Relationship between the Molecular Size of Poly I·Poly C and Its Biological Activity1

Seven polyinosinic·polycytidylic acid (poly I·poly C) preparations, ranging from 4.2 S to 21.2 S, prepared from various sizes of polyinosinate and polycytidylate, were examined for toxicity and interferon-inducing activity in mice. The increase in size of poly I·poly C was accompanied by increases both in the maximal amount of interferon produced and in the length of persistence of a high level of interferon in plasma. Toxicity of poly I·poly C was proportional to the molecular size within the range of 8 S to 16 S. The amount of interferon induced by 1/5 LD₅₀ of poly I·poly C depended on the size of the inducer, being increasingly lower with progressively smaller sizes. Next, activities of poly I·poly C in culture cells were examined. The resistance-inducing activity of poly I·poly C in primary chick embryo cells (CEC) increased with the size of the inducer (4.2 S to 11.6 S), whereas the activity in L cells was not so markedly dependent upon its molecular size as in CEC. In the presence of calf serum during induction of resistance the activity was lowered. The activities of preparations with small molecular sizes were affected by calf serum more markedly than those of large molecular sizes. The interferon-inducing activity in RK13 was not appreciably influenced by the size of poly I·poly C, especially in the presence of DEAE-dextran, while the activity in L cells was markedly dependent upon the size of the inducer. These results suggest that the influence of the molecular size of poly I·poly C upon the resistance-inducing and interferon-inducing activities varies among different kinds of cells, and alters in the presence of serum or DEAE-dextran.     

CORTICAL F‐ACTIN REORGANIZATION AND A CONTRACTILE RING‐LIKE STRUCTURE FOUND DURING THE CELL CYCLE IN THE RED CRYPTOMONAD,PYRENOMONAS HELGOLANDII1

Cortical F‐actin reorganization during the cell cycle was observed in Pyrenomonas helgolandii U. J. Santore (SAG 28.87) for the first time in Cryptophyta using fluorescein‐isothiocyanate (FITC)–phalloidin staining. In interphase, a number of F‐actin bundles were observed as straight lines running parallel to the long axis of the cell on the cell cortical region. They extended from an F‐actin bundle that runs along the margin of the vestibulum. Although the F‐actin bundles running parallel to the long axis of the cell disappeared during anaphase, they gradually reappeared in telophase. By contrast, the F‐actin bundle along the vestibulum margin remained visible during cytokinesis and dynamically changed following the enlargement of the vestibulum, suggesting that F‐actin was involved in the mechanism of vestibulum enlargement. F‐actins were not found in the cytoplasmic and nucleoplasmic regions throughout the cell cycle. In addition, a contractile ring‐like structure appeared at the cleavage furrow during cytokinesis. Treatment with cytochalasin B and latrunculin B significantly inhibited the formation of cleavage furrow, resulting in forming an abnormal cell with two nuclei, suggesting that cytokinesis in P. helgolandii is controlled by the contractile ring‐like structure constituted of F‐actin.     

New Plasmid-Mediated Phosphorylation of Gentamicin C in Staphylococcus aureus

A clinical isolate of Staphylococcus aureus resistant to gentamicin, kanamycin and 3′,4′-dideoxykanamycin B contained two enzymes capable of inactivating gentamicin, i.e., an aminoglycoside 2″-phosphotransferase and aminoglycoside acetyltransferase.     

Acridone alkaloids from Severinia buxifolia

Two new acridone alkaloids, severifoline and N-methylseverifoline along with the known alkaloids, N-methylatalaphylline, atalaphyllinine and 5-hydroxy-N-methylseverifoline, were isolated from the root bark of Severinia buxifolia. The structures of severifoline and N-methylseverifoline were established by chemical and spectroscopic methods.     

Copper elicits an increase in cytosolic free calcium in cultured tobacco cells

At concentrations greater than 0.1 mM, CuSO(4) provoked a rapid and sustained increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), in tobacco suspension culture cells expressing apoaequorin, a Ca(2+)-sensitive photoprotein. The increase was suppressed by treatment with LaCl(3), indicating that the increase is due to an influx of Ca(2+) from the apoplast through plasma membrane Ca(2+) channels. Although stimulation of H(2)O(2) production upon the CuSO(4) treatment (0.1 mM) was observed, treatment with catalase did not inhibit the increase in [Ca(2+)](cyt), and treatment with H(2)O(2) dose-dependently suppressed or delayed the increase. These results suggested that active oxygen species generated through copper-mediated reactions, or copper-mediated oxidative damages to plasma membrane, are not responsible for the increase. Treatment with sulfhydryl reagents, which alkylate or oxidize thiol groups, or acidification of the culture medium suppressed the increase in [Ca(2+)](cyt). These results demonstrated that copper causes an influx of Ca(2+) through plasma membrane Ca(2+) channels, and that plasma membrane thiol groups play an important role in activating the Ca(2+) channels.     

Purification and Characterisation of Keratinase from a Thermotolerant Feather-degrading Bacterium

Summary Isolation and identification of a thermotolerant feather-degrading bacterial strain from Thai soil as well as purification and properties of its keratinase were investigated. The thermotolerant bacterium was identified as Bacillus licheniformis. The keratinase was purified to homogeneity by three-step chromatography. The purified enzyme exhibited a high specific activity (218 U mg⁻¹) with 86-fold purification and 25% yield. The enzyme was monomeric and had a molecular mass of 35 kDa. The optimum pH and temperature for the enzyme were 8.5 and 60 °C, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA and iodoacetamide, but was stimulated by metal ions. It hydrolysed soluble proteins with a relative activity of 4–100% and insoluble proteins, including keratins, with a relative activity of 3–35%. Therefore, the enzyme could improve the nutritional value of meat- and poultry-processing wastes containing keratins, collagen and gelatin.     

Large-Scale Engineering of the Corynebacterium glutamicum Genome

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.