellular signal pathways after activation of the HGF receptor c-Met in primary cultured rat neocortical cells, in vitro kinase assays were performed. HGF stimulation enhances the phosphorylation of endogenous 80- and 45-kDa substrates. Studies with protein kinase inhibitors and phorbol 12-myristate 13-acetate showed that protein kinase C (PKC) is activated intracellularly. The 80-kDa protein was identified to be the major PKC substrate MARCKS. Although four PKC subspecies, PKCα, PKCε, PKCγ, and PKCλ, were expressed in the cells, only PKCα, PKCε, and PKCγ were selectively translocated in the plasma membrane after HGF stimulation. As expected from these three PKC subspecies, phosphorylation of phospholipase Cγ1 (PLCγ1) but not phosphatidylinositol 3-kinase was enhanced, although the stimulation of brain-derived neurotrophic factor induced phosphorylation of phosphatidylinositol 3-kinase. In contrast to the neocortical cells, HGF did not enhance phosphorylation of PLCγ1 in primary astrocytes. We also found that activated PKC(s) served as a major mitogen-activated protein kinase activator in this pathway. These findings suggest that HGF exerts neurotrophic effects through selective phosphorylation of PLCγ1 and activation of distinct PKC subspecies in neocortical cells, most likely neurons.     

Pfaffosides,nortriterpenoid saponins,from Pfaffia paniculata

Three new nortriterpene saponins having inhibitory effects on the growth of cultured tumor cells, named pfaffosides D, E and F, have been isolated from Pfaffia paniculata. Their structures have been established as 3β-O-[β-d-xylopyranosyl-(1 → 2)-β-d-(6-O-n-butyl) glucuronopyranosyl]-pfaffic acid-(28 → 1)-β-d-glucopyranosyl ester, 3β-O-[β-d-xylopyranosyl-(1 → 2)-β-d(6-O-methyl)glucuronopyranosyl]-pfaffic acid-(28 → 1)-β-d glucopyranosyl ester and 3β-O[β-d-glucuronopyranosyl]-pfaffic acid respectively, based on their chemical and spectroscopic properties     

Biosynthesis of shikonin in callus cultures of Lithospermum erythrorhizon

Administration of various supposed precursors to the callus cultures of Lithospermum erythrorhizon grown on the Linsmaier—Skoog medium supplemented with IAA and kinetin established that the constituent shikonin is formed via shikimic acid, p-hydroxybenzoic acid, m-geranyl-p-hydroxybenzoic acid and geranylhydroquinone. In a strain of callus culture lacking the capacity to synthesize shikonin and in callus cultures which have had this capacity but lost it due to cultivation on a medium supplemented with 2,4-D, substances up to m-geranyl-p-hydroxybenzoic acid in the biosynthetic sequence have been detected. Although illumination with white light also arrested shikonin production, traces of pigment were still formed presumably because light did not reach the innermost part of the callus cultures.     

Epithelial Metaplasia Induced Amnionic Ectoderm by the Dermis of Chicken Embryos1

Amnionic ectoderm of 6.8-day chicken embryos was associated with 6.8-day dorsal dermis or 13–15-day scale dermis and cultured on host chorio-allantoic membrane for 8 days. The amnionic ectoderm, recombined and cultured with the dorsal dermis, developed feather filaments consisted of a feather root, a horny sheath, and barb ridges. With several feather keratin-specific monoclonal antibodies (4E12 and 1F3), these structures in the induced feather filaments were shown to express feather-specific keratin antigens. The amnionic ectoderm, recombined and cultured with the shank dermis, became stratified squamous and developed scales. The scales were keratinized and their surface reacted only weakly with the monoclonal antibodies specific for the feather keratins. However, 1F3 reacted with two polypeptides in the cytoskeletal fraction of the scales, but not of the feather filaments. The results confirm our previous findings from in vitro experiments with the proamnionic ectoderm (Mizuno, 1970, 1972).     

Quantitative detection method of triglycerides in serum lipoproteins and serum-free glycerol by high-performance liquid chromatography

We have developed a simple and reliable method for quantitative detection of triglycerides (TG) in serum lipoproteins and serum-free glycerol (FG) by high-performance liquid chromatography (HPLC). After separation of serum constituents using a new gel-permeation column (TSK gel Lipopropak XL, Tosoh) and a new eluent (TSK eluent LP-2, Tosoh), TG and FG were detected by on-line reaction using a modified reagent which contained glycerol kinase, glycerol-3-phosphate oxidase and lipoprotein lipase. HPLC patterns showed five peaks corresponding to chylomicrons, very-low-density, low-density, high-density lipoproteins and FG. Absolute concentrations of TG in each lipoprotein fraction and serum FG were calculated from the corresponding peak areas using standard FG as a calibrator. Due to its very high sensitivity of peak detection, this method has become desirable for the analyses of lipoproteins of very low concentrations such as in cell culture systems. This technique will contribute to a better understanding of lipoprotein TG and serum FG distribution in human and nonhuman subjects.     

