Chemical Composition of Giant Cells Induced by Tunicamycin and Normal Mycelia of Penicillium citrinum

Growth of Penicillium citrinum was reduced in the presence of tunicamycin. Under this condition, reduction of yield of cell wall was greater than that of cellular protein.

Chitin content in the cell wall was several times higher in giant cells formed from conidia in the presence of tunicamycin than in normal mycelia, while reducing sugar content, presumably reflecting glucan content, did not significantly differ. Galactosamine, which was present in normal mycelia and absent in conidia, could not be detected in giant cells. The amino acid composition of the cell wall and whole cells of giant cells differed distinctly from that of normal mycelia.

Tunicamycin did not significantly inhibit the synthesis of DNA, RNA and protein as judged by incorporation of radioactive precursors, while cell wall synthesis, as judged by incorporation of radioactive N-acetylglucosamine, glucose and alanine into acid insoluble fraction, was inhibited by more than 40% in the presence of 10 μg/ml of tunicamycin. In fungi tunicamycin probably acts primarily as an inhibitor of cell wall glycoprotein synthesis and not of chitin synthesis.

Cyclic nucleotides level also differed distinctly between giant cells and mycelia.     

Studies on Host-selective Toxins Produced by a Pathotype of Alternaria citri Causing Brown Spot Disease of Mandarins

Two host-selective pathotoxins, ACTG-toxins A and B, and four related but less active toxins, C, D, E, and F, were isolated from the culture broth of Alternaria citri, a fungus that produces brown spot disease of Dancy tangerine (Citrus reticulata) and other mandarin cultivars. The basic common structural features of these toxins were the presence of a six-membered group bonded, via a methylene group, to a five-membered ring having an alkenyl substituent (Fig. 1). For Toxins A, B, C and F, the six-membered ring had an enolizable β-diketo group. For Toxin C, the five-membered ring was a tetrahydrofuran group. For Toxins D and E, an additional dihydropyran ring was formed by dehydration between a tertiary hydroxyl on the cyclopentene ring and an enolic hydroxyl group on the cyclohexane ring, and also the presence of a terminal hydroxymethyl group on the alkenyl substituent, instead of the methyl groups in Toxins A, B and C. In Toxin F, the terminal group was a formyl.     

Synthesis of (±)-8,16,26,34-Tetrahydroxy-6,10,14,18,24,28,32,36-octaoxohentetracontane as a Potent Synthetic Analog of Race T Toxin Produced by Helminthosporium maydis

A stereoisomeric mixture of (±)-8,16,26,34-tetrahydroxy-6,10,14,18,24,28,32,36-octaoxo-hentetracontane (C₄₁) was synthesized together with (±)-8,16-dihydroxy-6,10,14,18-tetraoxotri-cosane (C₂₃), utilizing bis addition of pentyl-l,5-dimagnesiumbromide to two moles of a C₁₈-alde-hyde.     

Oxidation of Polyamines by Fungal Enzymes

Polyamine oxidase was found in mycelia of fungi belonging to the genera of Aspergillus, Mucor, Penicillium, Rhizopus, Cylindrocarpon, Fusarium and Gibberella when they were grown in medium containing spermine or spermidine as the sole source of nitrogen. The maximal formation of the enzymes of Penicillium chrysogenum and Aspergillus terreus was observed in early stationary phase of growth, and thereafter, the enzymes disappeared with consumption of substrate. The oxidation products of spermine and spermidine by the two enzymes were identified as putrescine, 3-aminopropionaldehyde and H₂O₂. Therefore, the enzymes were characterized as a type of polyamine oxidase of rat liver.     

