Ubiquitylation and destruction of endogenous c-mycS by the proteasome: are myc boxes dispensable?

c-MycS proteins are truncated forms of the transcription factor which have been shown to be produced by translation initiation at internal methionines (101, 121, and 134) and to be functional in the regulation of gene expression, cell proliferation, and apoptosis. Treatment of human leukemia HL60 cells with lactacystin, a specific inhibitor of the proteasome, increased the steady-state levels of endogenous c-MycS proteins. The half-life of endogenous [(35)S]MycS was similar to that of c-Myc ( approximately 23 min) in HL60 cells. c-Myc(Delta2-143), which lacks the transcription regulatory domain, had a half-life which was similar to that of endogenous c-Myc in 293 and HL60 cells. Treatment of the cells with lactacystin stabilized [(35)S]Myc(Delta2-143) and [(35)S]Myc and caused multi-ubiquitin conjugates of c-Myc, c-MycS, and Myc(Delta2-143) to accumulate. These findings indicate that the Myc homology boxes and the rest of the transcription regulatory domain (the first 144 amino acids) are dispensable for ubiquitylation and rapid destruction of c-MycS and c-Myc by the proteasome.

Effect of a novel 5-lipoxygenase activating protein inhibitor, BAYx 1005, on asthma induced by cold dry air.

BACKGROUND: Leukotrienes have been implicated in the mediation of airway obstruction induced by hyperventilation of cold dry air in asthmatic subjects. The effect of a novel inhibitor of 5-lipoxygenase activating protein, BAYx 1005, on the bronchospastic response to cold dry air hyperventilation was investigated in asthmatic patients. METHODS: After a screening cold dry air hyperventilation challenge to document cold air responsiveness, 16 asthmatic subjects (baseline forced expiratory volume in one second (FEV1) > 60% of predicted) underwent cold air challenge three hours after receiving 750 mg of BAYx 1005 or placebo using a randomised, double blind, crossover design. Leukotriene synthesis inhibition was estimated by measuring the concentration of leukotriene B4 in whole blood stimulated with calcium ionophore A21387. RESULTS: Treatment with BAYx 1005 produced a 34% (95% CI 11 to 63) increase in the amount of cold air minute ventilation required for a 10% decrease in FEV1 (PD10VE) compared with placebo (mean (SE) 37.6 (1.12) 1/min compared with 28.0 (1.13) 1/min, p < 0.006). The PD20VE increased 19% (95% CI 8 to 31) after treatment with BAYx 1005 compared with placebo (57.3(1.10)1/min versus 48.1 (1.10) 1/min, p < 0.002). Treatment with BAYx 1005 produced a 15.4% decrease in ionophore-stimulated LTB4 production, while treatment with placebo produced a 7.1% increase in ex vivo LTB4 (p < 0.02). CONCLUSIONS: Treatment with BAYx 1005, a novel inhibitor of leukotriene synthesis, produced a significant blunting of cold dry air responsiveness consistent with the hypothesis that leukotrienes mediate part of the bronchoconstriction induced by hyperventilation of cold dry air.

Effects of temperature on the coupled activities of the vanadate-sensitive proton pump from maize root microsomes.

The mechanism by which proton transport is coupled to ATP hydrolysis by vanadate-sensitive pumps is poorly understood. The effects of temperature on the activities of the vanadate-sensitive ATPase from maize (Zea mays) roots were assessed to provide insight into the coupling mechanism. The initial rate of proton transport had a bell-shaped dependence on temperature with an optimal range between 20 and 30 degrees C. However, the rate of vanadate-sensitive ATP hydrolysis increased as the temperature was raised from 4 to 43 degrees C. The differential sensitivity of proton transport to temperatures above 30 degrees C was also observed when the ATPase was reconstituted into dioleoylphosphatidylcholine vesicles. Inhibition of proton transport with temperatures above 30 degrees C was associated with higher rates of proton leakage from the membranes. In addition, proton transport was more inhibited than ATP hydrolysis at temperatures below 10 degrees C. Reduced rates of proton transport at lower temperatures were not associated with higher rate of proton conductivity across the membranes. Therefore, the preferential inhibition of proton transport at temperatures below 10 degrees C may reflect an effect of temperature on the coupling between proton transport and ATP hydrolysis within the vanadate-sensitive ATPase.

