Construction of an ordered cosmid collection of the Escherichia coli K-12 W3110 chromosome.

A cosmid library of the Escherichia coli K-12 W3110 chromosome was constructed in which clones were assigned to locations on the chromosome map by hybridization and genetic marker complementation tests. Approximately 70% of the genome was represented by this library. The identified clones can be maintained in the homologous system and would facilitate genetic studies of E. coli.     

Synthesis of protein and mRNA is necessary for transition of suspension-cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G₁ phase by anisoniycio or cycloheximide caused the arrest of cells in the G₁ phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G₁ phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G₁ phase caused the arrest of cells in the G₁ phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G₁ phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G₁ phase, and that the mRNA(s) that encode these proteins are also synthesized at the G₁ phase.     

Ephaptic Transmission and Conduction Velocity of an Action Potential in Chara Internodal Cells Placed in Parallel and in Contact with One Another

When two separated Chara internodal cells were kept in contactover a length of 14 mm or more in moist air, an action potentialof one cell could be transmitted to the other cell in about40% of cases (ephaptic transmission). The action potential ofthe former cell was sometimes eliminated transiently when anew action potential was elicited in the latter cell. The newaction potential reactivated the former cell. The conductionvelocity of the action potential was reduced from 0.30?0.11cm/s (mean?SD) to 0.15?0.05 cm/s by ephaptic transmission ofthe action potential. Substitution of an artificial pond waterfor moist air or contact over a shorter length (4 mm insteadof 14 mm or more) between the two cells reduced the couplingratio, the ratio of the change in membrane potential of onecell to that of the other cell, and no transmission was observed.After ephaptic transmission, the action potentials of the twocells were conducted at almost the same velocity along the cells.The velocity was increased to 0.68?0.06 cm/s by simultaneousconduction of the action potentials in the two adjacent cells.When the simultaneously conducted action potentials reachedthe node of one cell, at the point at which another cell madecontact, they were able very frequently to trigger a new actionpotential in the adjoining cell. (Received September 28, 1989; Accepted April 5, 1990)     

Changes in the Membrane Properties of Nodal Cells of Chara braunii Induced by Exposure to Artificial Pond Water

Nodal cells of Chara braunii were exposed to artificial pondwater (APW) for a month or more, with removal of larger surroundingcells. The membrane resistance, resting potential, amplitudeof the action potential, and threshold voltage measured fromthe resting level were examined. Exposure to APW had markedeffects on membrane properties. (Received April 2, 0990; Accepted August 24, 1990)     

A flow injection analysis system involving immobilized NADH oxidase in column form for clinical analysis.

A highly sensitive FIA system for chemiluminometric determination of reduced coenzyme, NADH, was developed, using immobilized NADH oxidase from Brevibacterium ammoniagenes. The enzyme catalyzed the oxidation of NADH generating hydrogen peroxide which emitted chemiluminescence when mixed with luminol and potassium ferricyanide. The immobilized enzyme reactor was a mini-column, measuring 1 or 2 mm in inner diameter and 20 mm in length, and the sample volume was only 1 microliter per assay, with a feeding speed of one sample per min and a lowest detection limit of 10 pmol NADH. A FIA system was also developed for the determination of magnesium in human serum, using an enzyme column reactor with simultaneously coimmobilized hexokinase, D-glucose-6-phosphate dehydrogenase, and NADH oxidase. The performance of the system was as satisfactory as a routine colorimetric assay, but with much higher sensitivity.     

Canopy structures and its effect on shoot growth and flowering in subalpine forests

Changes in foliage density distribution with altitude and its effect on shoot growth and flowering were studied in forest section planes (profiles) of subalpine forests and scrubs (Krummholz) in Nepal and Japan.Patterns of foliage in forest canopy were evaluated by an analysis of variance. Foliage densities were very high at high altitude canopies, but the change in spatial patterns along altitude differs in both areas.The spatial pattern of new shoot production was similar to that of current foliage density and was affected by the amount of foliage above the sample probably through light condition. Flowering of tall trees occurred in the surface of the upper canopy, but a shrub species flowered even under tree canopies.     

Impairment of Mobility in Endodermal Cells by FAK Deficiency

Focal adhesion kinase (FAK) is a novel nonreceptor protein tyrosine kinase that localizes in focal adhesions. It is expressed in a variety of cell types, and we reported earlier that its deficiency causes a decrease of mobility in mesodermal cells with enhanced formation of focal adhesions. With embryoid bodies generated from embryonic stem cells, we also observed a decrease of mobility in FAK-deficient endodermal cells with enhanced focal adhesion formation.     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Expression of NADH-Dependent Glutamate Synthase in Response to the Supply of Nitrogen in Rice Cells in Suspension Culture

The effects of inorganic and organic nitrogen on the levelsof mRNA for NADH-dependent glutamate synthase (GOGAT) and theprotein were examined in rice cells in suspension culture. Asupply of NH⁺₄, NO^-₃, glutamine, or asparagine induced the accumulationof the protein and mRNA, but levels of mRNA for ferredoxin-GOGATwere hardly affected. ¹Present address: P.C. Center Wakuya-cho, Toda-gun, Miyagi,Japan.     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.