Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFA^ts) grown at 28°C (PL28), and at 42°C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19°C for PL28 and at 43°C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42°C) are in the state where the gel and liquid crystalline phases coexist.     

Direct fluorometric determination of a dissociation constant as low as 10(-10) M for the subtilisin BPN'--protein proteinase inhibitor (Streptomyces subtilisin inhibitor) complex by a single photon counting technique.

It was found that an increase in fluorescence intensity at 340 nm is observed on the binding of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' in the pH range 6--10. The dissociation constant, Ki, of the enzyme-inhibitor complex was determined as a function of pH and temperature by direct fluorometric titration utilizing the single photon counting technique in the protein concentration range of 10(-9) M. Ki values as low as 10(-10) M could be obtained with reasonable accuracy by this high-sensitivity detection method. From the temperature dependence of Ki, it was found that the binding is endothermic, and is entirely "entropy-driven" in nature. The effect of pH on Ki suggested the participation of an ionizable group with pKapp = 8.5 in the binding.     

Herbimycin A blocks IL-1-induced NF-kappa B DNA-binding activity in lymphoid cell lines.

The mechanism by which interleukin-1 alpha (IL-1 alpha) activates NF-kappa B DNA-binding activity is not completely understood. While it is well established that protein kinase C can activate NF-kappa B, neither protein kinase C nor protein kinase A appears to be critical in the induction of NF-kappa B by IL-1 alpha. Since a number of growth factors signal via protein tyrosine kinase, in this study we examined a possible involvement of protein tyrosine kinase in the IL-1 alpha-induced NF-kappa B. The results showed that in the murine pre-B cell line 70Z/3 and in the murine T cell line EL-4 6.1 C10 IL-1 alpha-induced NF-kappa B was associated with transient increase in protein tyrosine kinase activity. Pre-treatment of these cell lines with herbimycin A, an inhibitor of tyrosine kinase activity, blocked the IL-1 alpha-enhanced protein tyrosine kinase activity and the IL-1 alpha-induced NF-kappa B DNA-binding activity. Herbimycin A at concentrations sufficient to block IL-1 alpha-induced NF-kappa B did not block the phorbol 12-myristate 13-acetate (PMA)-induced NF-kappa B. The data suggest that IL-1 alpha and PMA activate NF-kappa B by different pathways and that induction of NF-kappa B DNA-binding activity by IL-1 might be dependent on protein tyrosine phosphorylation.     

Modulation of monocytic differentiation of HL-60 cells by inhibitors of protein tyrosine kinases.

We showed previously that the expressions of various src family protein tyrosine kinases (PTKs) were induced independently during the monocytic differentiation of HL-60 cells. The role of PTKs was further assessed in the present study by investigating the effects of PTK inhibitors on the differentiation. It was demonstrated that PTK inhibitors such as genistein and herbimycin A modulated monocytic differentiation of HL-60 cells; they inhibited the differentiation induced by TPA, while promoting that induced by vitamin D3 (D3). Immunoblotting analysis of protein molecules which had been phosphorylated on their tyrosine residues demonstrated that TPA induced phosphorylation of certain molecules different from those induced by D3 in HL-60 cells. PTK inhibitors blocked the phosphorylation and modulated differentiation driven by the inducers. These data suggest that PTKs are involved both promotively and suppressively in signaling events that induce monocytic differentiation of HL-60 cells.     

Both cyclooxygenase and lipoxygenase inhibitor partially restore the anorexia by interleukin-1 beta.

Since the peripheral prostaglandin synthetizing system may at least partly involved in the anorexia that follows central interleukin-1 beta (IL-1) administration, this study was undertaken to investigate the effect of ibuprofen (ip), selective cyclooxygenase blocker and AA 861, selective lipoxygenase inhibitor, on changes of food and water intake by a single injection of IL-1 (2 micrograms/rat, ip). We demonstrated that food and water intake were suppressed by peripheral administration of IL-1. Throughout the entire observation periods, suppressed food intake was partially restored to control levels by ibuprofen, while water intake completely restored. In addition, no significant differences about water/food intake were observed in the IL-1 + ibuprofen-treated groups, respectively. In the next experiment, IL-1 induced anorexia was also partially restored to the control level following pretreatment with AA 861. These results may suggest that other mechanism including lipoxygenase blocker besides prostaglandin production may be involved in IL-1 induced anorexia.     

Phosphorylation of coagulation factor II by phospholipid/Ca(2+)-dependent protein kinase (protein kinase C)

Prothrombin is a major constituent of the blood coagulation cascade and requires phospholipid and Ca2+ for its activation. We have found that phospholipid/Ca(2+)-dependent protein kinase (Protein kinase C) phosphorylates prothrombin and the associated apparent Km value for prothrombin (0.86 microM) is comparable to the Km value reported for most known substrates of protein kinase C. A 2-dimension separation analysis revealed that serine residue was apparently phosphorylated by PKC. The phosphorylation was inhibited by such phosphatidylserine- and/or Ca2+ competitive protein kinase C inhibitors as trifluoperazine, palmitoylcarnitine and gossypol. These results suggest that protein kinase C phosphorylation was involved in the regulation of blood coagulation.     

Actin cytoskeleton of resting bovine platelets

Actin filaments in resting discoid bovine platelets were examined by fluorescence and electron microscopy. Rhodamine-phalloidin staining patterns showed a characteristic wheel-like structure which consisted of a central small circle connected by several radial spokes to a large peripheral circle. This wheel-like structure was composed of actin filaments forming a characteristic arrowhead structure with heavy meromyosin from muscle. Actin filaments were densely arrayed in parallel with a marginal microtubule band and radiated out from the center to the periphery. Platelets treated with colchicine lost their marginal microtubule band but retained their wheel-like structure and normal discoid form. Cytochalasin B disrupted the wheel-like structure but not the marginal microtubule band or the normal discoid form. After simultaneous treatment with both cytochalasin B and colchicine, platelets lost their discoid shape. These results suggest that actin filaments and microtubules both play important roles in the maintenance of the discoid shape of resting bovine platelets.     

Inhibited insulin secretion by recombinant human interleukin-1 beta in adrenalectomized rats: involvement of prostaglandin

An involvement of prostaglandin synthesis in reduced insulin secretion by interleukin-1 was investigated in adrenalectomized (ADX) rats. The recombinant human interleukin-1 beta (IL-1) significantly reduced insulin secretion in ADX rats 2 and 4 hr after the injection, although IL-1 stimulated insulin secretion in intact rats. In ADX rat, IL-1 showed dose-dependent inhibition of pancreatic insulin secretion. In addition, insulin response to intravenous glucose loading was also attenuated in ADX rats with pretreatment by IL-1. At 4 hours after injection, ibuprofen (IBP; 0.5-50.0 mg/kg, ip), selective cyclooxygenase blocker, attenuated insulin inhibition by IL-1 in a dose-dependent manner. These data suggest that IL-1 may suppress in vivo insulin release at least in part through the mediation of prostaglandin synthesis in the absence of adrenal glands.