Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.     

Anticlastogenic effect of flavonoids against mutagen-induced micronuclei in mice.

14 flavonoids, including flavone and flavonol derivatives, were tested for their anticlastogenic effect against induction of micronuclei by benzo[a]pyrene in polychromatic erythrocytes of mice. When each flavonoid was administered orally, together with intraperitoneally administered benzo[a]pyrene, most flavonol derivatives showed an anticlastogenic effect. The data suggest that the 2,3-double bond and 3,5,7-hydroxyl groups in the flavonoid molecules may be essential to produce anticlastogenic effects against benzo[a]pyrene. Galangin, one of the active compounds, and (-)-epicatechin, a weak one, were administered to mice in order to compare their anticlastogenic effect against 3 different kinds of carcinogens: ethyl methanesulfonate, 7,12-dimethylbenz[a]anthracene, and adriamycin. Galangin showed a stronger anticlastogenic effect than (-)-epicatechin against ethyl methanesulfonate and 7,12-dimethylbenz[a]anthracene. However, there was no significant effect against adriamycin-induced micronuclei by both compounds. Our study indicates that most flavonoids are anticlastogenic agents. Their anticlastogenic effects are apparently independent of their own clastogenic activities. Furthermore, their anticlastogenic activities do not apply universally to all types of genotoxic chemicals.     

Changes in Esterification of the Uronic Acid Groups of Cell Wall Polysaccharides during Elongation of Maize Coleoptiles

Cell walls of grasses have two major polysaccharides that contain uronic acids, the hemicellulosic glucuronoarabinoxylans and the galactosyluronic acid-rich pectins. A technique whereby esterified uronic acid carboxyl groups are reduced selectively to yield their respective 6,6-dideuterio neutral sugars was used to determine the extent of esterification and changes in esterification of these two uronic acids during elongation of maize (Zea mays L.) coleoptiles. The glucosyluronic acids of glucuronoarabinoxylans did not appear to be esterified at any time during coleoptile elongation. The galactosyluronic acids of embryonal coleoptiles were about 65% esterified, but this proportion increased to nearly 80% during the rapid elongation phase before returning to about 60% at the end of elongation. Methyl esters accounted for about two-thirds of the total esterified galacturonic acid in cell walls of unexpanded coleoptiles. The proportion of methyl esters decreased throughout elongation and did not account for the increase in the proportion of esterified galactosyluronic acid units during growth. The results indicate that the galactosyluronic acid units of grass pectic polysaccharides may be converted to other kinds of esters or form ester-like chemical interactions during expansion of the cell wall. Accumulation of novel esters or ester-like interactions is coincident with covalent attachment of polymers containing galactosyluronic acid units to the cell wall.     

Inhibition of Sucrose Enhancer Effect of the Potato Proteinase Inhibitor II Promoter by Salicylic Acid

Effect of salicylic acid (SA) on the expression of the potato proteinase inhibitor (PI) II promoter was studied with transgenic tobacco plants (Nicotiana tabacum) carrying a gene fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) reporter. As previously observed, the PI-II promoter was inducible by wounding and the promoter activity was further enhanced by sucrose. Addition of SA did not influence the wound induction of the PI-II promoter but significantly inhibited the sucrose response. The 5′-deletion mutant −573 was unable to respond to wounding but did respond to sucrose and SA. The 3′-deletion analysis indicated the presence of a sucrose-responsive element between −574 and −520. A study of the insertion mutants revealed the function of another sucrose-responsive element between −522 and −500. Enhancer effects of these sucrose-responsive elements were inhibited by SA. These studies suggest that SA inhibits PI-II promoter activity by decreasing the sucrose response. Analysis of SA-related chemicals revealed that only acetyl-SA showed a similar inhibitory effect, and other hydroxybenzoic acids had little or no effect on the sucrose enhancer activity. Therefore, it seems that the interaction between SA and the receptor molecule is specific.     

Current concepts of cell-cycle regulation and its relationship to oocyte maturation, fertilization and embryo development

Genetic and biochemical approaches have contributed to an explosion of literature on cell-cycle control. Regulation of the cell-cycle is controlled by a series of kinases and phosphatases. Key control points are during the G(1)-S and G(2)-M transitions. During both transitions, cyclins interact with a specific kinase to allow a cell to pass through that phase. The meiotic maturation of oocytes, fertilization and embryo development are all events influenced by cell-cycle regulation. Understanding cell-cycle control should provide new ways for gamete and embryo biologists to approach culture and development problems.     

DNA structures contributing to the instability of recombinant plasmids inStreptococcus thermophilus

Summary Bifunctional shuttle vector (pBN183) and recombinant plasmids (pDCO2 and pDCO3) carrying aStreptomyces cholesterol oxidase gene (choA) were deleted to varying degrees inStreptococcus thermophilus. Restriction mapping of the deleted plasmids led to the identification of deletion prone regions in the transforming DNAs. Sequence analysis revealed that direct repeats and hairpin structures occurred in these regions, suggesting that they are deleterious to the stability of plasmids inS. thermophilus.     

Plasmid stability in a recombinant mammalian cell bioprocess

Summary A simple experimental method is devised to determine the fraction of plasmid-harboring cells in a bioprocess employing recombinant mammalian cells. The fraction of plasmid-harboring cells decreased as serum content in the growth medium decreased. The relatively higher increase in the generation time of the plasmid-harboring cell was primarily responsible for this decrease. The mathematical expression obtained for this fraction in terms of the two parameters, i.e. the generation time ratio and the plasmid-loss probability, could represent the experimental data extremely well. The numerical values of these parameters could show the inherent insight of the system. It was found that the data plot against time can draw us to a misleading conclusion of the absence of the effect of serum concentration.     

Continuous production of clinical dextran using two-stage reactor

Summary Clinical dextran with desired molecular weight was produced continuously in the two-stage reactor. Cells ofLeuconostoc meseteroides B512F cultivated in the first reactor were transferred to the second reactor where sucrose and primer were added for clinical dextran production. By using this two-stage reactor, the fraction of desired clinical dextran increased significantly when observed with gel permeation chromatography.     

Purification and some properties of an extracellular l-amino acid oxidase from Cellulomonas cellulans AM8 isolated from soil

Summary The soil isolate Cellulomonas cellulans AM8 produces an extracellular l-amino acid oxidase (L-AAO) with broad substrate specificity. The strain produced up to 0.35 unit (U)/ml of the extracellular L-AAO in a simple medium containing glycerol and yeast extract. The enzyme was easily purified up to 30 U/mg protein using Phenyl-Sepharose fast flow. The purified enzyme migrated as single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 55 kDa. On native PAGE the molecular mass was approx. 300 000 kDa, which may be due to aggregation. With the exception of glycine, proline, and threonine, all the amino acids normally constituting proteins were oxidized. The V _max values from 0.7 to 35.2 U/mg for aspartic acid and lysine, respectively, and the K _m values from 0.007 to 7.1 mm for cysteine and valine, respectively, were obtained at 25° C and pH 7.0 in oxygen-saturated solutions. The L-AAO had a pH optimum of 6.5–7.5. It was stable for several months at — 30° C and for some days at 35° C. Ferricyanide served as an electron acceptor with a V _max of 50 U/mg and K _m for 0.3 mm with phenylalanine as the substrate. Correspondence to: R. D. Schmid