Functional dissection of the gamma-tubulin complex by suppressor analysis of gtb1 and alp4 mutations in Schizosaccharomyces pombe

In fission yeast, gamma-tubulin (encoded by the gtb1+ gene), Alp4 (Spc97/GCP2), and Alp6 (Spc98/GCP3) are essential components of the gamma-tubulin complex. We isolated gtb1 mutants as allele-specific suppressors of temperature-sensitive alp4 mutations. Mutation sites in gtb1 mutants and in several alp4 alleles were determined. The majority of substituted amino acids were mapped to a small area on the predicted surface of the gamma-tubulin molecule that might directly interact with the Alp4 protein. The cold sensitivity of gamma-tubulin mutants was almost completely suppressed by an alpha-tubulin mutation and partially suppressed by a low concentration of thiabendazole, a microtubule assembly inhibitor. Other gtb1 mutants had increased resistance to this drug. Gel-filtration and immunoprecipitation analyses suggested that the mutant gamma-tubulin formed an altered gamma-tubulin complex with increased stability compared to wild-type gamma-tubulin. In most gtb1 mutants, sexual development was impaired, and aberrant asci that contained an irregular spore shape and number were produced. In contrast, spore formation was not appreciably damaged in some alp4 and alp6 mutants, even at temperatures where vegetative proliferation was substantially defective. These results suggested that the function of the gamma-tubulin complex or the requirement of each component of the complex is differentially regulated between the vegetative and sexual phases of the life cycle in fission yeast. In addition, genetic data indicated intimate functional connections of gamma-tubulin with several kinesin-like proteins.     

Instability of the apo form of aromatic L-amino acid decarboxylase in vivo and in vitro: implications for the involvement of the flexible loop that covers the active site

Pyridoxine deficiency caused a decrease in the amount of aromatic L-amino acid decarboxylase (AADC) in PC12 cells to less than 5% of the control. The degree of the enzyme saturation with the coenzyme pyridoxal 5'-phosphate (PLP) was around 90% for both the control and the pyridoxine-deficient cells, contrary to earlier reports by others. Mathematical analysis of the result indicated that the AADC apoenzyme is degraded at least 20-fold faster than the holoenzyme in the cells. To determine the mechanism of the preferential degradation of the apoenzyme, in vitro model studies were carried out. AADC has a flexible loop that covers the active site. This loop was easily leaved by proteases at similar rates for both the holoenzyme and the apoenzyme. However, in the presence of the substrate analog, dopa methyl ester, the holoenzyme was not cleaved by proteases, while the apoenzyme was cleaved similarly. These results indicated that the ligand that forms a Schiff base (aldimine) with PLP is fixed to the active site and stabilizes the flexible loop. The structure of the rat AADC-dopa complex modeled on the crystal structure of pig AADC showed that the flexible loop can fit in the concave surface at the entrance of the active site, its aliphatic and aromatic residues forming hydrophobic interactions with the substrate catechol ring. It was postulated that the flexible loop of the holoenzyme is stabilized in vivo by taking a closed structure that holds the PLP-substrate aldimine, while the apoenzyme cannot bind the substrate and its flexible loop is easily cleaved, leading to the preferential degradation of the apoenzyme.     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.     

A revision of the genus Mouretia (Eubiaceae)

Four species are recognised in Mouretia, a genus previously thought to be monospecific. Two new species M. vietnamensis and M. larsenii are described, and M. tonkinensis var_: inequalis is elevated to the rank of species. All species are illustrated. The chromosome number far M. larsenii is given. The character states for the genus are presented. A comparison between Mouretia and Mycetia show them to share most of their character states. It is suggested that the two genera form a monophyletic group, that should be included in the tribe Hedyotideae.     

Detection of DNA of Causative Agent of Spotted Fever Group Rickettsiosis in Japan from the Patient's Blood Sample by Polymerase Chain Reaction

The polymerase chain reaction (PCR) was applied for the etiological diagnosis of spotted fever group (SFG) rickettsiosis in Japan. Nucleotide primers derived from the 17-kDa antigen gene of Rickettsia rickettsii primed a rickettsia-specific 246-base-pair product for all of the Katayama, Abe, Misaka and Kojima strains, which we had isolated previously. Moreover, we were able to detect the same product by PCR amplification from the peripheral blood of a patient in the acute stage of the illness. The PCR method is considered to be useful for rapid etiological diagnosis of SFG rickettsiosis in Japan.     

New bis(macrocyclic) dinickel(II) complexes obtained by oxidation of bis(5,7-dimethyl-1,4,8,11-tetraazacyclotetradeca-4,7-dien-6-yl)dinickel(II) perchlorate

New bis(macrocyclic) dinickel(II) complexes with bis(Me₂[14]-4,7-dien-6-ylidene), 2a and 2b, were synthesized by oxidation of a dinickel(II) complex with an unsaturated bis(macrocyclic) ligand containing four CN bonds, bis(Me₂[14]-4,7-dien-6-yl) (1). Complex 2a was found to undergo intramolecular cyclization between the methyl group of one macrocycle and the carbon atom of the CN group of the other macrocycle to produce a bis(macrocyclic) dinickel(II) complex bridged by a fivemembered ring (3). The structures of 2b and 3 were determined by X-ray crystallography. The nonsymmetrical bis(macrocyclic) structure of the dinickel(II) complex 3 was reflected in its cyclic voltammogram and ¹H and ¹³C NMR spectra. The catalytic capabilities of these bis(macrocyclic) nickel(II) complexes in the reductive debromination of 1-bromo-4-tert-butylbenzene were also investigated.     

Antiviral properties of combination peptides of HIV-1 Rev NLS and NES

Nuclear translocation signal has been identified as a mediator of protein shuttling between nuclear and cytoplasm. Here we report that the combination of peptides from nuclear localization signal (NLS) and nuclear export signal (NES) of HIV-1 Rev have an antiviral activity against the Herpes virus of turkey and Marek's disease virus serotype 1.     

Shoots grafted into the upper crowns of tall Japanese cedar (Cryptomeria japonica D. Don) show foliar gas exchange characteristics similar to those of intact shoots

The lower foliar photosynthetic rates seen in shoots in the upper crowns of tall trees than those in seedlings could be caused by extrinsic factors, such as hydraulic conductance, for shoots or by irreversible intrinsic change in the meristems during tree development. To clarify which is most significant, we compared foliar gas exchange characteristics and water relations among scions of Japanese cedar (Cryptomeria japonica D. Don) grafted into the upper crowns of tall trees, rooted cuttings developed from scions of the same clones, and intact shoots in the upper crowns of the tall trees. Grafted shoots had the same water regime as intact shoots, as confirmed by their similar water potentials at the turgor loss point, which were more negative than those of the rooted cuttings. No significant difference was observed between the grafted and intact shoots in their light-saturated photosynthetic rate (P_max), stomatal conductance (g_s), photosynthetic capacity, carboxylation efficiency, ratio of intercellular to ambient CO₂ concentration (C_i/C_a), and carbon isotope composition (¹³C). Compared with the rooted cuttings, the grafted shoots showed significantly lower P_max, g_s, photosynthetic capacity, and carboxylation efficiency (to 49%, 31%, 68%, and 65%, respectively). The C_i/C_a and ¹³C indicated significantly stronger instantaneous and long-term stomatal limitation in the grafted shoots than in the rooted cuttings. These indicate that changes in extrinsic factors can reduce foliar photosynthetic rates in shoots in the upper crowns of tall trees as a result of stronger stomatal limitation and reduced photosynthetic activity, without irreversible intrinsic changes in the meristems.