Nucleotide sequence of the gene for the hole-forming toxin aerolysin of Aeromonas hydrophila.

The gene for the hole-forming toxin aerolysin from Aeromonas hydrophila was sequenced. Although most of the sequence seems unrelated to that of Staphylococcus aureus alpha-toxin, both proteins are very hydrophilic, and they each contain a nearly identical string of 10 amino acids.     

Bovine lens aldose reductase: identification of two enzyme forms

Two structurally different forms of bovine lens aldose reductase have been identified. Freshly prepared lens extracts contain an unactivated "b form" (ARb) which is sensitive to inhibition by Sorbinil. Upon incubation of the extracts with oxygen radical generating systems, ARb is converted to a more active "a form" (ARa), which is not inhibited by Sorbinil. ARa and ARb were purified to electrophoretic homogeneity.     

The temporal relationship between phospholipase activation, diradylglycerol formation and superoxide production in the human neutrophil.

Fluctuations in the amounts of choline, inositol 1,4,5-trisphosphate (IP3) and diradylglycerol have been used to monitor phospholipase activation in the human neutrophil. Stimulation of human neutrophils by formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) resulted in a rapid activation of both phosphatidylinositol 4,5-bisphosphate breakdown by phospholipase C and phosphatidylcholine breakdown by phospholipase D. Diradylglycerol accumulation occurred more slowly than that of either choline or IP3 and was inhibited by 30 mM-butanol, suggesting that the bulk was derived from the phospholipase D pathway via phosphatidate phosphohydrolase. Consistent with this is the observation that choline and diradylglycerol are produced in similar amounts. 1,2-Diacylglycerol (DAG) and 1-O-alkyl-2-acyl-sn-glycerol species accumulated with different time courses, indicating that one or more steps in the phospholipase D pathway was selective for the diacyl species. Superoxide production by fMet-Leu-Phe-stimulated neutrophils paralleled DAG accumulation over the first 5 min, but thereafter this production stopped, despite the fact that DAG remained elevated. We conclude that DAG derived from the phospholipase D pathway is only one of the second messengers important in controlling this functional response.     

Selectivity of protein kinase inhibitors in human intact platelets

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.     

The MgADP-induced decrease of the SH1-SH2 fluorescence resonance energy transfer distance of myosin subfragment 1 occurs in two kinetic steps

The fluorescence resonance energy transfer distance between 5-[2-[iodoacetyl)amino)ethyl]aminoaphthalene-1-sulfonic acid covalently attached to the SH1 thiol of myosin subfragment 1 as the energy donor and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide attached to SH2 as the energy acceptor has been found to decrease by about 7 A in the presence of MgADP (Dalby, R. E., Weiel, J., and Yount, R. G. (1983) Biochemistry 22, 4696-4706; Cheung, H. C., Gonsoulin, F., and Garland, F. (1985) Biochim. Biophys. Acta 832, 52-62). Fluorescence stopped-flow experiments on the same system have yielded biphasic traces which are resolvable into a fast and slow component, k1 and k2, respectively. Results of experiments in which k1 and k2 were measured as a function of excess ADP concentration showed: 1) a nonlinear dependence of the apparent rate constants on [ADP]; 2) k1 is a factor of 10 faster than k2. These results are consistent with the 3-step mechanism previously proposed for nucleotide binding to myosin S1 (Garland, F., and Cheung, H. C. (1979) Biochemistry 18, 5281-5289). Kinetic experiments in which the anisotropy of the donor was monitored show this quantity to be unchanged over the course of the reaction. The biphasic decrease of donor intensity is assigned to an increase in energy transfer efficiency which, from the above results, is due to a decrease in donor-acceptor distance, occurring in two steps. The fast step is associated with a 4-5-A decrease of the donor-acceptor separation, while the slow step is associated with a further decrease of approximately 2 A.     

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA. This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility, Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, MD 20205.     

Stimulation and inhibition of secretion by phorbol myristate acetate in different cell types

In washed human platelets and in HL60 granulocytes phorbol myristate acetate (PMA, 1-2000nM) synergised with threshold concentrations of secretogogues to induce a sustained maximum secretory response. Likewise, superoxide production from HL60 cells maintained a maximal response at PMA concentrations between 30-300nM. At concentrations up to 10nM PMA also augmented calcium ionophore, A23187, stimulated histamine release from rat peritoneal mast cells. However, in the mast cell PMA concentrations above 10nM reduced maximum histamine release in a dose-dependent manner.     

An investigation of the SH1-SH2 and SH1-ATPase distances in myosin subfragment-1 by resonance energy transfer using nanosecond fluorimetry

The separation between the two reactive thiols SH1 (Cys-704) and SH2 (Cys-694) and that between SH1 and the active site of myosin subfragment-1 were further investigated by F?rster energy transfer techniques. The SH1-SH2 distance was determined with the probe 5-[[2-[(iodoacetyl)amino]ethyl] amino]naphthalene-1-sulfonic acid (AEDANS) attached to SH1 as the energy donor and 5-(iodoacetamido)fluorescein (IAF) attached to SH2 as energy acceptor. The results derived from measurements of donor lifetimes yielded a donor-acceptor separation in the range 26-52 A, with the distance R(2/3) based on rapid and isotropic probe motions being 40 A. These parameters were not sensitive to added MgADP, in agreement with previous results obtained by using the steady-state method. The SH1-SH2 distance was also determined with AEDANS attached to SH1 and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) attached to SH2. The range in R for the AEDANS/DDPM pair was 12-36 A, with R(2/3) equal to 27 A. The transfer efficiency between these two probes increased by an average of 38% upon addition of MgADP. These results are in agreement with those previously reported (Dalbey, R.E., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696-4706), but the uncertainty in choosing an appropriate value of the orientation factor to describe the AEDANS-DDPM separation does not allow a unique interpretation of the observed increase in energy transfer because it could reflect either an increase in the average orientation factor or a decrease in the donor-acceptor separation. Nevertheless, the results are consistent with the notion that nucleotide binding induces structural perturbations that can be sensed by SH1 and SH2. The distance between SH1 and the ATPase site was determined with AEDANS linked to SH1 and the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) noncovalently bound to the active site as energy acceptor. The bound TNP-ADP was highly immobilized, with a depolarization factor approaching unity. The separation between AEDANS at SH1 and TNP-ADP at the active site was in the range 15-44 A. The actual minimal separation between SH1 and the active site is probably less than 15 A, which suggests that direct interaction between the two sites cannot be ruled out from energy transfer results.