The occurrence and properties of dihydrofolate reductase in pea seedlings

Dihydrofolate reductase (E.C. 1.5.1.3 [EC] ) was found in pea seedlingsand was partially purified by treatments with ammonium sulfate,protamine sulfate and by DEAE-cellulose column chromatography.Some properties of the enzyme were investigated. Optimum pHfor the reaction was 6.5. In the enzyme reaction, FAH₂ and NADPH₂were specifically required. MICHAELIS constants for FAH₂ andNADPH₂ were 4.3x10^–6 M and 4.0x10^–5 M, respectively.Folate antagonists such as aminopterin, methotrexate and pyrimethaminewere potent inhibitors of this enzyme. Enzyme activity was almostcompletely inhibited at a concentration of 10^–7 M of aminopterinand methotrexate and 10^–6 M of pyrimethamine. Growth of germinating pea seeds was inhibited by aminopterin,methotrexate and pyrimethamine, and it recovered significantlywith a tetrahydro-derivative of folate, CF, but not with dihydrofolicor folic acid. These results suggest that growth inhibitionof pea seedlings by these antagonists is due to inhibition ofdihydrofolate reductase in seedlings. ¹Studies on the enzymatic synthesis and metabolism of folatecoenzymes in plants IV. (For the previous paper, Part III, seeReference (21)) . Part of this paper was presented at the AnnualMeeting of the Agricultural Chemical Society of Japan held atTokyo on April 4, 1967 (Received October 8, 1969; )     

Biosynthesis of folic acid compounds in plants VI. The occurrence and properties of the dihydrofolate-synthesizing enzyme in pea seedlings

An enzyme, which catalyzes the formation of dihydrofolate fromdihydropteroic acid and L-glutamic acid, was found in pea seedlings.The enzyme was purified approximately 25-fold from the crudeextracts of pea seedlings, and its some properties were investigated.Optimum pH for the enzyme activity was found to be 8.8. Pteroicand tetrahydropteroic acids were not active as substrate. Theenzymatic reaction required as cofactors ATP, divalent (Mg²⁺or Mn²⁺) and univalent (K⁺, NH₄⁺ or Rb⁺) cations. The productwas characterized as dihydrofolic acid by bioautography. MICHAELIS constants for L-glutamic acid, ATP, dihydropteroicacid and Mg²⁺ were 7.0x10^–4, 9.0x10^–5, 3.5x10^–6and 1.2x10^–3 M, respectively. The MICHAELIS constant forMn²⁺ was 3.0x10^–4. The enzyme was inhibited by PCMB orsilver nitrate and, to some extent, by L-aspartic acid. Inhibitionby PCMB was completely reversed by addition of 2-mercaptoethanol.Enzyme activity was distributed widely among plants. The importanceof magnesium and potassium ions for enzyme catalysis is discussed. ¹For the previous paper, Part V, see Reference (30). (Received March 28, 1970; )     

CHANGES IN THE CAPACITY FOR CLONAL GROWTH AND DIFFERENTIATION IN VITRO OF THE VERTEBRAL CARTILAGE CELLS WITH EMBRYONIC DEVELOPMENT. 1. CULTURE IN THE STANDARD MEDIUM

When culturing the vertebral cartilage cells of the chick embryo, the youngest embryo from which the dissociated single cell of the cartilage gives rise to the differentiated cartilage colony (CMC) in the given conditions of the clonal cell culture is at st.26. At this stage, percentage of such cells to make CMC in vitro is less than 0.1% of the inoculated single cells. With development, percentage of such “stabilized” cells increases, finally reaching to 35% at st.40. In embryos from st.29 to st.36, many of the cultured single cells give rise to the regressing colonies (RGC) which show some proliferation at the early stage in vitro, but in due time cease to proliferate and regress.     

