Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

Identification of a 4-fluorobenzyl l-valinate amide benzoxaborole (AN11736) as a potential development candidate for the treatment of Animal African Trypanosomiasis (AAT)

Novel l-valinate amide benzoxaboroles and analogues were designed and synthesized for a structure-activity-relationship (SAR) investigation to optimize the growth inhibitory activity against Trypanosoma congolense (T. congolense) and Trypanosoma vivax (T. vivax) parasites. The study identified 4-fluorobenzyl (1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-l-valinate (5, AN11736), which showed IC₅₀ values of 0.15?nM against T. congolense and 1.3?nM against T. vivax, and demonstrated 100% efficacy with a single dose of 10?mg/kg against both T. congolense and T. vivax in mouse models of infection (IP dosing) and in the target animal, cattle, dosed intramuscularly. AN11736 has been advanced to early development studies.     

Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii- environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene

The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned; however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatching enzyme is considered to be well adapted to the natural hatching environment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the products alternatively spliced from the LCE gene are generated by the mutations in intronic regions responsible for splicing.     

A simple method for enrichment of uninucleate conidia of Aspergillus oryzae

Aspergillus oryzae produces multinucleate conidia, which makes the obtaining of homokaryons labor-intensive. Analysis of conidia by flow cytometry clarified the relationship that conidia of lower nuclear number were smaller in size. Based on this, we have developed a simple way to enrich uninucleate conidia with a membrane filter. Our results also suggest that the method is useful for elimination of heterokaryons.     

Thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) from Lenzites betulinus

A thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) was purified from the culture medium of Lenzites betulinus by ion exchange chromatography, gel filtration and isoelectric focusing chromatography. The MnP purified from L. betulinus (L-MnP) has a molecular mass of 40 kDa and its isoelectric point was determined to be 6.2. The first 19 amino acids at the N-terminal end of the L-MnP sequence were found to exhibit 74% identity with those of a Phlebia radiata MnP. L-MnP was proved to have the highest hydrogen peroxide tolerance among MnPs reported so far. It retained more than 60% of the initial activity after thermal treatment at 60°C for 60 min, and also retained more than 60% of the initial activity after exposure to 10 mM hydrogen peroxide for 5 min at 37°C.     

Galectin-9 Ameliorates Clinical Severity of MRL/lpr Lupus-Prone Mice by Inducing Plasma Cell Apoptosis Independently of Tim-3

Galectin-9 ameliorates various murine autoimmune disease models by regulating T cells and macrophages, although it is not known what role it may have in B cells. The present experiment shows that galectin-9 ameliorates a variety of clinical symptoms, such as proteinuria, arthritis, and hematocrit in MRL/lpr lupus-prone mice. As previously reported, galectin-9 reduces the frequency of Th1, Th17, and activated CD8⁺ T cells. Although anti-dsDNA antibody was increased in MRL/lpr lupus-prone mice, galectin-9 suppressed anti-dsDNA antibody production, at least partly, by decreasing the number of plasma cells. Galectin-9 seemed to decrease the number of plasma cells by inducing plasma cell apoptosis, and not by suppressing BAFF production. Although about 20% of CD19^−/low CD138⁺ plasma cells expressed Tim-3 in MRL/lpr lupus-prone mice, Tim-3 may not be directly involved in the galectin-9-induced apoptosis, because anti-Tim-3 blocking antibody did not block galectin-9-induced apoptosis. This is the first report of plasma cell apoptosis being induced by galectin-9. Collectively, it is likely that galectin-9 attenuates the clinical severity of MRL lupus-prone mice by regulating T cell function and inducing plasma cell apoptosis.     

A ganoderic acid derivative,a highly oxygenated lanostane-type triterpenoid from Ganoderma lucidum

Ganoderic acid C, a new lanostane-type triterpenoid was isolated from the fruit body of Ganoderma lucidum. The structure of ganoderic acid C was elucidated by spectroscopic data and X-ray analysis of methyl ganoderate C acetate.     

Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus

Transfer RNA (m7G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m7G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNAPhe and E. coli tRNAThr. It was confirmed that 5'-leader and 3'-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.     

Biotransformation of progesterone by suspension cultures of Digitalis purpurea cultured cells

It has been shown that the cultured cells of Digitalis purpruea are capable of transforming progesterone (I) to 5α-pregnane-3,20-dione (II), 5α-pregnan-3β-ol-20-one (III), its glucoside (IV), 5α-pregnane-3β,20α-diol (V), its glucoside (VI), 5α-pregnane-3β,20β-diol (VII), its glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one (IX), its glucoside (X), Δ-pregnen-20β-ol-3-one (XI) and its glucoside (XII). 5α-Pregnan-3β-ol-20-one glucoside (IV), 5α-pregnane-3β,20α-diol glucoside (VI), 5α-pregnane-3β,20β-diol glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one glucoside (X) and Δ⁴-pregnen-20β-ol-3-one glucoside (XII) have been found for the first time as new metabolises by plant tissue cultures. A scheme for the biotransformation of progesterone (I) has been proposed, and the reduction and glucosidation activities distinctly have been observed in these cultured cells.     

Production of theanine and other γ-glutamyl derivatives by Camellia sinensis cultured cells

The maximum theanine production by Camellia sinensis cultured cells was achieved by culturing in the modified MS medium containing 2 mg/l indole-3-butyric acid, 0.1 mg/l kinetin, 0 mM NH₄NO₃ and 39.6 mM KNO₃ with 40 mM ethylamine hydrochloride or 20 mM ethylamine hydrochloride and 10 mM L-glutamic acid. Other primary amines, such as methylamine, n-butylamine, 2-hydroxyethylamine, 2cyanoethylamine, aniline, benzylamine and phenylethylamine, were also biotransformed to N⁵-alkyl-L-glutamine derivatives by C. sinensis cultured cells.Abbreviations IBA indole-3-butyric acid - K kinetin - MS medium Murashige and Skoog's basal medium (1962) - NMR nuclear magnetic resonance Part 70 in the series Studies on Plant Tissue Culture. For Part 69 see Furuya et al. (1990).