The Conditions for Bacteriochlorophyll Formation and the Ultrastructure of a Methanol-utilizing Bacterium,Protaminobacter ruber,Classified as Non-photosynthetic Bacteria

A methanol-utilizing bacterium, Protaminobacter ruber, formed a green pigment, when it was grown on 1,2-propanediol as a sole carbon and energy source. The pigment was identified as bacteriochlorophyll a by the absorption spectrum resembling the pigment from photosynthetic bacteria and by the exact stoichiometric relationship among the original pigment, the pigment treated to remove magnesium (bacteriopheophytin) and magnesium ion obtained from the pigment. Bacteriochlorophyll formation was stimulated by the exposure to light during the relatively early stage of the growth, while the continuous light exposure completely prevented the pigment formation. Aeration was also necessary for the pigment synthesis as well as the bacterial growth. Electron micrographs of thin section of P. ruber cells cultured in the intermittent light showed the probable existence of a chromatophore-like structure.     

Occurrence of a New Enzyme Reducing Metmyoglobin in Dolphin Muscle

A new enzyme has been obtained in a crystalline state from the muscle of blue white dolphin. This enzyme resembles to methemoglobin reductase from erythrocyte with respect to (a) elution pattern of DEAE-Sephadex column chromatography, (b) absorption spectra, (c) molecular weight and (d) activity of reducing methemoglobin, metmyoglobin and ferric cytochrome c. However, distinct differences can be observed between two enzymes with regard to (a) sedimentation coefficient, (b) diffusion coefficient, (c) frictional ratio, (d) pH-mobility curve and (e) specific activity of reducing the above three substrates. It is advocated that enzyme is termed metmyoglobin reductase.     

A New Mint (Variety of Mentha aquatica L.) Containing (—)-Isopinocamphone as a Major Constituent of Essential Oil

Certain strains of Rhodotorula were found capable of utilizing L-phenylalanine as a sole carbon and nitrogen source and of accumulating ether-soluble metabolite in the cultured broth. The metabolite was isolated and identified as trans-cinnamic acid. The nonoxidative deamination of phenylalanine to trans-cinnamic acid was catalyzed by dried cells, acetone-dried cells or intact cells with surface active agents. The distribution of phenylalanine ammonia-lyase activity in yeasts was investigated. It was found that the enzyme activity specifically occurred in Rhodotorula and that the formation of enzyme was enhanced by culturing on the medium supplemented with phenylalanine.     

Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-Oxo-prostaglandin 13-reductase catalytic mechanism and a possible Src homology 3 domain binding loop

The bifunctional leukotriene B(4) 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB(4) 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B(4) (LTB(4)), and 15-oxo-lipoxin A(4) (15-oxo-LXA(4)). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB(4) 12-HD/PGR activity. Here we report the crystal structure of the LTB(4) 12-HD/PGR, the binary complex structure with NADP(+), and the ternary complex structure with NADP(+) and 15-oxo-PGE(2). In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE(2) contains two chains, but only the omega-chain of 15-oxo-PGE(2) was defined in the electron density map in the ternary complex structure. The omega-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2'-hydroxyl group of nicotine amide ribose of NADP(+) and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE(2) is consistent with a broad substrate specificity of LTB(4) 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB(4) 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the omega-chain.     

CuZn-SOD deficiency causes ApoB degradation and induces hepatic lipid accumulation by impaired lipoprotein secretion in mice

Elevated hepatic reactive oxygen species play an important role in pathogenesis of liver diseases, such as alcohol-induced liver injury, hepatitis C virus infection, and nonalcoholic steatohepatitis. In the present study, we investigated and compared the hepatic lipid metabolisms of liver-specific Sod2 (superoxide dismutase 2) knock-out (Sod2 KO), Sod1 knock-out (Sod1 KO), and Sod1/liver-specific Sod2 double knock-out mice (double KO). We observed significant increases in lipid peroxidation and triglyceride (TG) in the liver of Sod1 KO and double KO mice but not in the liver of Sod2 KO mice. We also found that high fat diet enhanced fatty changes of the liver in Sod1 KO and double KO mice but not in Sod2 KO mice. These data indicated that CuZn-SOD deficiency caused lipid accumulation in the liver. To investigate the molecular mechanism of hepatic lipid accumulation in CuZn-SOD-deficient mice, we measured TG secretion rate from liver using Triton WR1339. We found significant decrease of TG secretion in CuZn-SOD-deficient mice. Furthermore, we observed marked degradation of apolipoprotein B (apoB) in the liver and plasma of CuZn-SOD-deficient mice, indicating that degradation of apoB impairs secretion of lipoprotein from the liver. Our data suggest that oxidative stress enhances hepatic lipid accumulation by impaired lipoprotein secretion due to the degradation of apoB in liver.     