Detection of recombinant haplotypes in wild mice (Mus musculus) provides new insights into the origin of Japanese mice

Japanese house mice (Mus musculus molossinus) are thought to be a hybrid lineage derived from two prehistoric immigrants, the subspecies M. m. musculus of northern Eurasia and M. m. castaneus of South Asia. Mice of the western European subspecies M. m. domesticus have been detected in Japanese ports and airports only. We examined haplotype structuring of a 200 kb stretch on chromosome 8 for 59 mice from throughout Eurasia, determining short segments (≈ 370–600 bp) of eight nuclear genes (Fanca, Spire2, Tcf25, Mc1r, Tubb3, Def8, Afg3l1 and Dbndd1) which are intermittently arranged in this order. Where possible we identified the subspecies origin for individual gene alleles and then designated haplotypes for concatenated alleles. We recovered 11 haplotypes among 19 Japanese mice examined, identified either as ‘intact’ haplotypes derived from the subspecies musculus (57.9%), domesticus (7.9%), and castaneus (2.6%), or as ‘recombinant’ haplotypes (31.6%). We also detected recombinant haplotypes unique to Sakhalin. The complex nature of the recombinant haplotypes suggests ancient introduction of all three subspecies components into the peripheral part of Eurasia or complicated genomic admixture before the movement from source areas. ‘Intact’domesticus and castaneus haplotypes in other Japanese wild mice imply ongoing stowaway introductions. The method has general utility for assessing the history of genetic admixture and for disclosing ongoing genetic contamination.     

Radial electrogenic activity in the stem of Vigna sesquipedalis: involvement of spatially separate pumps

Abstract. The surface electric potential of Vigna sesquipedalis stem, relative to that of the root medium, was most negative in the elongating zone where the difference in pH between parenchyma (P) and xylem sap (X) was at a maximum. The surface electrical potential was found to be determined by the distribution of radial potential difference ( V _sx comprised two components; V_sx= V _px− V _ps.There were two electromotive forces, generating V _ps at the surface of the parenchyma symplast and V _px between P and X; both were partially dependent on respiration. The depolarization of V _px upon removal of O₂ was maximal in the elongating zone, suggesting that the activity of an H⁺-pump extruding protons from the parenchyma into the xylem in exchange for K⁺ was greatest in this zone.     

The Most Appropriate Position and Number for Absolute Anchorages for Orthodontic Tooth Movements

Absolute anchorages proved to be very effective for orthodontic tooth movements. We used a 3D digitizer to record each tooth on pre-treatment diagnostic and post-treatment predictive setup models and then 3D coordinate system conversion was performed to make the coordinate values comparable. An arithmetic calculation of vector and moment based on the orthodontic forces and the tooth displacement under preliminary premises undertaken to decide the most favorable pOsition and number for absolute anchorages. Position-For two-dimensional and three-dimensional calculations, the most appropriate positions for absolute anchorages should theoretically be on the line of resultant force (2D) and the plane (3D) where the total moment effect tends to be zero. Number-As for the number of the absolute anchorages needed, it depends on the number of target teeth. Different combinations of target teeth provide different sets of results.     

PRECISE MEASUREMENT OF THE CHANGE OF STATISTICAL DISTRIBUTION OF CELL SIZE OCCURRING DURING THE SYNCHRONOUS CULTURE OF CHLORELLA

Change of statistical distribution of cell size occurring inthe synchronous culture of Chlorella ellipsoidea was followedby using a COULTER counter. The culture was started using apopulation of D-cells which showed a sharp distribution of cellsize. During the growth phase, there occurred characteristicchanges in the height and shape of the distribution curve; namely,the height first decreased with broadening of the distributionand then increased to the level even higher than the originalpeak. The broadening of the curves, which indicates the loweringof degree of homogeneity of population, occurred during theperiod of most active growth, and the homogeneity was restoredat later stages of ripening where the growth became sluggish.When the ripened cells (L₃) were transferred to the darknesstheir volume decreased to some extent before the occurrenceof cellular division. It was assumed that the shrinkage of cellsobserved may be partly due to exudation of water from the cellsand partly to the consumption of fuel material caused by enhancedrespiration, both having been shown to occur at the stage ofcell maturation. (Received June 12, 1964; )