Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes.

Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

Cleavage of cyclin A at R70/R71 by the bacterial protease OmpT.

Previous work has shown that cyclin A can be cleaved at Arg-70/Arg-71 by a proteolytic activity present in an in vitro-coupled transcription/translation system by using rabbit reticulocyte lysate programmed by plasmid DNA encoding p27(KIP1), a cyclin-dependent kinase inhibitor, but not by plasmid DNAs encoding other cyclin-dependent kinases inhibitors. Here we report that cyclin A is also cleaved by translation product programmed by plasmid DNA encoding cyclin B. Several findings indicate that the cleavage activity in this assay is provided by the bacterial protease OmpT, which cofractionates with cyclin B and p27(KIP1) plasmid DNAs and is thus carried over into the coupled in vitro transcription/translation reactions. (i) Cleavage activity appeared even when transcription or translation of the cyclin B or p27(KIP1) was blocked. (ii) Activity resembling OmpT, a serine protease that cleaves between dibasic residues, routinely copurifies with p27(KIP1) and cyclin B plasmid DNAs. (iii) Both cyclin A cleavage activity and OmpT activity are heat stable, resistant to denaturation, and inhibited by Zn(2+), Cu(2+), or benzamidine. (iv) Cyclin A cleavage activity is detected when using lysates or DNAs prepared from Escherichia coli strains that contained OmpT but not with strains lacking OmpT. (v) Purified OmpT enzyme itself cleaves cyclin A at R70/R71. These data indicate that OmpT can be present in certain DNA preparations obtained by using standard plasmid purification protocols, and its presence can potentially affect the outcome and interpretation of studies carried out using in vitro-translated proteins.

Identification of XPR-1, a progesterone receptor required for Xenopus oocyte activation.

Quiescent full-grown Xenopus oocytes remain arrested at the G(2)/M border of meiosis I until exposed to progesterone, their natural mitogen. Progesterone triggers rapid, nontranscriptional responses that lead to the translational activation of stored mRNAs, resumption of the meiotic cell cycles, and maturation of the oocyte into a fertilizable egg. It has long been presumed that progesterone activates the oocyte through a novel nontranscriptional signaling receptor. Here, we provide evidence that a conventional transcriptional progesterone receptor cloned from Xenopus oocytes, XPR-1, is required for oocyte activation. Overexpression of XPR-1 through mRNA injection increases sensitivity to progesterone and accelerates progesterone-activated cell cycle reentry. Injection of XPR-1 antisense oligonucleotides blocks the ability of oocytes to respond to progesterone; these oocytes are rescued by subsequent injection of XPR-1 or the human progesterone receptor PR-B. Antisense-treated oocytes can be activated in response to inhibition of protein kinase A, one of the earliest known changes occurring downstream of progesterone stimulation. These results argue that the conventional progesterone receptor also functions as the signaling receptor that is responsible for the rapid nontranscriptional activation of frog oocytes.

Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA.

Full-grown Xenopus oocytes arrest at the G2/M border of meiosis I. Progesterone breaks this arrest, leading to the resumption of the meiotic cell cycles and maturation of the oocyte into a fertilizable egg. In these oocytes, progesterone interacts with an unidentified surface-associated receptor, which induces a non-transcriptional signalling pathway that stimulates the translation of dormant c-mos messenger RNA. Mos, a mitogen-activated protein (MAP) kinase kinase kinase, indirectly activates MAP kinase, which in turn leads to oocyte maturation. The translational recruitment of c-mos and several other mRNAs is regulated by cytoplasmic polyadenylation, a process that requires two 3' untranslated regions, the cytoplasmic polyadenylation element (CPE) and the polyadenylation hexanucleotide AAUAAA. Although the signalling events that trigger c-mos mRNA polyadenylation and translation are unclear, they probably involve the activation of CPEB, the CPE binding factor. Here we show that an early site-specific phosphorylation of CPEB is essential for the polyadenylation of c-mos mRNA and its subsequent translation, and for oocyte maturation. In addition, we show that this selective, early phosphorylation of CPEB is catalysed by Eg2, a member of the Aurora family of serine/threonine protein kinases.

Cell cycle-regulated proteolysis of mitotic target proteins.

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1-S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.

Cdc20 associates with the kinase aurora2/Aik.