Conformational analysis of chitobiose and chitosan

The relaxed potential energy surfaces of chitobiose were calculated based on the MM3-force field by optimizing dimer structures on a 10° grid spacing of the torsional angles about the glycosidic bonds (Φ,Ψ). The 36 conformations; the four combinations of the hydroxymethyl group orientations coupled with the nine of the secondary group ones— were assumed for each Φ,Ψ conformation. The four conformations, each differing in the hydroxymethyl group orientations, were considered for the whole Φ,Ψ space, and all the 36 conformations, for the restricted space of low energy. While the resulting energy map and the structures of the energy minima were similar to those proposed for cellobiose in many respects, more restricted energy profile was suggested for the relaxed map of chitobiose where differences in the energy level between the global minimum and the local minima were within 5.4 kcal/mol, compared with the equivalent value of 3.6 kcal/mol for cellobiose. Further depression of the global minimum occurred when the acidic residue was used. The Monte Carlo samples of the chitosan chain were generated based on the relaxed map to predict the unperturbed coil dimension in solution. The chitosan chains showed Gaussian behavior at x = 500 (x, degree of polymerization) and gave the characteristic ratio C_x, of about 70, which was much larger than the experimental values observed for the chitosan and cellulosic chains. © 1994 John Wiley & Sons, Inc.     

A self-organizing model of walking patterns of insects

Insects generate walking patterns which depend upon external conditions. For example, when an insect is exposed to an additional load parallel to the direction in which it is walking, the walking pattern changes according to the magnitude of the load. Furthermore, even after some of its legs have been amputated, an insect will produce walking patterns with its remaining legs. These adaptations in insect walking could not previously be explained by a mathematical model, since the mathemati cal models were based upon the hypothesis that the relationship between walking velocity and walking patterns is fixed under all conditions. We have produced a mathematical model which describes self-organizing insect walking patterns in real-time by using feedback information regarding muscle load (Kimura et al. 1993). As part of this model, we introduced a new rule to coordinate leg movement, in which the information is circulated to optimize the efficiency of the energy transduction of each effector orga n. We describe this mechanism as ‘the least dissatisfaction for the greatest number of elements’. In this paper, we introduce the following aspects of this model, which reflect adaptability to changing circumstances: (1) after one leg is exposed to a transient perturbation, the walking pattern recovers swiftly; (2) when the external load parallel to the walking direction is continuously increased or decreased, the pattern transition point is shifted according to the magnitude of the load increme nt or decrement. This model generates a walking pattern which optimizes energy consumption at a given walking velocity even under these conditions; and (3) when some of the legs are amputated, the model generates walking patterns which are consistent with experimental results. We also discuss the ability of a hierarchical self-organizing model to describe a swift and flexible information processing system. Received: 8 February 1993/Accepted in revised form: 12 November 1993     

Diversity in Molecular Mass of the Common EDTA-Soluble Antigens of Clostridium chauvoei and Clostridium septicum

Common EDTA-soluble antigens of Clostridium chauvoei and C. septicum were examined by indirect-immunofluorescence (IFA) and immunoblot analysis. The monoclonal antibodies (mAbs) specific for the 35 kDa antigen of C. chauvoei strain ATCC 10092 were used. These mAbs reacted with all 11 strains, 6 of C. chauvoei and 5 of C. septicum, in IFA. In immunoblot analysis with the mAbs, the bands at molecular mass of 35 kDa were found in all C. chauvoei strains, while the bands at 36 kDa were found in 4 of 5 strains of C. septicum. These results indicate that the 35 kDa antigen of C. chauvoei and the 36 kDa antigen of C. septicum possess a similar epitope recognized by the mAb.     

Anionic sites in glomerular basement membrane of rats with serum sickness nephritis: quick-freezing and deep-etching study

Summary Serum sickness nephritis was induced in male Fisher 344/JCL rats by injecting egg albumin into the foot pads and peritoneal cavity. The alteration of anionic sites in the glomerular basement membrane (GBM) of the rats with significant proteinuria was studied with a quick-freezing and deep-etching method using polyethyleneimine as a cationic probe. In control rats, anionic sites were located around the fibrils of the lamina rara externa, which radiated perpendicularly from the lamina densa to podocyte cell membranes. In the glomeruli of proteinuric rats, many electron-dense deposits were observed in the subepithelial side of the GBM, where the fibrils of the lamina rara externa were usually obscured and anionic sites around them could not be recognized. However, in some areas, a clear boundary could be observed between deposits and the lamina densa. Electron micrographs of freeze-fractured deposits showed that the fibrils radiated perpendicularly from the lamina densa and that anionic sites around them had been preserved. These results suggest that some of the deposits simply passed through the GBM and masked transiently the fibril structures of the GBM, but others probably destroyed these fibril structures, including anionic sites.