Purification and characterization of a novel lactonohydrolase, catalyzing the hydrolysis of aldonate lactones and aromatic lactones, from Fusarium oxysporum.

A novel lactonohydrolase, an enzyme that catalyzes the hydrolysis of aldonate lactones to the corresponding aldonic acids, was purified 10-fold to apparent homogeneity, with a 61% overall recovery, from Fusarium oxysporum AKU 3702, through a purification procedure comprising DEAE-Sephacel, octyl-Sepharose CL-4B and hydroxyapatite chromatographies and crystallization. The molecular mass of the native enzyme, as estimated by high-performance gel-permeation chromatography, is 125 kDa, and the subunit molecular mass is 60 kDa. The enzyme contains 15.4% (by mass) glucose equivalent of carbohydrate, and about 1 mol calcium/subunit. The enzyme hydrolyzes aldonate lactones, such as D-galactono-gamma-lactone and L-mannono-gamma-lactone, stereospecifically. Furthermore, it can catalyze the asymmetric hydrolysis of D-pantoyl lactone, which is a promising chiral building block for the chemical synthesis of D-pantothenate. These reactions are reversible, and the reaction equilibrium at pH 6.0 has a molar ratio of nearly 1:1 with D-pantoyl lactone and D-pantoic acid. The Km and Vmax for D-galactono-gamma-lactone are 3.6 mM and 1440 U/mg, respectively, and those for D-galactonate are 52.6 mM and 216 U/mg, respectively. The enzyme also irreversibly hydrolyzes several aromatic lactones, such as dihydrocoumarin and homogentisic-acid lactone.     

Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.

Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.     

Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP)

Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A⁴³²⁴ in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes.Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.     

A new DNA profiling system for cell line identification for use in cell banks in Japan

Summary Using the polymorphic DNA probes, ChdTC-15, ChdTC-114, pYNH24, and λTM-18, a DNA profiling system was developed that verified identities of individual cultured cell lines collected in the Japanese cell banks, JCRB, RCB, and IFO. These highly polymorphic DNA probes include both VNTR (Variable Number of Tandem Repeats) sequences and substantial lengths of unique regions. In the mixed probe system, several distinct bands from four to eight can be used for cell line identification. These bands were widely spread in a range of molecular sizes, and were stable and reproducible under stringent conditions of Southern blot hybridization. Because the DNA profile was specific for each individual human cell line, it is useful not only to authenticate many existing cultured cell lines but also to monitor their identity during propagation in a laboratory, and to confirm newly established lines as unique.     

Immunotherapeutic potential in guinea-pig tumor model of deoxyribonucleic acid from Mycobacterium bovis BCG complexed with poly-L-lysine and carboxymethylcellulose.

In vivo antitumor activity of a deoxyribonucleic acid fraction obtained from Mycobacterium bovis BCG (named MY-1) increased when it was complexed with poly-L-lysine (poly LL) solubilized by addition of carboxymethylcellulose (CMC). The complex of MY-1 and poly LL/CMC induced interferon in vivo at a low dose of MY-1 which alone exerted no IFN induction. With Line 10 hepatoma (L10) which is syngeneic with strain 2 guinea pigs, it was demonstrated that repeated intralesional injections of the complex resulted in delay of tumor growth and complete cure of animals from L10 tumor inoculated. Similar treatment of the animals with the same amount of MY-1 or poly LL/CMC alone had little therapeutic effect on the tumor growth.     

Stereospecific Reduction of Ethyl 2′ -Ketopantothenate to Ethyl D-(+)-Pantothenate with Microbial Cells as a Catalyst

A novel enzymatic process for the synthesis of D-(+)-pantothenic acid through the asymmetric reduction of the 2′ -ketopantothenate ester is described. Candida macedoniensis AKU 4588 was found to convert ethyl 2′ -ketopantothenate (80 mg/ml) almost specifically to ethyl D-(+)-pantothenate (>98% enantiomeric excess), with a molar yield of 97.2%.     

Gas chromatographic method for the determination of urinary acetylpolyamines

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N¹-acetylspermidine and N⁵-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1, 10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.     

Disintegration of Rhodospirillum rubrum chromatophore membrane into photoreaction units, reaction centers, and ubiquinone-10 protein with mixture of cholate and deoxycholate.

1. The membrane of Rhodospirillum rubrum chromatophores was disintegrated with mild detergents (cholate and deoxycholate) in order to study the spatial arrangement of the functional proteins in the photochemical apparatus and the electron transport system in the membrane. 2. The components solubilized from the membrane by a mixture of cholate and deoxycholate (C-DOC) were separated into four fractions by molecular-sieve chromatography in the presence of C-DOC; they were designated as F1, F2, F3, and F4 in the order of elution. The fractions were further purified by repeated molecular-sieve chromatography in the presence of C-DOC until each fraction was chromatographically homogeneous. 3. F1 appeared to be conjugated forms of F2. 4. The purified F2 was composed of a rigid complex having a weight of 7 X 10(5) daltons, containing approximately 10 different kinds of protein species with molecular weights of 3.8 X 10(4), 3.6 X 10(4), 3.5 X 10(4), 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), 1.3 X 10(4), 1.2 X 10(4), 1.1 X 10(4), and 1.0 X 10(4). The complex contained 33 bacteriochlorophylls, 4 iron atoms, and 90 phosphates, but no cytochrome, ubiquinone, or phospholipid. It showed the same reaction center activity as chromatophores, indicating that the complex was a unit of the photochemical apparatus (photoreaction unit). Each chromatophore of average size was estimated to possess about 24 photoreaction units. 5. The purified F3 showed an absorbance spectrum characteristic of reaction centers, and contained 3.4 bacteriochlorophylls, 2.0 bacteriopheophytins, and 1.9 acid-labile iron atoms, but no cytochrome or ubiquinone (C-DOC reaction center). It had a weight of 1.2 X 10(5) daltons, and the main components were 4 protein species with molecular weights of 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), and 1.0 X 10(4). 6. The purified F4 showed a molecular weight of about 11,000, and contained one mole of ubiquinone-10 per mole (ubiquinone-10 protein). 7. The reaction center activity of C-DOC reaction centers was stimulated by ubiquinone-10 protein. In addition, the reaction center oxidized reduced cytochrome c2 in the light, provided that ubiquinone-10 protein was present (photo-oxidase activity).     

Phototropism in Vaucheria geminata II. The mechanism of bending and branching

The mechanism of phototropic bending and branching in Vaucheriageminata was analyzed. Using the half-side-illumination methodit was shown that phototropic bending is initiated by the formationof a new growth center on the illuminated side of the apicaldome of the tube, not by the difference in growth rate betweenthe lighted and shaded sides of the tip. Branching was alsoinitiated when the part proximal to the apical dome was illuminated.Blue light was effective for branching as it was for phototropicbending. The refractive indices measured at the growing tipand an area other than the tip were 1.36 and 1.34 respectively.A hyaline patch resembling the hyaline cap, which appeared priorto the onset of tip-growth in the apical dome, was invariablyformed shortly before the initiation of a new branch at theilluminated locus. (Received December 20, 1974; )