Cloning and sequencing of a ligninase gene from a lignindegrading basidiomycete,Phanerochaete chrysosporium

Summary A ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3 and 5 intron boundaries. The putative eukaryotic regulatory sequences, i.e. CAAT and TATA box-like sequences, are present in the 5 flanking region.     

A case of diverticular disease of the colon in a Japanese monkey (Macaca fuscata)

Diverticular disease of the colon was detected in a female Japanese monkey by X-ray examination. The monkey was 15 years old and had been kept under captive conditions for nine years. Lack of appetite and activity, and constipation were observed. The monkey was given fiber-rich vegetables and wild plants, and its appetite and activity then improved. Based on a consideration of various factors, it is suggested that one possible cause of the diverticulosis in this case was a low dietary fiber intake.     

Stacking and hydrogen bonding interactions between phenylalanine and guanine nucleotide: crystal structure of L-phenylalanine-7-methylguanosine-5'-monophosphate complex

The crystal structure of title complex has been analyzed by X-ray diffraction method as a model for elucidating the possible interaction between the phenylalanyl residue of proteins and the N7-protonated or methylated guanine base of nucleic acids. The guanine base is associated with the benzene ring of phenylalanine by stacking interaction, and further connected with the carboxyl group by the formation of a pair of hydrogen bonds. These two interaction modes are suggested to be responsible for the specific recognition of base sequence by protein.     

Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin

Upon irradiation with 365-nm light, copper(II)-camptothecin significantly produced single- and double-strand breaks of DNA and also induced a marked inactivation of bacteriophage. The nucleotide sequence analysis exhibited considerably random DNA cleavage. The DNA strand scission by the camptothecin-Cu(II)-UV light system, as well as the phage inactivation, was strongly suppressed by bathocuproine and catalase, indicating participation of cuprous species and hydrogen peroxide in the reaction. The present results suggest that (1) Cu(II) ion may play an important role as a cofactor in antitumor action of camptothecin and (2) the combination of copper-camptothecin plus long-wave ultraviolet light is useful against certain cancer treatment as a new photochemotherapy.     

Adenosine 3′,5′-Cyclic Monophosphate-Deficient Mutants of Vibrio cholerae

Two adenosine 3',5'-cyclic monophosphate (AMP)-deficient mutants of Vibrio cholerae (biotype El Tor) were successfully isolated by nitrosoguanidine treatment followed by pencillin screening for pleiotropic sugar-negative clones. Exogenous cyclic AMP is required for the fermentation of sucrose, trehalose, fructose, maltose, and mannose but not of glucose, as well as for the formation of normal flagella and specific somatic antigens. A striking characteristic of the mutants is their growth behavior at higher temperatures. They cannot grow on TCBS selective plates at 37 C or higher unless they are provided with a supply of exogenous cyclic AMP, although they are capable of producing colonies on the same medium, even without cyclic AMP, at temperatures lower than 30 C. Since the mutants are converted to spheroplasts, spindle forms, and spiral filaments in cyclic AMP-free media at 37 C, and this phenomenon is stopped by the addition of cyclic AMP or a combination of 20% sucrose and 0.2% magnesium chloride, it is assumed that cyclic AMP is essential for the synthesis of the cell wall of V. cholerae at higher temperatures.     

Isolation and characterization ofPleurotus ostreatus mutant strains resistant to a carboxin-derived fungicide,flutolanil

To develop a dominant genetic marker inPleurotus ostreatus, mutant strains resistant to a carboxin-derived fungicide, flutolanil, were isolated. These mutants included strains which showed resistance to 50-fold higher concentration of fluotolanil than the wild-type strain, even after successive cultivations in the absence of the drug. Dominance of the phenotype was confirmed by back-crossing between the resistant and wild-type monokaryons. The flutolanilresistance was also shown to be stably inherited by the basidiospore-derived progenies of the mutant strains.     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

Effects of fungal pretreatment and steam explosion pretreatment on enzymatic saccharification of plant biomass

The effects of consecutive treatments by a lignin-degrading fungus Phanerochaete chrysosporium and by steam explosion for the enzymatic saccharification of plant biomass were studied experimentally, and the optimal operational conditions for obtaining the maximum saccharification were evaluated. Beech wood-meal was treated by the fungus for 98 days and then by high steam temperatures of 170-230 degrees C with steaming times of 0-10 min. The treatment of the wood-meal by fungus prior to steam explosion enhanced the saccharification of wood-meal. The treated wood-meal was separated into holo-cellulose, water soluble material, methanol soluble lignin, and Klason lignin. The saccharification decreased linearly with the increase in the amount of Klason lignin. It was estimated by the equation for the saccharification of exploded wood-meal expressed as a function of steam temperature and steaming time that the maximum saccharification of wood-meal was obtained by consecutive treatments such as fungal treatment for 28 days and then steam explosion at a steam temperature of 215 degrees C and a steaming time of 6.5 min. (c) 1995 John Wiley & Sons, Inc.     

In vitro neurotoxicity of amyloid β-peptide cross-linked by transglutaminase

Transglutaminase catalyzes the intermolecular cross-linking of peptides between Gln and Lys residues, forming an -(-glutamyl) lysine bond. Amyloid -peptide, a major constituent of the deposits in Alzheimer disease, contains Lys16, Lys28, and Gln15 which may act as substrates of transglutaminase. Transglutaminase treatment of amyloid -peptide (1–28) and amyloid -peptide (1–40) yielded cross-linked oligomers. Transglutaminase-treated A retarded neurite extension of PC12 cells, and rat cultured neurons of hippocampus and septum, brain areas severely affected by Alzheimer disease, and subsequently caused cell death, whereas the transglutaminase-untreated counterparts did not show harmful effects. The transglutaminase-catalyzed oligomers of amyloid -peptide and their neurotoxicity may be involved in two characteristics in Alzheimer disease, neuronal degeneration and formation of the insoluble deposits.Abbreviations: AD – Alzheimer disease, A – amyloid -peptide, DMEM – Dulbecco's modified Eagle's medium, DMEM/F–12–1:1 mixture of DMEM and Ham's F–12 medium, FCS – fetal calf serum, HS – horse serum, PAGE – polyacrylamide gel electrophoresis, MTT – 3-(4,5-dimethylthiazol–2-yl)–2,5-diphenyltetrazolium bromide, NGF – nerve growth factor, TGase – transglutaminase.