Auxin transport in suspension-cultured soybean root cells : I. Characterization.

The kinetic parameters of auxin transport in suspension-cultured soybean (Glycine max [L.] Merr.) root cells were investigated. The same processes that are responsible for polar indoleacetic acid (IAA) transport in other plant tissues were found to occur in soybean root cells. These include (a) passive diffusion of the undissociated auxin molecule across the plasma membrane, (b) uptake via a specific, saturable carrier, and (c) phytotropin-sensitive efflux. Metabolism of exogenously added IAA was rapid; at the end of a 15-minute uptake period >80% of the IAA taken up had been converted to other compounds. The time course of [(14)C]IAA uptake in the first 90 seconds revealed two phases, the first corresponding to a rate of uptake approximately twice as large as the second phase. The transition to the second phase was delayed in the presence of the phytotropins triiodobenzoic acid or naphthylphthalamic acid, suggesting that an increase in the efflux of label as IAA accumulates in the cytoplasm is responsible for the transition. Carrier-mediated uptake contributes between 50 and 60% to the total rate of auxin uptake from a 0.28 micromolar IAA solution, with passive diffusion accounting for the remainder. Kinetic analysis of carrier-mediated uptake revealed a pH optimum of 5.0 and a Michaelis-Menten constant of 0.4 micromolar at pH 5.5. Because phytotropins had no effect on the initial rate of uptake, the efflux carrier does not appear to be involved in the uptake process.

Auxin Transport in Suspension-Cultured Soybean Root Cells : II. Anion Effects on Carrier-Mediated Uptake.

To test the hypothesis that the carrier-mediated component of the indoleacetic acid (IAA) influx involves an electrogenic proton/IAA anion symport, the effects on the IAA influx of salts expected to depolarize the membrane potential were examined in suspension-cultured soybean (Glycine max [L.] Merr.) root cells. Although KCl does inhibit carrier-mediated uptake, the effect is specific to the anion at low concentrations and not due to more general processes such as changes in ionic or osmotic strength. Other anions such as bromide, iodide, and fluoride inhibit the carrier more strongly. Because potassium iminodiacetate, which is also expected to depolarize the membrane potential, has no inhibitory effect on the IAA influx, there is no evidence for the involvement of the membrane potential in carrier-mediated uptake. It is therefore most likely that in soybean cells, if carrier-mediated uptake occurs via a proton symport, the H(+):IAA- stoichiometry is 1:1. At concentrations greater than 70 millimolar, sorbitol, a nonionic osmoticum, inhibits carrier-mediated IAA uptake. The effects of specific anions and osmotic potential on the uptake carrier necessitates the reevaluation of other auxin transport studies in which KCl was routinely used as an agent with which to depolarize the membrane potential.

Should psychiatrists administer anesthesia for ECT?

Whether psychiatrists are qualified to give anesthesia for ECT is controversial. At the authors' hospital, over a 9-year period ECT resulted in no mortality and minimal morbidity; in 98.8% of the treatments, anesthesia was given by psychiatrists. The average nursing time required for cases in which anesthesiologists administered anesthetic was longer than that for psychiatrists' cases. This difference may be related to succinylcholine dose and efficacy of ECT. The authors' surveys indicated that psychiatrists and anesthesiologists have differing opinions on whether psychiatrists should administer anesthesia for ECT and that few psychiatry residency programs which teach ECT provide training in anesthesia.

Immersion fixation of rapidly frozen, untreated tissues and suspensions of cells including spermatozoa.

Small pieces of tissue, and cell suspensions in plastic artificial insemination (AI) 'straws', were frozen rapidly in Freon-12 at -155 degrees C, without pre-treatment. Peripheral fragments were thawed directly in 2% glutaraldehyde at 0 degrees C, and processed for transmission electron microscopy (TEM) studies. Preservation of ultrastructure was satisfactory, and freezing artifacts were minimal.