Arbuscular mycorrhizal fungi on adjacent semi-natural grasslands with different vegetation in Japan

Arbuscular mycorrhizal (AM) fungi on Japanese semi-natural grasslands were investigated at three adjacent sites with different vegetation. The predominant grasses at the three sites were 1)Pleioblastus chino, 2)Miscanthus sinensis andArundinella hirta (M. sinensis/A. hirta), and 3)Zoysia japonica, respectively. The degree of colonization was higher inM. sinensis/A. hirta than inP. chino andZ. japonica. AM fungi were recovered by spore extraction and by pot cultures started from soil inoculum or from transplanting of field plants. Total spore number obtained by the spore extraction method was highest in the rhizosphere ofM. sinensis/A. hirta and lowest in that ofP. chino. AGlomus sp. resemblingG. geosporum predominated in association withM. sinensis/A. hirta andP. chino. FromZ. japonica, three species,Acaulospora gerdemannii, Glomus leptotichum, and a species resemblingG. clarum, were isolated by pot culture from soil and two species,A. longula andScutellospora cerradensis, by pot culture from transplanting ofZ. japonica. FromM. sinensis/A. hirta, one species,A. longula, was found by pot culture from soil. FromP. chino, no AM fungus was detected by either method. Single-spore culture confirmed thatG. leptotichum andA. gerdemannii are conspecific.     

The induction of reproductive cell formation of Ulva pertusa Kjellman (Ulvales, Ulvophyceae)

Synchronous zooid formation in Ulva pertusa Kjellman was induced in excised disks maintained in sterilized seawater at 20°C, 12:12 h L:D cycle and fluorescent light at 100 μmol photons m ²s ¹. Zooids were reieased from mature disk tissue on the morning of the second or the third day after excision. The degree of zooid formation was found to be dependent on disk size and the region of the mother thalius from which the disk tissue was excised. Zooid formation was induced in more than 90% of small disks (0.9 mm in diameter) which were taken from the margins of the Ulva thalli. When disks were incubated together with a perforated mother thalius, the disks remained sterile. The presence of maturation inhibitors in vegetative thalli is suggested.     

Development of a sensitive liquid chromatography–electrospray ionization tandem mass spectrometry method for the measurement of 7-cyanoquinocarcinol in human plasma

A sensitive method for the determination of an anti-cancer agent, DX-52-1 (7-cyanoquinocarcinol, I) and quinocarmycin (II) which is formed from I either by metabolism or degradation, in human plasma has been developed utilising liquid chromatography electrospray–ionization tandem mass spectrometry (LC–ESI-MS–MS). The procedure involves solid-phase extraction at pH 2 and low temperature (4–6°C) to prevent the decomposition of I to II, the separation by reversed-phase HPLC and the multiple reaction monitoring (MRM) by ESI-MS–MS. The mean precision and accuracy at the lower limit of quantitation (LLOQ) of I, 0.25 ng ml⁻¹, were 8.7% and −10.8%, respectively. Since an interfering peak eluting slightly earlier than II was observed on the HPLC of blank plasma, the LLOQ of II was set at 5 ng ml⁻¹ where the mean precision and accuracy were 15.6% and −9.8%. The results suggested that the method is useful for the simultaneous monitoring of Iand II in the clinical trials of I.     

Subcellular Distribution and Phosphorylation of the Nuclear Localization Signal Binding Protein, NBP60

We previously purified a nuclear localization signal binding protein, NBP60, from rat liver (1993,J. Biochem.113, 308–313). In this study, the subcellular localization of NBP60 was examined using anti-NBP60. Most NBP60 was found to be localized in the nuclear envelope fraction of rat liver obtained on cell fractionation followed by immunoblotting. Staining of the nuclei of cultured cells by the antibody was observed on immunofluorescence microscopy. NBP60 was widely detected in rat nuclear fractions prepared from other tissues and also in nuclei of cultured cells derived from other species. It was shown by immunoelectron microscopy that most NBP60 is present in the nuclear envelope and at least some of that is present on nuclear pore complexes. Although NBP60 was localized in the nuclear envelope in interphase cells, it diffused into the cytoplasm in the mitotic phase. The purified NBP60 was highly phosphorylated by a cdc2 mitotic kinase, whereas nuclear pore proteins p144, p62, p60, and p54 were not phosphorylated by the kinase directly. NBP60 was also phosphorylated by protein kinase A, calmodulin-dependent protein kinase II, and casein kinase II. The phosphorylation of NBP60 by cdc2 kinase and/or the other kinases may be related to the change in the protein's location during the mitotic phase.     

