The formation of flavonoids in cell suspension cultures ofPrunus x yedoensis matsum

Two lines of the red and pale yellow cell suspension cultures, prepared fromPrunus x yedoensis Matsum. callus induced by Murashige and Skoog's (1962) basal medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D, 1.0 mg/l), kinetin (0.1 mg/l) and sucrose (30 g/l), were maintained on Schenk and Hildebrandt medium as modified by Mitchell and Gildow (1975). The red cell suspension culture produced cyanidin 3-monoglucoside, 5, 4′-dihydroxy-7-methoxyisoflavone 4′-glucoside (prunetrin), isoquercitrin, catechin, epicatechin, and procyanidin B-1, B-2, B-3 and B-4, while the pale yellow cells produced only a small amount of catechin and epicatechin as main flavonoids. These flavonoid compounds found in the red cell culture were present also in maturePrunus leaves. Maximum growth and maximum amount of total phenol and proanthocyanidin (procyanidins) were obtained with 0.3 mg/l of both 2,4-D and kinetin. Maximum concentration of anthocyanin was also obtained with 0.3 mg/l 2, 4-D regardless of kinetin concentration. Accumulation of proanthocyanidin was markedly stimulated by low concentrations of phosphate, which reduced growth by about half, and also by high concentrations of inorganic nitrogen. Production of both anthocyanin and proanthocyanidin was reduced by lowered nitrogen levels. Cell growth and production of all phenolics were inhibited when ammonium ion replaced nitrate in the medium.     

A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity

A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus. These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity.     

Quantitative analysis of serum IL-6 and its correlation with increased levels of serum IL-2R in HIV-induced diseases

We have devised a luminescence sandwich ELISA for the quantification of IL-6 in both sera and cell culture supernatants, which had a detection limit of 100 fg/ml of test sample. By using the luminescence sandwich-ELISA, low but measurable levels of IL-6 (9.5 pg/ml on average) were found in the sera from normal individuals. The serum levels of IL-6 were elevated in HIV-seropositive asymptomatic carriers (55.5 pg/ml on average), and the IL-6 levels were correlated with the degree of HIV-induced disease progression (AIDS-related complex 106.8 pg/ml on average and (AIDS 283 pg/ml). IL-6 immunoreactivity in the sera of AIDS patients eluted at a 25,000 m.w. major peak, which was biologically active and heat-stable, and a 500,000 m.w. minor peak in size-exclusion HPLC. Interestingly, a significant correlation was observed between the serum IL-6 levels and soluble IL-2R levels. In vitro, HIV infection of PHA-activated PBMC led to enhanced release of IL-6 into the culture supernatants. Moreover, soluble IL-2R release was markedly increased by adding exogenous IL-6, whereas it was decreased by adding the neutralizing anti-IL-6 mAb to the cultures. These results demonstrate that increased IL-6 levels are significantly associated with sIL-2R levels, and suggest a cause of the increased levels of this receptor in patients with HIV infection. Furthermore, both serum IL-6 and serum IL-2R levels in HIV infection reflect the stage of the HIV-induced disease.     

Intravesical treatment of bladder cancer with recombinant human interferon-β

Summary In order to examine its clinical efficacy, recombinant human interferon- (rIFN-) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3–36) × 10⁶ U rIFN- on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3–36) × 10⁶ U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 10⁶ U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN- varies with treatment protocol. Frequent and longer exposure to rIFN- may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN- is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.     

A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.     

Reduction of tetrazolium salts by sulfate-reducing bacteria

Abstract The reduction of tetrazolium salts by the sulfate-reducing bacteria, Desulfovibrio desulfuricans and Desulfotomaculum orientis , was examined. D. desulfuricans and D. orientis reduced triphenyltetrazolium chloride (TTC) and 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyltetrazolium chloride (INT) forming intracellular formazan deposits. The reduction rate of INT was higher than that of TTC. INT reduction was not inhibited by the addition of sulfate or molybdate, and sulfate uptake was inhibited by the addition of both INT and molybdate. The ratio of intracellular formazan forming cells to acridine orange direct counts in both strains decreased with culture age and starvation time.     

Induction of cyclo-oxygenase synthesis in human promyelocytic leukaemia (HL-60) cells during monocytic or granulocytic differentiation.

Cyclo-oxygenase (COX) production in human promyelocytic leukaemia (HL-60) cells was studied during monocytic differentiation induced by 1 alpha, 25-dihydroxyvitamin D3 (24 nM; 3 days) or phorbol 12-myristate 13-acetate (100 nM; 1 day), or during granulocytic differentiation induced by retinoic acid (1 microns; 4 days). Undifferentiated or differentiated HL-60 cells were labelled with [35S]methionine, and membrane-bound COX was solubilized and quantified by SDS/PAGE. Immunoprecipitated 35S-labelled COX from cells induced to differentiate into monocytic or granulocytic lineage were clearly detected on the autoradiograms as a protein of approx. 70 kDa molecular size, whereas only a very faint COX band was detected in untreated HL-60 cells. During both monocytic and granulocytic differentiation, COX activity (measured by the conversion of exogenous arachidonic acid into prostaglandin E2) was dramatically increased. In addition, thromboxane synthesis was preferentially enhanced during monocytic differentiation. HL-60 cells, induced to differentiate into the monocytic or granulocytic lineage, provide a useful tool to investigate the cellular mechanisms involved in regulation of the synthesis of individual prostanoid-metabolizing enzymes.