Establishment of a Sensitized Immunoblotting Method for Measuring Plant Tubulin Content

A sensitized immunoblotting method was established for measuringsmall amounts of plant tubulin. The method involves electrophoretictransfer of protein including tubulin from SDS-polyacrylamidegels onto nitrocellulose paper, successive incubation of thenitrocellulose paper with a mouse monoclonal antibody to - orß-tubulin of chicken brain, an antibody to mouse IgGas the second antibody and the radioactive iodinated proteinA, and determination of the radioactivities of the bands onthe nitrocellulose paper thus probed. The radioactivities werelinearly proportional to the amounts of - or ß-tubulinfrom dark-grown Vigna mungo seedlings within a range of 4 to56 ng or of 4 to 32 ng, respectively. This method was used to estimate the tubulin contents of severalplant species using Vigna tubulin as a standard. -Tubulin contentsthus estimated were 25, 9, 19 and 11 µg-equivalents ofVigna tubulin per mg protein for Vigna seedlings, Daucus suspensioncells, Catharanthus suspension cells and Mougeolia cells, respectively.ß-Tubulin contents of Vigna, Daucus, Catharanthusand Mougeotia cells were 29, 10, 13 and 5 µg-equivalentsof Vigna tubulin per mg protein, respectively. (Received August 6, 1985; Accepted December 5, 1985)     

Effects of Nutrient Limitation and {gamma}-Irradiation on Tracheary Element Differentiation and Cell Division in Single Mesophyll Cells of Zinnia elegans

The effects of nutrient limitation and -irradiation on trachearyelement differentiation and cell division were investigatedusing single cells isolated from the mesophyll of Zinnia elegans.When the phosphate concentration of the medium was reduced to10 µM (1/50 of Fukuda and Komamine's medium, 1980a), thefrequency of cell division during 4 days of culture decreased,while the frequency of tracheary element differentiation wasunaffected. -Irradiation with a dose of 92 Gy at 36 h of culturepreferentially and thoroughly suppressed cell division withoutreducing the number of tracheary elements formed. The appearanceof secondary cell wall thickenings was delayed by irradiation,but synchrony was maintained. Thus the Zinnia system previouslyreported [Fukuda and Komamine (1980a) Plant Physiol. 65: 57]was improved to give a more useful system for the study of cytodifferentiation,in which tracheary element formation occurred from single cellswithout cell division. ¹Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted February 22, 1986)     

Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence

Structures of glycolipids present in the human embryonal carcinoma cell PA1, were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide, sialosylgangliotriaosylceramide, sialylated and nonsialylated lacto-N-neotetraosylceramide, and the following glycolipids with a blood group type 1 sequence: (formula; see text) The two former glycolipids, lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present in many of the human embryonal carcinoma cells but not in a variety of other cell lines, suggesting that sialylated but not fucosylated blood group type 1 sequences are characteristic markers for human embryonal carcinoma cells and malignant teratocarcinomas.     

Isolation and characterization of a new endo-beta-galactosidase from Diplococcus pneumoniae

An endo-beta-galactosidase, which hydrolyzes the internal beta-galactosidic linkages of R----GlcNAc (or GalNAc) beta 1----3Gal beta 1----4GlcNAc (or Glc), was isolated from the culture supernatant of Diplococcus pneumoniae. The enzyme, named endo-beta-galactosidase DII, hydrolyzed linear N-acetyllactosamine repeating structures in glycolipids and glycopeptides to release oligosaccharides. The specificity of endo-beta-galactosidase DII is the same as that of Escherichia freundii endo-beta-galactosidase as far as described above, but the following differences between these two enzymes were found: Branched lactosaminyl glycolipids and H-antigenic glycolipids were resistant to endo-beta-galactosidase DII, even when linear structure was present at the inner part. Throughout the enzymic hydrolysis, endo-beta-galactosidase DII released mostly small oligosaccharides (tetra-, tri-, and disaccharides) from substrates, suggesting that the enzyme split off the oligosaccharides stepwise from the nonreducing terminal. Lactosaminoglycans were partially hydrolyzed by endo-beta-galactosidase DII to produce small oligosaccharides as the major product and residual glycopeptides. The residual glycopeptides were readily hydrolyzed by E. freundii endo-beta-galactosidase to produce various sizes of oligosaccharides. Keratan sulfate was not degraded by endo-beta-galactosidase DII. These properties of endo-beta-galactosidase DII characterize it as a new endo-beta-galactosidase with a unique specificity.     

