Analysis of lipid components in zooplankter individuals by reactive pyrolysis-gas chromatography in the presence of organic alkali.

It has been eagerly requested to develop a highly-sensitive method to characterize extremely minute amounts of natural organic materials occluded in meteorites and/or space dusts in order to confirm the existence of life in the extraterrestrial space. In this article, the reactive pyrolysis-gas chromatography (Py-GC) applied to the analysis of the lipid components contained in every zooplankter individual is introduced for the sake of its potential extension to the characterization of trace amounts of the extraterrestrial organic materials. Here, Py-GC was applied to 1) the discriminative analysis among zooplankter individuals cultured in different food concentrations, and 2) the correlation analysis between the distributions of fatty acid components in the lipids of zooplankter individuals and ingested algae phytoplankton.     

Evidence for an essential histidyl residue at the active site of pyridoxamine (pyridoxine)-5'-phosphate oxidase from rabbit liver.

Pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) from rabbit liver is inactivated by diethylpyrocarbonate in an all-or-none fashion with first order kinetics with respect to modifier concentration. The rate of inactivation increases with pH and reflects a group with a pKa of 7.5. Inactivated enzyme is in the holo form with intact FMN. Four histidyls and a cysteinyl residue are modified by excess reagent. The restoration of enzymatic activity by hydroxylamine, the spectrophotometric and colorimetric amino acid analyses, and our previous studies on cysteine modification (Tsuge, H., and McCormick, D.B. (1979) in Flavins and Flavoproteins (Yamano, T., and Yagi, K., eds) Japan Scientific Societies Press, Tokyo, in press) all suggest that inactivation occurs solely by modification of histidine. Analyses by kinetic and statistical methods indicate that three histidines are modified slowly and are not critical for activity, while one histidine is modified nine times more rapidly and accounts for the observed inactivation. Inactivated enzyme shows no significant perturbations in structure, as evidenced by absorption, CD, fluorescence, and gel filtration, but is unable to bind the product, pyridoxal 5'-phosphate. Furthermore, the substrate-competitive inhibitor, pyridoxal 5'-phosphate oxime, protects from inactivation. Hence, diethylpyrocarbonate inactivates this enzyme by modifying a crucial histidyl residue at the substrate/product-binding site.     

A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3

Two distinguishable activity bands for dye-linked l-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an alpha4beta4 structure, while PDH2 has an alphabetagammadelta structure. PDH2 was found to be similar to the dye-linked l-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the beta subunit bound FAD and exhibited proline dehydrogenase activity, while the alpha subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the alpha and beta subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the alpha subunit of PDH1 clearly differs from that in the alpha subunit of PDH2. It thus appears that a second novel dye-linked l-proline dehydrogenase complex is produced in P. horikoshii.     

Multiple large segment deletion method for Corynebacterium glutamicum

A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering.     

Isolation and characterization of a native composite transposon, Tn14751, carrying 17.4 kilobases of Corynebacterium glutamicum chromosomal DNA

A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 x 10(2) transformants per mug of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.     

Crystal growth and solid-state structure of poly(lactide) Stereocopolymers

Solid-state structure and melting behavior for random stereocopolymers of L-lactide with meso-lactide (P(L-LA-co-meso-LA)) with different meso-LA compositions of 0, 2, 4, and 10 mol % were investigated under various isothermal crystallization conditions. The crystalline morphology of P(L-LA-co-meso-LA) samples changed from the spherulitic aggregates to hexagonal lamellae stacking with a rise in crystallization temperature. Under each crystallization condition, P(L-LA-co-meso-LA) samples formed alpha-crystal modifications for homopolymer of L-LA. By using the atomic force microscopy and small-angle X-ray scattering, the stacking structure of lamellar crystals was examined for the isothermally crystallized P(L-LA-co-meso-LA) thin films. The lamellar thickness of P(L-LA-co-meso-LA) ranged from 6.2 to 15.5 nm, and the values increased with crystallization temperature. Melting profiles of crystalline regions were examined by the differential scanning calorimetry (DSC) for the P(L-LA-co-meso-LA) samples. Distinct two melting peaks were detected in the DSC thermograms of several samples. Investigations on the time-dependent changes in lamellar structure and melting temperature of the P(L-LA-co-meso-LA) samples under isothermal crystallization conditions provided the evidence that a small amount of D-lactyl units was trapped in the crystalline regions during early stage of crystallization process under the certain crystallization condition. In addition, it was found that the D-lactyl units trapped in crystalline regions were excluded from crystalline lamellae to form the thermally stable crystals without changes in crystal thickness during further isothermal storage at a crystallization temperature. The equilibrium melting temperature (T(m)0) of P(L-LA-co-meso-LA) samples was estimated by using modified Hoffman-Weeks methods, and the obtained values decreased from 215 to 184 degrees C as the meso-LA composition was increased from 0 to 10 mol %. Furthermore, the crystal growth kinetics of the P(L-LA-co-meso-LA) samples was analyzed by using the secondary nucleation theory. Transitions of crystalline regime both from regime III to regime II and from regime II to regime I were detected for each sample. The transition temperature from regime II to regime I of each of the P(L-LA-co-meso-LA) samples was very close to the temperature region revealed the morphological changes in the crystalline aggregates from the spherulitic aggregates to hexagonal lamellae stacking.     

Stabilization of cauliflower mosaic virus P3 tetramer by covalent linkage

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein (P3) that is indispensable for viral infectivity. Although P3 has been shown to be a prerequisite for CaMV aphid transmission, its role in viral replication remains unknown. We previously showed that P3 forms a tetramer in planta and that P3 tetramer co-sediments with viral coat protein on sucrose gradient centrifugation, suggesting that a tetramer may be the functional form of P3. We presumed that disulfide bonds were involved in tetramer formation because 1) the tetramer was detected by Western blotting after electrophoresis under non-reducing conditions, and 2) the cysteine-X-cysteine motif is well conserved in CaMV P3 and P3 homologues among Caulimoviruses. Therefore we mutated either or both of the cysteine residues of CaMV P3. The mutant viruses were infectious and accumulated to a similar extent as the wild-type. An analysis of mutant proteins confirmed that the wild-type P3 molecules in the tetramer are covalently bound with one another through disulfide bonds. It was also suggested that mutant proteins are less stable than wild-type protein in planta. Furthermore, sedimentation study suggested that the disulfide bonds are involved in stable association of P3 with CaMV virions or virion-like particles, or both. The mutant viruses could be transmitted by aphids. These results suggested that the covalent bonds in P3 tetramer are dispensable for biological activity of P3 under experimental situations and may have some biological significance in natural infection in the field.