Discrimination between ubiquitin-dependent and ubiquitin-independent proteolytic pathways by the 26S proteasome subunit 5a.

The 26S proteasome subunit 5a binds polyubiquitin chains and has previously been shown to inhibit the degradation of mitotic cyclins. Presumably inhibition results from S5a binding and preventing recognition of Ub-cyclin conjugates by the 26S proteasome. Here we show that S5a does not inhibit the degradation of full-length ornithine decarboxylase (ODC) consistent with previous reports that the enzyme is degraded in an antizyme-dependent, but ubiquitin-independent reaction. S5a does, however, inhibit degradation of short ODC translation products generated by internal initiation events. Because in vitro translation often produces some shortened products, the existence of ubiquitin conjugated to a 35S-labeled protein is not necessarily evidence that the full-length protein is a substrate of the Ub-dependent proteolytic pathway.

Methadone maintenance in pregnancy: a reappraisal.

OBJECTIVE: The purpose of this study is to evaluate women receiving methadone maintenance during pregnancy. STUDY DESIGN: Thirty-two pregnancies in women receiving methadone maintenance were matched by gestational age to women with a positive urine screen for cocaine at delivery and to drug-free controls. Pregnancy outcome variables were compared, including birth weight and neonatal morbidity. Analysis was by chi2 and t test with significance set at .05. RESULTS: Birth weight of methadone-exposed infants was 2748 g versus 2925 g for cocaine and 3032 g for controls, P = not significant. Birth weight comparison with a 50-mg maternal methadone cutoff dose was not different. A head circumference for methadone infants of 32.4 +/- 4.7 cm was significantly less than controls, 33.5 +/- 4.0 cm, P < .04, but not different from infants of cocaine users, 32.8 +/- 3.1 cm. Women using cocaine had a significantly higher incidence of meconium in labor compared with methadone and controls. Of women taking methadone 27 of 32 (84.3%) were positive for other drugs of abuse in the last screen before or at delivery. Cocaine 12 of 32 (37.5%), other opiates 13 of 32 (40.6%), and marijuana 14 of 32 (43.7%) were the most prevalent. Neonatal withdrawal occurred in 23 of 32 (72%) women taking methadone. The neonates of women using < 50 mg of methadone were as likely to withdraw as those women using > or = 50 mg, 61.5% versus 79.0%, P not significant. Three neonates in the methadone group (9.3%) had major congenital anomalies, with 2 of the 3 (66.6%) resulting in mortality. CONCLUSIONS: Birth outcome is not significantly different between methadone and cocaine users. Women receiving methadone maintenance are likely to abuse other illicit drugs.

MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity.

Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling Xenopus laevis egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.

Breathing when chest wall muscle are tonically contracted for isometric, non-respiratory tasks.

We have previously shown that regional chest wall impedance increases when the chest wall muscles are tonically contracted to perform isometric, non-respiratory tasks. To test how this affects breathing, we measured respiratory frequency, tidal volume, end-tidal PCO2, electromyographic activity (EMG) at four points on the chest wall surface, and regional displacements across six planes of the chest wall during maintenance of three different postures that necessitated strong tonic respiratory muscle contraction. These postures included a static push-up, a bilateral leg-lift and a partial sit-up. The subjects (n = 8) were able to maintain the postures for 1.5-2.5 min, and strong tonic EMG activity was observed in each posture at all points measured. The rate and depth of breathing and pattern of regional chest wall displacements were variable within the group of subjects and among the three postures. However, minute ventilation increased and end-tidal PCO2 decreased in each subject during each posture (P < 0.05). In six of the eight subjects, transdiaphragmatic pressure (Pdi) was measured during 1 min of the same exercises. The ratio of the breathing fluctuation in Pdi to tidal volume was at least twice as high compared with rest, except for two subjects during the leg-lifts. We conclude that strong tonic contraction of the chest wall muscles impedes, but does not limit, breathing, and that there is no single breathing strategy used during such conditions.

Inhibition of ubiquitin-mediated proteolysis by the Arabidopsis 26 S protease subunit S5a.

