Reduction of 3-methoxytyramine concentrations in the caudate nucleus of rats after exposure to high-energy iron particles: evidence for deficits in dopaminergic neurons

Exposure to low doses of high-energy iron particles can alter motor behavior. The ability of rats to hang from a wire has been reported to be significantly degraded after exposure to doses as low as 0.5 Gy. In addition, deficits in the ability of acetylcholine to regulate dopamine release in the caudate nucleus (an area in the brain important for motor function) have been found. The concentrations of 3-methoxytyramine (3-MT), a metabolite of dopamine whose concentrations reflect dopamine release in vivo, were measured after rats were exposed to different doses of high-energy iron particles to gain further information about the effect of radiation on the dopaminergic system. Concentrations of 3-MT were significantly reduced 3 days after exposure to 5 Gy but returned to control values by 8 days. After 6 months, concentrations were again less than control values. Exposure to 5 Gy of high-energy electrons or gamma photons had no effect 3 days after exposure. Very high doses of electrons were needed to alter 3-MT concentrations. One hundred grays of electrons decreased 3-MT 30 min after irradiation but levels returned to control values by 60 min. Gamma photons had no effect after doses up to 200 Gy. These results provide further evidence that exposure to heavy particles can degrade motor behavior through an action on dopaminergic mechanisms and that this can occur after doses much lower than those needed for low-LET radiation.     

Involvement of prostaglandins and histamine in radiation-induced temperature responses in rats

Exposure of rats to 1-15 Gy of gamma radiation induced hyperthermia, whereas exposure to 20-150 Gy produced hypothermia. Since radiation exposure induced the release of prostaglandins (PGs) and histamine, the role of PGs and histamine in radiation-induced temperature changes was examined. Radiation-induced hyper- and hypothermia were antagonized by pretreatment with indomethacin, a cyclooxygenase inhibitor. Intracerebroventricular administration of PGE2 and PGD2 induced hyper- and hypothermia, respectively. Administration of SC-19220, a specific PGE2 antagonist, attenuated PGE2- and radiation-induced hyperthermia, but it did not antagonize PGD2- or radiation-induced hypothermia. Consistent with an apparent role of histamine in hypothermia, administration of disodium cromoglycate (a mast cell stabilizer), mepyramine (H1-receptor antagonist), or cimetidine (H2-receptor antagonist) attenuated PGD2- and radiation-induced hypothermia. These results suggest that radiation-induced hyperthermia is mediated via PGE2 and that radiation-induced hypothermia is mediated by another PG, possibly PGD2, via histamine.     

Effect of vascular cannulation on serum concentrations of LH, FSH, prolactin and cortisol in prepubertal gilts

Two experiments were conducted to determine whether cannulation of the jugular vein in gilts alters serum concentrations of LH, FSH, prolactin (PRL) or cortisol (C). In Experiment 1, 12 crossbred prepubertal gilts weighing 95 +/- 1.3 kg were immobilized by snaring, and tygon tubing was threaded into the anterior vena cava through a 12-gauge needle inserted into the jugular vein. Five hours later, blood samples were drawn at 20-min intervals for 4 h (Day 0). Samples were also drawn at 20-min intervals for 4-h periods 24 h (Day 1) and 48 h (Day 2) after cannulation. Serum concentrations of LH were similar (P=0.26) among Day 0 (0.40 ng/ml), Day 1 (0.39 ng/ml) and Day 2 (0.34 ng/ml). Serum PRL was similar (P=0.07) among Day 0 (4.10 ng/ml), Day 1 (3.87 ng/ml) and Day 2 (3.43 ng/ml). Serum concentrations of C were greater (P < 0.001) on Day 0 (8.32 ng/ml) than Day 1 (4.48 ng/ml) or Day 2 (3.54 ng/ml). In Experiment 2, cannulas were placed in 29 prepubertal gilts. Two days after initial cannulation, six blood samples were drawn at 20-min intervals. Gilts were then immobilized by snaring, and a second cannulae was inserted into the contralateral vein. Five blood samples were taken at 2-min intervals during the second cannulation and then six samples were drawn at 20-min intervals. Serum LH and FSH were not altered by cannulation or elevated during the subsequent 2-h sampling period (P>0.05). In contrast, serum concentrations of PRL rose slowly (P<0.05) during cannulation and remained elevated for 60 min before returning to baseline. Serum concentrations of C rose within 6 min of cannulation, remained elevated for 30 min, and then declined over the next 90 min. From these two experiments, it appears that secretory patterns of LH and FSH can be accurately assessed immediately after cannulation in prepubertal gilts. Measurements of serum PRL and C that reflect nonstressed conditions, however, cannot be obtained until at least 2 h or 1 d after cannulation, respectively.     

Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association.

Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.     

The 'destruction box' of cyclin A allows B-type cyclins to be ubiquitinated, but not efficiently destroyed.

The destruction of mitotic cyclins by programmed proteolysis at the end of mitosis is an important element in cell cycle control. This proteolysis depends on a conserved motif of nine residues known as the 'destruction box', which is located 40-50 residues from the N-terminus. The sequences of the A- and B-type destruction boxes are slightly different, which might account for the differences in timing of their destruction. When the cyclin A-type destruction box was substituted for the normal one in cyclin B1 or B2, however, the resulting constructs were unexpectedly stable, although the converse substitution of B-type destruction boxes in cyclin A permitted normal degradation. We compared the ubiquitination of various cyclin constructs, and found that whereas mutation of the highly conserved residues in the destruction box strongly reduced the level of ubiquitinated intermediates, the stable destruction box 'swap' constructs did form such adducts. Thus, while ubiquitination is probably necessary for cyclin destruction, it is not sufficient. We also found that poly-ubiquitinated cyclin derivatives are still bound to p34cdc2, which is not detectably ubiquitinated itself, raising the questions of how cyclin and cdc2 dissociate from one another, and at what stage, in the process of degradation.     