Application of N-terminally Truncated DNA Polymerase from Thermus thermophilus ({Delta}Tth Polymerase) to DNA Sequencing and Polymerase Chain Reactions: Comparative Study of {Delta}Tth and Wild-Type Tth Polymerases

N-Terminally truncated DNA polymerase from Thermus thermophilus(Tth polymerase) lacking 5'-3' exonuclease activity was usedfor DNA sequencing and polymerase chain reaction (PCR). In contrastto the high background of the sequencing ladder observed withthe wild-type Tth polymerase, Tth polymerase gave readable sequencingpatterns which extend up to more than 500 bases from the primersite on cycle sequencing and automated sequencing. The Tth polymerasewas used for the standard and mutagenic PCR, and net amplificationof the DNA and the mutations accumulated during PCR were analyzed.Under mutagenic PCR, the mutation rates were 7.0 x 10^–4(Tth) and 8.3 x 10^–4 (Tth) per nucleotide per cycle ofamplification, which were 4–9 times higher than the ratesunder standard PCR.     

Evidence for polymorphism of MB3 antigens among three HLA-D clusters associated with HLA-DR4

In a previous study, we showed that the three hitherto serologically indistinguishable HLA-D specificities associated with HLA-DR4, HLA-DYT, HLA-DKT2, and HLA-Dw4 can be distinguished on the basis of their reactivity with two distinct la-like-specific monoclonal antibodies, HU-18 and HU-23. In this study, we attempted to identify and characterize Ia-like molecules recognized by HU-18 and HU-23 on a molecular level because la subsets (HLA-DR, MB, MT, or SB) identified by them remained unknown. The results of sequential coprecipitation assays and two-dimensional gel analyses showed that both HU-18 and HU-23 recognize antigenic determinants borne on M133 but not on HLA-DRw6.2 molecules. Because the two monoclonal antibodies, specific for determinants carried on MB3 molecules, show distinct reactivity against homozygous typing cells defining HLA-DYT, HLA-DKT2, and HLA-Dw4, all of which share DR4-MB3, the data indicate that these three HLA-D clusters associated with HLA-DR4 possess distinct MB3 molecules, suggesting the existence of polymorphism in MB3 antigens.     

A cluster of mutations in HLA-A2 α2 helix abolishes peptide recognition by T cells

In order to investigate the regions of HLA-A2 that control peptide-specific cytotoxic T lymphocyte (CTL) recognition, 37 HLA-A2 genes coding for 50 point mutations that span the 2 helix were synthesized by the technique of saturation mutagenesis. Twenty-nine of these genes, which code for 41 point mutations, were transfected into C1R cells and used as targets in cytotoxicity assays, in the presence of influenza-A matrix peptide 58–68 with specific CTL as effectors. All the transfectants were recognized fully by matrix peptide-specific CTL apart from those with amino acid substitutions at positions 152, 154, 155, 156, or 161, which led to a total loss of recognition and those with mutations at residue 27 or a double mutation at 138 and 150, which were recognized in an intermediate manner. The clustering of the crucial residues that emerges may reflect direct interaction of their side-chains with peptide or the CTL receptor. Address correspondence and offprint requests to: R. J. Moots.     

Change of Cyclic AMP Level in Synaptosomes from Cerebral Cortex; Increase by Adenosine Derivatives

The endogenous level of cyclic AMP in incubated synaptosomes from cerebral cortex of guinea pigs was investigated after the addition of various agents to the incubation medium. It appeared that the synaptosomal suspension already contained exogenous adenosine. Preincubation with theophylline or with adenosine deaminase (ADase) decreased both the exogenous level of adenosine and the intrasynaptosomal level of cyclic AMP. The level of cyclic AMP was reincreased by the addition of adenosine agonists, especially 2-chloroadenosine. This increase was antagonized by deoxyadenosine and was not inhibited by dipyridamole. These results suggest that the adenosine derivatives in the synaptic cleft regulate the level of cyclic AMP in nerve terminals through adenosine receptor on the presynaptic membrane. ADP, ATP, dopamine, and histamine also stimulate the formation of cyclic AMP in the ADase-treated synaptosomes.     

Long term continuous ATP regeneration by enzymes of the alcohol fermentation pathway and kinases of yeast

Summary Continuous ATP regeneration from adenosine using enzymes of the alcohol fermentation pathway, adenosine kinase and adenylate kinase of baker's yeast was investigated using a reactor equipped with a semipermeable membrane. The addition of DTT, which protected the thiol groups of some enzymes against oxidation, increased the duration of the period of ATP formation from 42 h (previously the longest period) to 100 h. With the addition of a yeast extract containing intermediates of alcohol fermentation, a stable steady state was attained in which a yield of more than 75% ATP continued for 2 weeks. These results suggest that the long term continuous ATP formation attained might be due to the protection and stabilization of enzymes by the yeast extract which was added to prevent inactivation.