On the occurrence of a high content of cytokinins in rice callus tissues

Rice callus tissues contained at least three active cytokinincompounds: zeatin, its riboside and N⁶-(²-isopentenyl) adenosine.These butanol-extractable cytokinins were identified by theirchromatographic mobilities in Sephadex LH-20, paper and gaschromatography. Zeatin, the apparent major cytokinin, was presentat concentrations of 0.7 to 1.0 µg/g fresh tissue and1.3 to 1.7 µg/g fresh tissue in 10^–7 M and 10^–55M 2,4-D callus, respectively. On the basis of these and earlierresults, the induction and growth of rice callus tissue is discussedin relation to the occurrence of cytokinins in the tissue. (Received December 27, 1978; )     

Gas chromatographic determination of the hypoglycaemic agent gliclazide in plasma

A gas chromatographic method has been developed that permits the accurate and specific determination of the hypoglycaemic agent gliclazide in plasma. Gliclazide is extracted with chloroform and, after clean-up, derivatized with diazomethane followed by heptafluorobutyric anhydride to form N-methyl-N′-heptafluorobutyrylgliclazide, which is assayed on a gas chromatograph equipped with a flame ionization detector, an electron-capture detector or a nitrogen—phosphorus sensitive detector.Accurate determinations are possible with flame ionization detection over a concentration range of 1–15 μg/ml of gliclazide in plasma with a relative standard deviation of 5.2%. The minimum detectable concentration with electron-capture detection is 0.02 μg per sample. Plasma levels of gliclazide in dogs following single oral administration (40 mg per dog) have also been determined.     

Formation of ribonucleotide 2'',3''-cyclic carbonates during conversion of ribonucleoside 5''-phosphates to diphosphates and triphosphates by the phosphorimidazolidate procedure.

Ribo- and 2'-deoxyribonucleoside 5'-di- or triphosphates are commonly synthesized by reaction of inorganic phosphate or pyrophosphate with phosphorimidazolidates obtained by reaction of nucleoside 5'-phosphates with 1,1'-carbonyldiimidazole. The latter reaction, however, converted UMP, CMP, IMP, GMP, and AMP in high yield to the 2',3'-cyclic carbonate derivatives of their phosphorimidazolidates. Acidic treatment of the product from AMP gave AMP 2',3'-cyclic carbonate dihydrate; this was characterized by its uv, ir, and pmr spectra and by its conversion to adenosine 2',3'-cyclic carbonate by acid phosphatase and to AMP by basic hydrolysis. ADP or ATP synthesized by the phosphorimidazolidate method contained equal or greater amounts of their respective 2',3'-cyclic carbonates. The latter could be quantitatively converted to ADP and ATP, respectively, by 4-hr hydrolysis at pH 10.5, 22 degrees. ADP or ATP can be synthesized without concomitant 2',3'-cyclic carbonate formation by reaction of AMP with phosphorimidazolidates of inorganic phosphate or pyrophosphate.     

Binding of aluminium to DNA of DNP in pea root nuclei

The association of absorbed aluminium (Al) with nuclei in Alaskapea roots was shown by zonal centrifugation of isolated nuclei.In nuclei, 73% of the total incorporated Al was detected inthe DNP fraction. Furthermore, Al in DNP was found to be boundspecifically to DNA by gel filtration of dissociated DNP. (Received March 19, 1977; )     

Control of normal differentiation of myeloid leukemic cells. XIII. Inducibility for some stages of differentiation by dimethylsulfoxide and its disassociation from inducibility by MGI.

There are clones of myeloid leukemic cells that can be induced to differentiate by the normal differentiation-inducing protein MGI to form Fc and C3 rosettes, mature macrophages and granulocytes. One of these clones (MGI+DMSO+) was also inducible by dimethylsulfoxide (DMSO) for C3 but not Fc rosettes, and for mature macrophages but not for mature granulocytes. Other clones (MGI+DMSO-) were inducible by MGI but not DMSO and a third type of clone (MGI-DMSO-) was not inducible by either compound. Clones that differed in their inducibility by DMSO showed a similar inhibition of cell multiplication by DMSO. The results indicate, that some stages of differentiation can be induced by DMSO in an appropriate clone of myeloid leukemic cells and that there are different cellular sites for induction by DMSO and MGI.     

Expression of cDNA for batroxobin, a thrombin-like snake venom enzyme

The cloned cDNA for batroxobin has been expressed in E. coli. Batroxobin could only be obtained as intracellular aggregates of fusion proteins, fused with a small peptide. To obtain the mature batroxobin, the recognition sequence for thrombin was inserted between the peptide and the mature batroxobin. This fusion protein accumulated in an insoluble form and could easily be purified. After site-specific cleavage of the fusion protein with thrombin, recombinant batroxobin was isolated by preparative electrophoresis. Batroxobin with enzymatic activity was obtained by the refolding of recombinant batroxobin.     

Two genes, atpC1 and atpC2, for the gamma subunit of Arabidopsis thaliana chloroplast ATP synthase.

Arabidopsis thaliana has two genes (atpC1, atpC2) coding for gamma subunits of chloroplast ATP synthase. The atpC1 and atpC2 were cloned and sequenced. They had no introns within the reading frames and coded for proteins of 373 and 386 amino acid residues, respectively, including putative transit sequences (50 and 60 amino acid residues, respectively). In contrast, the spinach gamma subunit gene had two introns within the reading frame. The mature sequences coded by the two genes of A. thaliana (atpC1, 323 residues; atpC2, 326 residues) were homologous with that of spinach (J. Miki, M. Maeda, Y. Mukohata, and M. Futai (1988) FEBS Lett. 232, 221-226): the homologies of gamma subunits coded by atpC1 and atpC2 were 72%, those of the subunits coded by atpC1 and spinach cDNA were 84%, and those of the proteins coded by atpC2 and spinach cDNA were 71%. Like the spinach subunit, the gamma subunits coded by the two genes had unique regulatory domains not found in mitochondrial or bacterial subunits. Poly(A)+ mRNAs corresponding to atpC1 (1.5 kilobases) and atpC2 (2.5 kilobases) were detected in illuminated plants, the amount of the former being at least 140 times that of the latter. The atpC1 mRNA was not found in dark-adapted plants. Nuclear protein(s) specifically bound to the upstream region of atpC1 was detected by gel shift assay and its binding was shown to be inhibited by the GT-1 element of the gene encoding the ribulose-1,5-bisphosphate carboxylase small subunit, which is expressed under illumination (P. J. Green, S. A. Kay, and N. H. Chau (1987) EMBO J. 6, 2543-2549). Consistent with these findings, an increased amount of the gamma subunit was detected immunochemically in illuminated plants.     

One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters.

About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes. The recombinant plasmid pBWU13 carries the unc operon for F0F1. When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential. Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis. F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification.