Nucleosidediphosphate kinase from Ehrlich ascites tumor cells

A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.     

Isolation of functional cDNA clones for human thymidylate synthase

Thymidine auxotrophic mutants of mouse FM3A cells due to thymidylate synthase deficiency can be transformed into prototrophs by DNA-mediated gene transfer using total human DNA (Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., and Seno, T. (1983) J. Biol. Chem. 258, 48-53). From one such transformed cell clone, cloned recombinant lambda phages containing DNA fragments were obtained recently that were concluded by circumstantial genetic evidence to have been derived from the human thymidylate synthase gene (Takeishi, K., Ayusawa, D., Kaneda, S., Shimizu, K., and Seno, T. (1984) J. Biochem. (Tokyo) 95, 1477-1483). Using a DNA segment derived from the cloned genomic DNA fragment and free of repetitive sequences as a probe, functional cDNA corresponding to thymidylate synthase mRNA could be cloned from a cDNA library of SV40 transformed human fibroblasts constructed by Okayama and Berg (Okayama, H. and Berg, P. (1983) Mol. Cell. Biol. 3, 280-289). The cloned cDNA plasmid containing an insert of approximately 1.7-kilobase transformed mouse thymidine auxotrophic mutant cells to thymidine prototrophic cells at a frequency of 2-3 transformants/micrograms of DNA/10(5) cells, a value almost comparable to the highest so far reported. The resultant transformants retained the introduced cDNA and expressed human thymidylate synthase protein sufficient for supporting normal growth of otherwise auxotrophic mouse cells.     

Folic acid as a dietary factor affecting the wing morph of the planthopper, Laodelphax striatellus (hemiptera, delphacidae)

A factor effecting the production of brachypterous adults in Laodelphax striatellus was studied by comparing a diet on which brachypterous adults sometimes developed and a diet on which they never developed. By substituting components distinctive of the latter diet into the former, or by varying the concentration of certain components of the latter diet, the production of brachypterous adults was found to vary with the concentration of a vitamin, folic acid. Brachypterous adults were produced only when the diet contained folic acid at concentrations between 0.5 mg/l and 7.5 mg/l.

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Résumé Un facteur efficace pour la production d'adultes brachyptères chez Laodelphax striatellus a été recherché en comparant une nourriture sur laquelle des adultes brachyptères se développent parfois et une nourriture sur laquelle ils ne se développent jamais. En substituant les constituants spécifiques de cette dernière par ceux de la première ou en variant les concentrations de certains des constituants de la dernière on a montré que la production d'adultes brachyptères varie avec la concentration d'une vitamine, l'acide folique. Ceux-ci sont produits seulement lorsque l'alimentation contient de l'acide folique à des concentrations comprises entre 0,5 mg/l et 7,5 mg/l.

     

Necessity of IgE antibodies and mast cells for manifestation of resistance against larval Haemaphysalis longicornis ticks in mice

Genetically mast cell-deficient WBB6F1-W/Wv mice showed an apparent defect in manifestation of the resistance against larval Haemaphysalis longicornis ticks, but their serum IgE levels increased more than 100-fold after the second tick infestation. Immune sera obtained from the WBB6F1-W/Wv mice were adoptively transferred to the other WBB6F1-W/Wv mice which had received intracutaneous injections of WBB6F1-+/+ mouse-derived cultured mast cells. Because the resistance against ticks was detectable only when both mast cells and IgE antibodies were available, immediate hypersensitivity reaction appeared to have a physiologic role in the manifestation of the resistance against H. longicornis ticks.     

Utilization of Anhydrous Aluminum Phosphate as a Sole Source of Phosphorous by a Selected Carrot Cell Line

Two variants of carrot (Daucus carota L. cv. MS Yonsun) celllines, which had been selected with Al-phosphate as a sole sourceof phosphorous, were characterized on their mechanisms of phosphate-utilizationfrom Al-phosphate. Both cell lines excreted citrate into themedium. The amount of citrate excretion was highly correlatedwith cell growth in the presence of Al-phosphate. There wasabout a 1 to 1 correlation between solublized-Al and excreted-citratein the medium during cell growth. These results suggest that1) the citrate could chelate with Al, at a 1 to 1 ratio, inAl-phosphate, 2) the citrate-chelated Al remains outside thecells, and 3) solublized phosphate from Al-phosphate is utilizedfor the growth of carrot cells. The characteristics of the selectedcells were very stable, since the rate of citrate-excretionshowed no change after subculturing 25 passages without Al-phosphate. (Received August 7, 1989; Accepted October 19, 1989)     

Demonstration of Antigenic and Genotypic Variation in Orientia tsutsugamushi Which Were Isolated in Japan,and Their Classification into Type and Subtype

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.     

Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing

Factors affecting viability of IVF-derived bovine blastocysts after freezing and thawing were investigated. A total of 1,101 ova matured and fertilized in vitro were cultured under 2 different conditions, 1) in TCM-199 on granulosa cell monolayers at 5% CO(2) in air and 2) in synthetic oviduct fluid (SOF) medium without somatic cell support at 5% CO(2), 5% O(2), 90% N(2). All blastocysts that developed from the 2 different culture systems were individually classified into 4 grades of embryo quality and were then frozen by conventional slow freezing. Developmental rates of the IVF-derived ova to blastocysts and the survival rates of the frozen-thawed blastocysts were not different between the SOF medium (16 and 49%) and the co-culture system (13 and 61%, respectively). Survival of frozen-thawed blastocysts was affected by embryo quality in both the SOF and co-culture systems (P<0.001). Blastocysts produced in vitro were also individually classified into 3 developmental stages and were then cultured for 3 d in the co-culture system with granulosa cells after freezing and thawing. There was a difference in the survival rate of frozen-thawed embryos between blastocyst developmental stages (early vs mid, P<0.05; mid vs expanded, P<0.01; early vs expanded, P<0.001). The post-thawing survival rate of blastocysts frozen at Day 7 (62%) of culture was higher compared with that of Day 8 (45%), but there was no difference in survival rate between Day 7 and 8 of culture. The results indicate that the quality and developmental stage of blastocysts are important factors influencing their survival after freezing and thawing.