Quantification of Clostridium botulinum type A toxin and organisms in the feces of a case of infant botulism and examination of other related specimens

Bacteriological examinations were performed on the first case of infant botulism in Japan (an infant boy aged 79 days at onset of illness). Clostridium botulinum type A toxin and organisms were detected continually in the stools of the infant for at least 31 days and 39 days, respectively. The highest levels of the toxin and of the population of the organisms, 7.8 X 10(4) LD50/g and 1.3 X 10(6) colony forming units (cfu)/g, were detected in the stool specimen taken on the 20th day of illness. Type A organisms were detected also in the honey fed to the infant before onset of illness, teats of his feeding bottle, soil specimens taken at the house entry and the vacuum-cleaner dust. Fecal excretion of the toxin and organisms was no longer detected from the 68th day of illness and he recovered.     

Establishment of Agrobacterium tumefaciens-Mediated Transformation of an Oleaginous Fungus,Mortierella alpina 1S-4, and Its Application for Eicosapentaenoic Acid Producer Breeding

Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5⁻ strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for ω3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/10⁸ spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal ω3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.Two decades ago, a filamentous zygomycete fungus, Mortierella alpina 1S-4, was isolated from soil as a potent producer of polyunsaturated fatty acids (PUFAs) in our laboratory and was utilized for commercial production of arachidonic acid (AA) (20:4n-6) (21). Breeding of mutants derived from the wild strain led to the production of dihomo-γ-linolenic acid (20:3n-6) and Mead acid (20:3n-9) (10-12) (Fig. ​(Fig.1).1). Furthermore, we attempted to produce other PUFAs synthesized in M. alpina 1S-4, since some fatty acids (e.g., 18:2n-9, 18:4n-3, and 20:4n-3) have limited natural sources and could have promising beneficial physiological effects (9). In particular, for microbial production of n-3 PUFAs, currently prepared from fish oil, it is necessary to achieve stable productivity and quality; however, mutation treatment caused low activity of the specific enzymes involved in PUFA biosynthesis, which is unsuitable for industrial application. In addition, gene manipulation tools have not been sufficiently developed for metabolic control of the PUFA synthetic pathway. Genetic manipulation is a new means of molecularly breeding industrial strains, analyzing their physiological properties, and clarifying the biosynthetic pathway to PUFAs. A comprehensive transformation system for this fungus has been fundamentally established. It involves a uracil-auxotrophic mutant (ura5⁻ strain) as a host strain, a homologous ura5 gene as a selectable marker gene, and transformation through the biolistic method, which is the only effective method (24).Open in a separate windowFIG. 1.Putative biosynthetic pathway of PUFAs in Mortierella alpina 1S-4. OA, oleic acid; LA, linoleic acid; ALA, α-linolenic acid; GLA, γ-linolenic acid; SDA, stearidonic acid; EDA, n-9 eicosadienoic acid; DGLA, dihomo-γ-linolenic acid; ETA, n-3 eicosatetraenoic acid; MA, Mead acid. Open and black arrows indicate elongase and desaturase reactions, respectively.Agrobacterium tumefaciens-mediated transformation (ATMT) has been employed for a wide range of plants (7, 27). Recently, it was reported that A. tumefaciens is also able to transfer its DNA to various fungi, including ascomycetes, basidiomycetes, zygomycetes, and oomycetes, as well as to plants (2, 5, 16). Additionally, this bacterium can transform intact cells and spores as well as protoplasts. Under mild conditions, the ATMT system generates a large number of stable transformants, which show vigorous growth, indicating that the ATMT system can be an efficient tool for molecular manipulation of M. alpina 1S-4. Moreover, the frequency of homologous recombination was higher than that with conventional transformation methods (8). In this study, we evaluated the external gene transfer system using the ATMT system and determined the optimal conditions for M. alpina 1S-4. Furthermore, we overexpressed the ω3-desaturase gene to improve n-3 PUFA productivity in an industrial n-6-PUFA-producing strain, M. alpina 1S-4 (18, 20), using ATMT.     

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.