Induction of protein-tyrosine-phosphatase activity by interleukin 6 in M1 myeloblastic cells and analysis of possible counteractions by the BCR-ABL oncogene.

Interleukin 6 (IL-6) induces in M1 myeloblastic cells growth arrest and terminal differentiation toward monocytes. It is reported here that IL-6 reduced by 5- to 20-fold the tyrosine phosphorylation of cellular proteins in these cells. The same-fold reduction was also observed in M1 cells that were transfected with the BCR-ABL deregulated protein kinase. In these stable clones, the levels of tyrosine phosphorylation of cellular proteins were 30- to 100-fold higher than in the parental cells. IL-6 did not reduce the expression levels or the inherent tyrosine kinase activity of BCR-ABL p210. By measuring the protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in crude cell lysates, we found that protein dephosphorylation resulted, at least partially, from induction of PTPase activity by IL-6. The induction of PTPase in the BCR-ABL-transfected clones was not sufficient to confer the minimal protein phosphorylation levels characteristic of IL-6-treated cells. Yet, the transfected M1 clones showed normal growth and differentiation responses to IL-6. None of the gene responses to IL-6 including suppression in the levels of c-myc, c-myb, and cyclin A mRNA; junB and c-jun mRNA induction; and dephosphorylation of retinoblastoma protein were rescued by the BCR-ABL oncogene. The functional relevance of PTPase induction by IL-6 is discussed.

Inability of toxin inhibitors to neutralize enhanced toxicity caused by bacteria adherent to tissue culture cells.

Toxicity to Y-1 adrenal mouse cells caused by heat-labile toxin secreted by an enterotoxigenic strain of Escherichia coli (H-10407-p) was 40-fold enhanced in mixtures containing organisms capable of adhering to the Y-1 cells compared with monolayers exposed to organisms whose adherence was inhibited by mannoside. Severalfold the concentrations of anti-heat-labile toxin antibodies required to neutralize the toxicity of nonadherent bacteria were unable to neutralize the toxicity caused by adherent bacteria. The cytolytic activity toward tissue culture cells and mouse peritoneal macrophages caused by streptolysin S carried by Streptococcus pyogenes was severalfold increased in mixtures containing organisms capable of adhering to the target cells compared with mixtures containing nonadherent bacteria. The ability of trypan blue and RNA core to inhibit the cell-bound streptolysin S was determined in tissue culture cells containing adherent streptococci and mixtures of streptococci randomly colliding with erythrocytes. Both inhibitors were markedly less effective in neutralizing cytolysis than in their ability to neutralize hemolysis. We conclude that compared with toxins produced by nonadherent bacteria, those produced by bacteria adherent to cells are targeted more efficiently and become relatively inaccessible to neutralization by toxin inhibitors.

Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells.

Inhibition of bacterial adherence to bladder cells has been assumed to account for the beneficial action ascribed to cranberry juice and cranberry juice cocktail in the prevention of urinary tract infections (A. E. Sobota, J. Urol. 131:1013-1016, 1984). We have examined the effect of the cocktail and juice on the adherence of Escherichia coli expressing surface lectins of defined sugar specificity to yeasts, tissue culture cells, erythrocytes, and mouse peritoneal macrophages. Cranberry juice cocktail inhibited the adherence of urinary isolates expressing type 1 fimbriae (mannose specific) and P fimbriae [specific for alpha-D-Gal(1----4)-beta-D-Gal] but had no effect on a diarrheal isolate expressing a CFA/I adhesin. The cocktail also inhibited yeast agglutination by purified type 1 fimbriae. The inhibitory activity for type 1 fimbriated E. coli was dialyzable and could be ascribed to the fructose present in the cocktail; this sugar was about 1/10 as active as methyl alpha-D-mannoside in inhibiting the adherence of type 1 fimbriated bacteria. The inhibitory activity for the P fimbriated bacteria was nondialyzable and was detected only after preincubation of the bacteria with the cocktail. Cranberry juice, orange juice, and pineapple juice also inhibited adherence of type 1 fimbriated E. coli, most likely because of their fructose content. However, the two latter juices did not inhibit the P fimbriated bacteria. We conclude that cranberry juice contains at least two inhibitors of lectin-mediated adherence of uropathogens to eucaryotic cells. Further studies are required to establish whether these inhibitors play a role in vivo.

Growth advantage and enhanced toxicity of Escherichia coli adherent to tissue culture cells due to restricted diffusion of products secreted by the cells.

This study was undertaken to examine whether Escherichia coli adherent to tissue cells gain advantages over nonadherent bacteria due to their proximity to the cells. We used tissue culture cells and isogenic derivatives of a proline auxotrophic strain of E. coli that were fimbriated (Fim+) or nonfimbriated (Fim-), and were heat-labile enterotoxin producing (Tox+) or toxin nonproducing (Tox-). We found that the Fim+ bacteria; which were capable of adhering to tissue culture cells, initiated growth much sooner than did nonadherent Fim- bacteria; the adherent bacteria used tissue cell-derived proline, which was available at high concentrations only in the zone of bacterial adherence. Likewise, cyclic AMP secreted by adherent (Fim+) bacteria was maintained at high concentration on the tissue cell surfaces. As few as 2 X 10(5) adherent Fim+ Tox+ bacteria exert toxic activity upon Y1 adrenal cells, whereas toxin secreted in the medium by 6 X 10(6) Fim- Tox+ bacteria was undetectable. The results suggest that the growth advantage and enhanced toxicity of adherent E. coli is due to restricted diffusion of products secreted by the tissue culture and bacterial cells, respectively.

Amino acid precursors of Myxococcus xanthus antibiotic TA.

The production of Myxococcus xanthus antibiotic TA was stimulated by addition of alanine, serine and glycine to Casitone medium. These three amino acids served as the major biosynthetic precursors of the antibiotic. Alanine and serine were incorporated via acetate. In Casitone medium supplemented with alanine and serine, 29 to 30 of the 34 carbon atoms of antibiotic TA were derived from these two amino acids. Both carbon atoms of glycine were incorporated into antibiotic TA by a mechanism not involving acetate as an intermediate. Antibiotic TA was split into two fragments by alkaline hydrolysis followed by periodate oxidation. Radioactive alanine was incorporated into both fragments, whereas glycine was incorporated only into the smaller, polar fragment.

Mode of action of Myxococcus xanthus antibiotic TA.

Antibiotic TA inhibited incorporation of diaminopimelic acid and uridine diphosphate-N-acetylglucosamine into Escherichia coli cell walls without altering the ratio of cross-linked to uncross-linked peptidoglycan. Formation of the lipid intermediate was not blocked by TA, suggesting that TA interferes with polymerization of the lipid-disaccharide-pentapeptide.