Structure of cytochrome c551 from Pseudomonas aeruginosa refined at 1.6 Å resolution and comparison of the two redox forms

The molecular structures of ferri- and ferrocytochrome c₅₅₁ from Pseudomonas aeruginosa have been refined at a resolution of 1.6 Å, to an R factor of 19.5% for the oxidized molecule and 18.7% for the reduced. Reduction of oxidized crystals with ascorbate produced little change in cell dimensions, a 10% mean change in F_obs, and no damage to the crystals. The heme iron is not significantly displaced from the porphyrin plane. Bond lengths from axial ligands to the heme iron are as expected in a low-spin iron compound. A total of 67 solvent molecules were incorporated in the oxidized structure, and 73 in the reduced, of which four are found inside the protein molecule. The oxidized and reduced forms have virtually identical tertiary structures with 2 ° root-mean-square differences in main-chain torsion angles φ and ψ, but with larger differences along the two edges of the heme crevice. The difference map and pyrrole ring tilt suggest that a partially buried water molecule (no. 23) in the heme crevice moves upon change of oxidation state.Pseudomonas cytochrome c₅₅₁ differs from tuna cytochrome c in having: (1) a water molecule (no. 23) at the upper left of the heme crevice; that is, between Pro62 and the heme pyrrol 3 ring on the sixth ligand Met61 side, where tuna cytochrome c has an evolutionary invariant Phe82 ring; (2) a string of hydrophobic side-chains along the left side of the heme crevice, and fewer positively charged lysines in the vicinity; and (3) a more exposed and presumably more easily ionizable heme propionate group at the bottom of the molecule. A network of hydrogen bonds in the heme crevice is reminiscent of that inside the heme crevice of tuna cytochrome c. As in tuna, a slight motion of the water molecule toward the heme is observed in the oxidized state, helping to give the heme a more polar microenvironment. The continuity of solvent environment between the heme crevice and the outer medium could explain the greater dependence of redox potential on pH in cytochrome c₅₅₁ than in cytochrome c.     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

Controlled-release system of single-stranded DNA triggered by the photothermal effect of gold nanorods and its in vivo application

Gold nanorods have strong absorption bands in the near-infrared region, in which light penetrates deeply into tissues. The absorbed light energy is converted into heat by gold nanorods, the so-called 'photothermal effect'. Hence, gold nanorods are expected to act not only as on-demand thermal converters for photothermal therapy but also as controllers of a drug-release system responding to irradiation by near-infrared light. To achieve a controlled-release system that can be triggered by light irradiation, double-stranded DNA (dsDNA) was modified on gold nanorods. When the dsDNA-modified gold nanorods were irradiated by near-infrared light, the single-stranded DNA (ssDNA) was released from gold nanorods due to the photothermal effect. The amount of released ssDNA was dependent upon the power and exposure time of light irradiation. Release of ssDNA was also observed in tumors grown on mice after light irradiation. Such a controlled-release system of oligonucleotide triggered by the photothermal effect could expand the applications of gold nanorods that have unique optical characteristics in medicinal fields.     

Exclusively NOESY-based automated NMR assignment and structure determination of proteins

A fully automated method is presented for determining NMR solution structures of proteins using exclusively NOESY spectra as input, obviating the need to measure any spectra only for obtaining resonance assignments but devoid of structural information. Applied to two small proteins, the approach yielded structures that coincided closely with conventionally determined structures.     

Carcinoma infection and immune systems of Ehrlich ascites carcinoma-bearing mice treated with structurally similar sulfonium compounds

The effects of intraperitoneal administration of dimethylsulfonioacetate (DMSA), dimethlsulfoniopropionate (DMSP), and methylmethionine (MeMet) solutions (10 mM each) on the body weights and the hematological parameters (red and white blood cells) of Ehrlich ascites carcinoma (EAC)-bearing mice were examined for up to 10 d. Body weights significantly increased in the EAC-bearing mice treated with and without MeMet in contrast to those with DMSA and DMSP. This increase was attributed to the increased amounts of ascitic fluid. EAC-bearing mice with and without MeMet both showed abnormal values of hematological parameters, while those with DMSA and DMSP exhibited almost normal levels on the 10th day.     

Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3''-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.