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.     

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.     

Ontogenetic and individual variation in size, shape and speed in the Australian agamid lizard Amphibolurus nuchalis

The present study investigates relationships among size, shape and speed in the Australian agamid lizard Amphibolurus nuchalis . Maximal running speed, body mass, snout-vent length, tail length, fore- and hind limb spans and thigh muscle mass were measured in 68 field-fresh individuals spanning the entire ontogenetic size range (1.3 48 g). Relative lengths of both foreand hind limbs decrease with increasing body mass (= negative allometry), whereas relative tail length and thigh muscle mass increase with body mass (= positive allometry). Repeatable and significant differences in maximal running speed exist among individuals. Maximal running speed scales as (body mass)^0.161, and 59% of the variation in maximal speed was related to body mass. Based on the results of the present and previous studies, data on scaling of body proportions alone appear inadequate to infer scaling relationships of functional characters such as top speed.
Surprisingly, individual variation in maximal speed is not related to individual variation in shape (relative limb, tail and body lengths). These components of overall shape are not independent; individuals tended to have either relatively long or relatively short limbs, tails and bodies for their body mass. Even the significant difference in multivariate shape between adult males and females has no measurable consequences for maximal speed. Speeds of field-fresh animals did not vary on a seasonal basis, and eight weeks of captivity had no effect on maximal running speeds. Gravid females and long-term (obese) captive lizards were both approximately 12% slower than field-fresh lizards.