A variety of protease inhibitors have been used to study ubiquitin-dependent proteolysis by the 26 S protease. However, these inhibitors lack complete specificity and thus affect ubiquitin-independent pathways as well. We recently identified an Arabidopsis protein, MBP1, that is homologous to subunit 5a (S5a) of the human 26 S protease complex. MBP1 and S5a bind multiubiquitin chains with high affinity and presumably facilitate the recognition of ubiquitin conjugates by the 26 S protease. We show here that free MBP1 can be a potent inhibitor of ubiquitin-dependent proteolysis in several cell-free systems. When added to reticulocyte lysates or to Xenopus egg extracts, the plant protein effectively blocked the degradation of multiubiquitinated lysozyme and cyclin B, respectively. MBP1 did not enhance the removal of ubiquitin from lysozyme or affect the ability of the 26 S complex to hydrolyze fluorogenic peptides. These data suggest that the plant protein specifically interferes with the recognition of ubiquitin conjugates by the 26 S protease. Thus MBP1, human S5a, and their homologs should prove to be valuable reagents for investigating cellular events mediated by ubiquitin-dependent proteolysis.

Ubiquitination of full-length cyclin.

Mitotic cyclins are key cell-cycle regulators that are relatively stable through most of the cell-cycle then rapidly degraded at mitosis. We have detected ubiquitin conjugates of full-length Xenopus cyclin B2 strongly suggesting that ubiquitination rather than a proteolytic cleavage is the initiating event in cyclin destruction. The highest levels of ubiquitin conjugates correlate with the phase of rapid proteolysis. This result supports previous findings that implicate the ubiquitin system in cyclin proteolysis. However, we also observe cyclin-ubiquitin conjugates in both cytostatic factor arrested and interphase extracts where cyclin is more stable. The physiologic role of ubiquitinated cyclin under these conditions is unclear.

Ubiquitin metabolism in cycling Xenopus egg extracts.

Xenopus egg extract is capable of supporting mitosis in vitro, which makes it ideal for biochemical analysis of the cell cycle. Since several studies have implicated the ubiquitin system in cell cycle progression, we have measured ubiquitin conjugation rates, proteolysis of ubiquitin-lysozyme conjugates, and rates of isopeptidase activity in cycling Xenopus egg extracts. Although ubiquitin conjugation in cytostatic factor arrested extract was half that in activated extract, there were no changes in rates of ubiquitin conjugation during the cell cycle. Ubiquitin conjugates are degraded by a 26 S ATP-stimulated protease. The ability of the 26 S protease to degrade ubiquitin-lysozyme conjugates and a fluorigenic peptide also remained constant across the cell cycle. In contrast to previously characterized systems, isopeptidase activity in Xenopus egg extract is energy-dependent. Glycerol gradient fractionation of Xenopus egg extract separated two ATP-dependent isopeptidases. On co-sedimented with the 26 S protease; the other sedimented slower and was not associated with any additional proteolytic activity. As found for rates of Ub conjugation and conjugate proteolysis, there was little or no variation in isopeptidase activity during the cell cycle.

Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity.

Coated pits will assemble onto purified plasma membranes that are attached to a poly-L-lysine coated substratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558-563; Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108, 1615-1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 subunits from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached membranes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (Kd approximately 3 x 10(-8) M) to salt stripped membranes. Binding is saturable and abolished by treating membranes with less than 20 micrograms/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer-washed membranes that bear endogenous AP-2 bound triskelions with high affinity (Kd approximately 3 x 10(-9) M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.

Coat proteins isolated from clathrin coated vesicles can assemble into coated pits.

Isolated human fibroblast plasma membranes that were attached by their extracellular surface to a solid substratum contained numerous clathrin coated pits that could be removed with a high pH buffer (Moore, M.S., D.T. Mahaffey, F.M. Brodsky, and R.G.W. Anderson. 1987. Science [Wash. DC]. 236:558-563). When these membranes were incubated with coat proteins extracted from purified bovine coated vesicles, new coated pits formed that were indistinguishable from native coated pits. Assembly was dependent on the concentration of coat protein with half maximal assembly occurring at 7 micrograms/ml. Assembly was only slightly affected by the presence of divalent cations. Whereas normal appearing lattices formed in a low ionic strength buffer, when assembly was carried out in a low pH buffer, few coated pits were evident but numerous small clathrin cages decorated the membrane. Coated pits did not form randomly on the surface; instead, they assembled at differentiated regions of membrane that could be distinguished in carbon/platinum replicas of frozen and etched membranes by the presence of numerous particles clustered into patches the size and shape of a coated pit.

Assembly of clathrin-coated pits onto purified plasma membranes.

