Biosynthesis of brassinolide from castasterone in cultured cells of Catharanthus roseus

Feeding experiments with tritium- and deuterium-labeled castasterone (CS) were conducted with three cell lines of Catharanthus roseus, including crown gall cells and nontransformed cells. In all three cell lines, the conversion of CS to brassinolide (BL) was observed and unequivocally confirmed by gas chromatography/mass spectrometry (GC/MS). This is the first conclusive evidence that CS is the biosynthetic precursor of BL.Biosynthesis of brassinosteroids in Catharanthus roseus. Part II. Part I of this series: Yokota et al. (1990a).     

Evaluation of Superoxide Scavenging Activities of Hamamelis Extract and Hamamelitannin

Hamamelitannin, which is a component of bark extract of hamamelis (Hamamelis virginior L.), was found to be a potent scavenger of superoxide anion radicals. Superoxide anion scavenging activity of the compound was evaluated by ESR-spin trap method using DMPO (5,5'-dimethyl-1-pyrroline-N-oxide) as a spin trapping agent. The IC₅₀ value (the concentration producing 50% inhibition of superoxide anion radicals) of hamamelitannin was found to be 1.38 ± 0.06 μM much lower than that of ascorbic acid (23.31 ± 2.23 μM). Supporting the superoxide scavenging activity of hamamelitannin, the compound showed both suppresive ability against depolymelization of hyaluronic acid and protective ability against cytotoxicity induced by superoxide anion radicals. Hamamelitannin increased the survival rate of fibroblast to 85.5 ± 3.3%, compared with that of control (27.2 ± 4.3%).     

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.     

Role of the outer tissue in abscisic acid-mediated growth suppression of etiolated squash hypocotyl segments

Exogenously applied abscisic acid (ABA) substantially suppressed the elongation of hypocotyl segments of etiolated squash ( Cucurbita maxima Duch. cv. Houkou-Aokawaamaguri) after a 3 h lag period, without changes in the osmolalities of the apoplastic and symplastic solutions in the segment.
Segments with the outer tissues removed elongated more rapidly than unpeeled segments (whole segments). ABA did not suppress the elongation of peeled segments. When the segments were incubated in [¹⁴C]-glucose, radioactivity was more effectively incorporated into the cell wall fractions of the outer than into those of the inner tissue. ABA significantly inhibited the incorporation of radioactivity into hermicellulose and cellulose of the outer tissue prior to the suppression of segment elongation, but it did not inhibit the incorporation into the pectic traction of the outer tissue or into any of the cell wall fractions of the inner tissue. These results indicate that ABA primarily affected the outer tissue, in which it specifically reduced the synthesis of hemicellulose and cellulose prior to the ABA-mediated suppression of growth.     

Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin.

Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.     

Possible involvement of ethylene-activated {beta}-cyanoalanine synthase in the regulation of cocklebur seed germination

A possible involvement of ß-cyanoalanine synthase(CAS: EC 4.4.1.9 [EC] ) in germination processes of seeds was demonstratedusing pre-soaked upper seeds of cocklebur (Xanthium pennsylvanicumWallr.). Pretreatment in anoxia not only with KCN but also cysteine,as the substrates for CAS, stimulated the subsequent germinationof cocklebur seeds in air. However, the effect of cysteine wasmanifested even in air when applied together with C₂H₄, andits effect was further enhanced in combination with KCN. Thegermination-stimulating effect of KCN was intensified by C₂H₄only when 0₂ was present. In contrast, serine, another substrateof CAS, was effective in air only when combined with C₂H₄ and/orKCN. The addition of cysteine greatly reduced the cyanogenicglycoside content of seeds, but increased HCN evolution. Onthe other hand, glutathione did not have any effect on cockleburseed germination, HCN evolution or bound cyanogen content, suggestingthat cysteine is not acting as a reducing reagent. It is suggestedthat CAS regulates the process of cocklebur seed germinationby the dual action of enlarging the pool of amino acids andsupplying sulphydryl bases, the latter being more determinatelyimportant. Serine is effective only via the former action, whilecysteine would act via both. Key words: Cyanide, cyanogenic glycoside, ß-cyanoalanine synthase, seed germination, Xanthium pennsylvanicum     

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.     

Initiation of Cyclin B Degradation by the 26S Proteasome upon Egg Activation

Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH₂ terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.     

Prevention of Vascular Graft Infection by Sisomicin Incorporated into Fibrin Glue

To examine the efficacy of sisomicin (SISO) incorporated into fibrin glue (FG) for the prevention of graft infection in animal models, the susceptibility to infection of Dacron grafts (control) and SISO-FG Dacron grafts following the inoculation of Staphylococcus aureus or S. epidermidis was compared. The results showed that SISO-FG Dacron grafts displayed resistance to graft infection.