Embryonal lactosaminoglycan. The structure of branched lactosaminoglycans with novel disialosyl (sialyl alpha 2----9 sialyl) terminals isolated from PA1 human embryonal carcinoma cells

Lactosaminoglycan glycopeptides were isolated from human PA1 embryonal carcinoma cells and their structures were elucidated. The glycopeptides were digested by Escherichia freundii endo-beta-galactosidase before and after the modifications by exoglycosidases. The core glycopeptides and oligosaccharides thus obtained and the intact glycopeptides were analyzed by methylation, fast atom bombardment-mass spectrometry, and high-performance liquid chromatography. Based on these experiments, the structures of PA1 lactosaminoglycans were found to have the following unique features. 1) Three lactosaminoglycan fractions of different molecular weights were isolated by Sephadex G-50 gel filtration. Lactosaminoglycans of the highest molecular weight (GpI) have tetra-antennary cores, those of intermediate molecular weight (GpII) have triantennary cores and those of low molecular weight (GpIII) have triantennary and tetra-antennary cores. 2) GpI is composed of 22-26 lactosaminyl units and 7-9 branched galactose residues, GpII is composed of 16-22 lactosaminyl units and 5-7 branched galactose residues, and GpIII is composed of 12-16 lactosaminyl units and 3-4 branched galactose residues. 3) Each branch is short and is composed of the Gal beta 1----4GlcNAc beta 1----6 structure. 4) Sialic acid is preferentially linked to nonreducing terminal regions and a significant amount of the novel disialosyl structure, NeuNAc alpha 2----9NeuNAc alpha 2----3/6Gal, is present at the terminals of the longer polylactosaminyl side chains. 5) These lactosaminoglycans are carried by cell surface glycoproteins of Mr = 80,000 approximately 120,000, as evidenced by lectin-agarose chromatography.     

Structure of the carbohydrate units of human amniotic fluid fibronectin

Human amniotic fluid fibronectin was found to contain three types of carbohydrates: complex-type N-glycosidic glycans, lactosaminoglycans, and O-glycosidic glycans. The structures of the complex-type glycans were established by carbohydrate and methylation analysis, Smith degradation, sequential exoglycosidase treatments, lectin chromatography, and DEAE-Sephadex chromatography. Lactosaminoglycans were analyzed by fast atom bombardment mass spectrometry, and the O-glycosidically-linked oligosaccharides by gas-liquid chromatography-mass spectrometry and high-pressure liquid chromatography. The results show that amniotic fluid fibronectin contains 2 mol of biantennary and 2-3 mol of triantennary, complex-type N-glycosidic glycans. Unlike the N-glycosidic glycans of human adult plasma fibronectin, which contain only traces of fucose and are completely sialylated, the glycans from amniotic fluid fibronectin are fucosylated and only partially sialylated. The complex-type N-glycosidic glycans present in amniotic fluid fibronectin also include a fractional amount (0.1 mol) of glycans with a polylactosaminyl structure. In addition, 4 mol of O-glycosidic oligosaccharides, which have not previously been described in fibronectins, were found in amniotic fluid fibronectin. The major oligosaccharides in this fraction have the structures Gal beta 1----3GalNAcol, NeuNAc alpha 2----3Gal beta 1----3GalNAcol and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcol. O-glycosidically linked oligosaccharides were also detected in human adult plasma fibronectin but in smaller amounts than in amniotic fluid fibronectin. These results show that amniotic fluid fibronectin differs from plasma fibronectin with regard to the number of glycans attached to the polypeptide and that the glycans present in these two fibronectins differ in structure.     

Effect of indomethacin, tiaprofenic acid and dicrofenac on rat gastric mucosal damage and content of prostacyclin and prostaglandin E2

Gastric ulcerogenicity and depletion of endogenous prostaglandins (PGs) content induced by tiaprofenic acid, dicrofenac and indomethacin were examined using the same antiinflammatory effective doses. Male Wistar rats were given each of these drugs intragastrically 24, 18, and 3 hrs before sacrifice in the following doses (mg/kg): indomethacin (0.8, 4 and 20); tiaprofenic acid (1.2, 6 and 30); dicrofenac (0.8, 4 and 20). Endogenous prostacyclin (PGI2) and PGE2 in fundic mucosa were determined by radioimmunoassay. The three compounds produced fundic mucosal lesions in a dose-dependent manner. However, tiaprofenic acid and dicrofenac were both less potent than indomethacin in producing gastric mucosal lesions at similar antiinflammatory doses. Mucosal PGE2 content was abolished by the three compounds in the following doses (mg/kg): indomethacin (4 and 20); tiaprofenic acid (6 and 30); dicrofenac (20). Mucosal PGI2 was maintained around 50% of the control value in rats given tiaprofenic acid in a dose of 6 mg/kg or dicrofenac in a dose of 4 mg/kg, while indomethacin in a dose of 4 mg/kg markedly reduced mucosal PGI2 to 17% of the control value. In larger doses, tiaprofenic acid and dicrofenac were also significantly less potent in reducing mucosal PGI2 than indomethacin. These results suggest that the difference in ulcerogenicity between indomethacin and the other two compounds was closely related to their potency in decreasing PGI2 in the gastric (fundic) mucosa.     

Phenotype character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and application to breeding wild-type yeast strains

The gene responsible for the methylglyoxal resistance of Saccharomyces cerevisiae was cloned, and its phenotypic characteristics were investigated. S. cerevisiae cells with the gene could accumulate large amounts of glutathione in the medium and should remarkably high resistance to various toxic compounds such as methylglyoxal, tetramethylthiuram disulfide, iodoacetamide, and heavy-metal ions. The gene was also expressed in Escherichia coli cells, and the resistance of E. coli cells to toxic compounds also increased as observed for S. cerevisiae cells. The phenotypic characteristics of the gene were applicable to the selection of the transformants of wild-type yeast strains having no genetic markers.     

Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.