During receptor-mediated endocytosis, coated pits invaginate to form coated vesicles, clathrin and associated proteins dissociate from the vesicle membrane, and these proteins form new coated pits at the cell surface. As a means of elucidating molecular mechanisms that govern the function of coated pits, the assembly phase of this cycle was reconstituted by incubating purified membranes that were treated to remove endogenous coated pits with cytoplasm extracted from cultured cells. The in vitro assembly of coated pits on these membranes satisfactorily mimics many features of coated pit formation in the intact cell. These studies indicate that: the membranes contain a limited number of coated pit assembly sites that bind clathrin with high affinity; the half-time for assembly is 5 minutes both at 4 degrees C and 37 degrees C; during assembly, proteins with molecular sizes of 180, 110, and 36 kilodaltons are recruited to the plasma membrane; and assembly is not dependent on adenosine triphosphate, but this nucleotide triggers a temperature-dependent loss of coated pits that are assembled in the absence of adenosine triphosphate.

Terminal CNV in the absence of motor response.

We addressed the question of whether the terminal CNV, or E-wave, can be obtained in the absence of a motor response. In our design, the stimuli served only to reduce uncertainty with respect to a prior prediction, so that no response to S2 was required. In one experiment, the location of uncertainty reducing information was manipulated: in S1 alone or in S2 alone. When S2 reduced uncertainty, the pre-S2 E-wave was larger than when S2 did not reduce uncertainty. Similarly, when S1 reduced uncertainty, a pre-S1 negativity (whose topography did not differ from that of the pre-S2 E-wave) was larger than when S1 did not reduce uncertainty. The pre-S1 results also indicated that a prior experimenter-generated warning signal is not necessary for a non-motoric negativity to be obtained. In another experiment, the S1-S2 interval was manipulated (1 sec versus 3 sec). For the pre-S2 E-wave, onset was later and duration was longer for the longer S1-S2 interval. Peak amplitude and time of termination after S2 did not differ for short and long duration E-waves. Apparently, the timing of the E-wave is related to when in time the process it reflects is 'needed.' The dependence of the amplitude and timing of the pre-stimulus negativity on the temporal location of information, in conjunction with its independence of motor response requirement, suggests that the pre-stimulus negativity reflects some operation in the domain of expectancy, anticipation or 'mental preparation' for the informational stimulus.

Temperature-sensitive aspects of evoked and spontaneous transmitter release at the frog neuromuscular junction.

1. The temperature dependence of presynaptic processes involved in neuromuscular transmission was studied by rapidly increasing the temperature of cooled frog neuromuscular junctions by 4--10 degrees C using pulses from a neodymium laser. The temperature elevation was complete within 0.5 msec, and decayed back to control levels with a time constant of about 7--8 sec. 2. Temperature jumps completed before nerve stimulation increased the quantal content and decreased the latency of the end-plate potential (e.p.p.). The Q10 for e.p.p. quantal content in low [Ca2+] Ringer averaged about 3.9 over the range 1--18 degrees C. 3. Temperature jumps occurring during the synaptic delay (the interval between the presynaptic action potential and the onset of the e.p.p.) also increased the quantal content and decreased the latency of the e.p.p. These effects diminished as the onset of the temperature jump was moved closer to the expected onset of the e.p.p. Temperature jumps applied after the onset of the e.p.p. immediately accelerated the time course of the e.p.p. but did not significantly alter quantal content. These results demonstrate that the magnitude and timing of evoked release are influenced by temperature-sensitive processes that operate both during and shortly after the presynaptic nerve action potential, but are largely complete before the onset of release. 4. Temperature jumps were applied at various times during the interval between two nerve stimuli. The amplitude of the second e.p.p. decreased as the temperature jump was moved earlier in the interstimulus interval, suggesting that the rise in temperature following the first nerve stimulus accelerates the decay of facilitation. When the temperature jump was moved from 10 msec after to 10 msec before the onset of the first e.p.p., the amplitude of the second e.p.p. either decreased or showed no change. The fact that the second e.p.p. did not increase suggests that the temperature-sensitive processes that increase the quantal content of the conditioning e.p.p. do not greatly increase the facilitation following that e.p.p. 5. Temperature jumps immediately accelerated the time course of spontaneous miniature end-plate potentials (m.e.p.p.s) and increased their frequency. Experiments using slow temperature changes revealed that the Q10 for m.e.p.p. frequency in normal Ringer is about 10 over the range 10--20 degrees C. M.e.p.p. frequency was much less sensitive to temperature changes below about 10 degrees C. When the nerve terminal was depolarized by 20 mM-K+ in the presence of Ca2+, the Q10 for the rate of spontaneous release over the range 10--20 degrees C decreased to about 4, similar to the Q10 for e.p.p. quantal content. In the absence of extracellular Ca2+ the Q10 for m.e.p.p. frequency in 20 mM-K+ remained near 10. 6. The marked difference in Q10S for spontaneous transmitter release under different experimental conditions suggests that not all transmitter release uses identical mechanisms...