The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe.

A cell-free system derived from Xenopus eggs was used to identify the 'destruction box' of the Schizosaccharomyces pombe B-type cyclin, Cdc13, as residues 59-67: RHALDDVSN. Expression of indestructible Cdc13 from a regulated promoter in S.pombe blocked cells in anaphase and inhibited septation, showing that destruction of Cdc13 is necessary for exit from mitosis, but not for sister chromatid separation. In contrast, strong expression of a polypeptide comprising the N-terminal 70 residues of Cdc13, which acts as a competitive inhibitor of destruction box-mediated proteolysis, inhibited both sister chromatid separation and the destruction of Cdc13, whereas an equivalent construct with a mutated destruction box did not. Appropriately timed expression of this N-terminal fragment of Cdc13 overcame the G1 arrest seen in cdc10 mutant strains, suggesting that proteins required for the initiation of S phase are subject to destruction by the same proteolytic machinery as cyclin.     

The Kinetics of N-Ethylmaleimide Inhibition of a Vacuolar H+-ATPase and Determination of Nucleotide Dissociation Constants

All eukaryotic vacuolar (V-type) ATPases share the property of being inhibited by low concentrations (1-2 [mu]M) if N-ethylmaleimide (NEM). This distinguishes them from P-type ATPases, which are inhibited by higher concentrations of NEM (0.1-1 mM), and F-type ATPases, which are virtually resistant to inhibition by NEM. Using tonoplast vesicles from Beta vulgaris we have determined the kinetics of NEM inactivation of the V-type ATPase to be pseudo-first order. The concentration dependence of the reaction indicates interaction with a single class of inhibitory site with a rate constant of 4.1 x 104 M-1 min-1. Nucleotides protect against inactivation with an efficacy that agrees with their capacity to act as enzyme substrates. The dissociation constant for MgATP has been determined from protection experiments to be 0.44 mM, which is close to the observed Km for hydrolysis (0.39 mM). Likewise, the dissociation constant for protection by MgADP (127 [mu]M) is close to its inhibition constant as a competitive inhibitor (110 [mu]M). Taken together, these findings suggest that NEM inactivation is associated with nucleotide protectable exposure of a single cysteine residue on the catalytic subunit and confirm the utility of this residue for the determination of ligand dissociation constants through protection of maleimide inhibition.     

Suppression and Restoration of Lesion Formation in Arabidopsis lsd Mutants

Systemic acquired resistance (SAR) is a broad-spectrum, systemic defense response that is activated in many plant species after pathogen infection. We have previously described Arabidopsis mutants that constitutively express SAR and concomitantly develop lesions simulating disease (lsd). Here, we describe two new mutants, lsd6 and lsd7, that develop spontaneous necrotic lesions and possess elevated levels of salicylic acid (SA) as well as heightened disease resistance, similar to the previously characterized lsd and accelerated cell death (acd2) mutants. Genetic analysis of lsd6 and lsd7 showed that the mutant phenotypes segregated as simple dominant traits. When crossed with transgenic Arabidopsis plants containing the SA-degrading enzyme salicylate hydroxylase, the F1 progeny showed suppression of both SAR gene expression and resistance. In addition, salicylate hydroxylase suppressed lesion formation in the F1 progeny, suggesting that SA or some SA-dependent process may have a role in pathogen-associated cell death. Surprisingly, lesions were restored in the lsd6 F1 progeny after the application of either 2,6-dichloroisonicotinic acid or SA. Lesions were not restored by treatment with either compound in the lsd7 F1 plants. Our findings demonstrate that steps early in the signal transduction pathway leading to SAR and disease resistance are potentiated by later events, suggesting feedback control of lesion formation.     

The force exerted by a single kinesin molecule against a viscous load.

Kinesin is a motor protein that uses the energy derived from the hydrolysis of ATP to power the transport of organelles along microtubules. To probe the mechanism of this chemical-to-mechanical energy transduction reaction, the movement of microtubules across glass surfaces coated with kinesin was perturbed by raising the viscosity of the buffer solution. When the viscosity of the solution used in the low density motility assay was increased approximately 100-fold through addition of polysaccharides and polypeptides, the longer microtubules, which experienced a larger drag force from the fluid, moved more slowly than the shorter ones. The speed of movement of a microtubule depended linearly on the drag force loading the motor. At the lowest kinesin density, where dilution experiments indicated that the movement was caused by a single kinesin molecule, extrapolation of the linear relationship yielded a maximum time-averaged drag force of 4.2 +/- 0.5 pN per motor (mean +/- experimental SE). The magnitude of the force argues against one type of "ratchet" model in which the motor is hypothesized to rectify the diffusion of the microtubule; at high viscosity, diffusion is too slow to account for the observed speeds. On the other hand, our data are consistent with models in which force is a consequence of strain developed in an elastic element within the motor; these models include a different "ratchet" model (of the type proposed by A. F. Huxley in 1957) as well as "power-stroke" models.     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait low brood-viability resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.