Sialosylcholesterol Effects on Reconstitution of Microfilament and Glia Filament

Abstract: The effects of α-sialosylcholesterol (α-SC) on formation of either microfilament or glia filament of rat astrocytes were investigated using a reconstitution system. Polymerization of the depolymerized microfilament preparation that had been extracted from a crude cytoskeletal fraction of rat astrocytes, in the presence of 100 m M KCI and 10 m M MgCI₂, was suppressed in a dose-dependent manner by α-SC. α-SC inhibited polymerization of G-actin in a similar manner. The intensity of a-SC inhibition of G- actin polymerization was as great as that of microfilament polymerization, suggesting that the inhibition of microfilament polymerization by α-SC was due to the direct action of α-SC on actin, the main component of microfilament. α-SC depolymerized partly the polymerized microfilament preparation, which resembled F-actin (microfilament-like filaments). α-SC suppressed, in a dose-dependent manner, polymerization of a glia filament preparation that had been extracted from astrocyte cytoskeletons in the presence of phalloidin. An increase in the amount of added α-SC (up to 15 n M ) decreased the amount of the larger glia filament-like filaments, which were 10 nm thick and centrifuged down at 16,000 g for 30 min, and increased that of smaller ones precipitated only after centrifugation at 100,000 g for 1 h. The lower the concentration of the depolymerized glia filament extract, the greater was the inhibition by α-SC of the polymerization. α-SC repressed polymerization of vimentin, the dominant component of glia filament. Vimentin polymerization was more strongly inhibited by α-SC than polymerization of glia filament was. The findings suggested that α-SC suppressed polymerization of glia filament through a direct action on vimentin and that the glia filament-associated proteins increased its structural stability in the presence of α-SC.     

Presence of β-cyanoalanine synthase in unimbibed dry seeds and its activation by ethylene during pre-germination

Evolution of HCN from both rice ( Oryza sativa ) and cocklebur ( Xanthium pennsylvanicum ) seeds increased during a pre-germination period and preceded the evolution of (C₂H₄). These two species were adopted as the representatives of starchy and fatty seeds, respectively. Ethylene promotes seed germination of many species. However, HCN evolution declined abruptly when the radicles emerged and before the peak in C₂H₄ evolution. More-over, both rice and soybean ( Glycine max ) seeds showed some activity of β-cyanoalanine synthase (CAS, EC 4.4.1.9) even in the unimbibed dry state. The activities of CAS in the lower seed of cocklebur and in soybean seeds increased rapidly after emergence of the radicle. However, the CAS of rice seeds, with high activity in the dry state, exhibited a bimodal change, gradually decreasing until radicle emergence had occurred, but then increaing. It is thus likly that HCN evolution during initial imbibition may be derived from cyanogenic reserves and controlled by both pre-existing and subsequently-developing CAS. The exogenous application of C₂H₄ stimulated the activities of CAS in both rice and upper cocklebur seeds and reduced their cyanogen contents. Therefore, the decline of HCN evolution after germination seems to be due to the increased activities of CAS by endogenously produced C₂H₄.     

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.     

Secretion and localization of invertase and inulinase in recombinant Saccharomyces cerevisiae

Summary Secretion of invertase and inulinase produced by recombinant Saccharomyces cerevisiae cells were investigated under derepression conditions of GALI promoter. Secreted invertase mainly localized in the periplasmic space, but most of inulinase was found in the extracellular culture medium. This high level of extracellular secretion of inulinase was not dependent on the growth phase in which derepression of GALI promoter occurs. Our results indicate that the inulinase polypeptide itself may have a function for the protein secretion into the culture medium.     

Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.     

β-Glucosidase activities and HCN liberation in unimbibed and imbibed seeds, and the induction of cocklebur seed germination by cyanogenic glycosides

In many seed species, the major source of HCN evolved during water imbibition is cyanogenic glycosides. The present investigation was performed to elucidate the role of endogenous cyanogenic glycosides in the control of seed germination and to examine the involvment of β-glucosidase in this process. All seed species used here contained some activities of β-glucosidase already in the dry state before imbibition. in the decreasing order of Malus pumila, Daucus carota, Hordeum vulgare, Chenopodium album and so on. β-Gluosidase activity in upper and lower seeds of cocklebur (Xanthium pennsylvanicum Wallr.) decreased with imbibition, and in lower seeds the activity disappeared when they germinated. On the contrary, in caryopses of rice (Oryza sativa L. cv. Sasanishiki) β-glucosidase increased during imbibition, and this increase continued even after germination. β-Glucosidase in cocklebur seeds was more active in the axial than in the cotyledonary tissue. Amygdalin, prunasin and linamarin could all serve as substrattes for the β-glucosidase(s) from both cocklebur and rice. Amygdalin, prunasin and linamarin as well as KCN, were effective in stimulating the germination of upper cocklebur seeds. The seeds evolved much more free HCN gas when they were exposed to the cyanogenic glycosides than when the glycosides were absent. Moreover, the application of the cyanogenic glycosides or of KCN caused accumulation of bound HCN in the seeds. Carbon monoxide, which stimulated cocklebur seed germination only slightly, did not cause accumulation of bound HCN. We suggest that a balance between the cytochrome and the alternative respiration pathways, which is adequate for germination (Esashi et al. 1987. Plant Cell Physiol. 28: 141–150), may be brought about by the action of endogenous HCN; a large portion of which is liberated from cyanogenic glycosides via the action of β-glucosidase. In addition to the partial suppression of the cytochrome path and unlike carbon monoxide, the HCN thus produced may act to supply cyanide group(s) to unknown compounds necessary for germination.     

In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction.

A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm. Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal. When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+). Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase. Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus. This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.