Cdc20/fizzy family proteins are involved in activation of the anaphase-promoting complex/cyclosome, which catalyzes the ubiquitin-dependent proteolysis of cell cycle regulatory proteins such as anaphase inhibitors and mitotic cyclins, leading to chromosome segregation and exit from mitosis. Previous work has shown that human Cdc20 (hCdc20/p55CDC) associates with one or more kinases. We report here that Cdc20-associated myelin basic protein kinase activity peaks sharply in early M phase (embryonic cells) or in G2 phase (somatic cells). In HeLa cells, Cdc20 is associated with the kinase aurora2/Aik. Aurora2/Aik is a member of the aurora/Ipl1 family of kinases that, like Cdc20, previously has been shown to be localized at mitotic spindle poles and is involved in regulating chromosome segregation and maintaining genomic stability. The demonstration that Cdc20 is associated with aurora2/Aik suggests that some function of Cdc20 is carried out or regulated through its association with aurora2/Aik.

Obstetrics anyone? How family medicine residents' interests changed.

OBJECTIVE: To determine family medicine residents' attitudes and plans about practising obstetrics when they enter and when they graduate from their residency programs. DESIGN: Residents in each of 4 consecutive years, starting July 1991, were surveyed by questionnaire when they entered the program and again when they graduated (ending in June 1996). Only paired questionnaires were used for analysis. SETTING: Family medicine residency programs at the University of Toronto in Ontario. PARTICIPANTS: Of 358 family medicine residents who completed the University of Toronto program, 215 (60%) completed questionnaires at entry and exit. MAIN OUTCOME MEASURES: Changes in attitudes and plans during the residency program as ascertained from responses to entry and exit questionnaires. RESULTS: Analysis was based on 215 paired questionnaires. Women residents had more interest in obstetric practice at entry: 58% of women, but only 31% of men were interested. At graduation, fewer women (49%) and men (22%) were interested in practising obstetrics. The intent to undertake rural practice was strongly associated with the intent to practise obstetrics. By graduation, residents perceived lifestyle factors and compensation as very important negative factors in relation to obstetric practice. Initial interest and the eventual decision to practise obstetrics were strongly associated. CONCLUSIONS: Intent to practise obstetrics after graduation was most closely linked to being a woman, intending to practise in a rural area, and having an interest in obstetrics prior to residency. Building on the interest in obstetrics that residents already have could be a better strategy for producing more physicians willing to practise obstetrics than trying to change the minds of those uninterested in such practice.

The cyclin-dependent kinase inhibitor p27(Kip1) induces N-terminal proteolytic cleavage of cyclin A.

Progression through the cell cycle is regulated in part by the sequential activation and inactivation of cyclin-dependent kinases (CDKs). Many signals arrest the cell cycle through inhibition of CDKs by CDK inhibitors (CKIs). p27(Kip1) (p27) was first identified as a CKI that binds and inhibits cyclin A/CDK2 and cyclin E/CDK2 complexes in G1. Here we report that p27 has an additional property, the ability to induce a proteolytic activity that cleaves cyclin A, yielding a truncated cyclin A lacking the mitotic destruction box. Other CKIs (p15(Ink4b), p16(Ink4a), p21(Cip1), and p57(Kip2)) do not induce cleavage of cyclin A; other cyclins (cyclin B, D1, and E) are not cleaved by the p27-induced protease activity. The C-terminal half of p27, which is dispensable for its kinase inhibitory activity, is required to induce cleavage. Mechanistically, p27 does not appear to cause cleavage through direct interaction with cyclin/CDK complexes. Instead, it activates a latent protease that, once activated, does not require the continuing presence of p27. Mutation of cyclin A at R70 or R71, residues at or very close to the cleavage site, blocks cleavage. Noncleavable mutants are still recognized by the anaphase-promoting complex/cyclosome pathway responsible for ubiquitin-dependent proteolysis of mitotic cyclins, indicating that the p27-induced cleavage of cyclin A is part of a separate pathway. We refer to this protease as Tsap (pTwenty-seven- activated protease).

EWS/FLI1 up regulates mE2-C, a cyclin-selective ubiquitin conjugating enzyme involved in cyclin B destruction.

The EWS/FLI1 fusion gene found in Ewing's sarcoma and primitive neuroectodermal tumor, is able to transform certain cell lines by acting as an aberrant transcription factor. The ability of EWS/FLI1 to modulate gene expression in cells transformed and resistant to transformation by EWS/FLI1, was assessed by Representational Difference Analysis (RDA). We found that the cyclin selective ubiquitin conjugase murine E2-C, was up regulated in NIH3T3 cells transformed by EWS/FLI1 but not in a nontransformed NIH3T3 clone expressing EWS/FLI1. We also found that mE2-C is upregulated in NIH3T3 cells transformed by other genes including activated cdc42, v-ABL and c-myc. We demonstrated that expression of mE2-C in both the EWS/FLI1 transformed and parent NIH3T3 lines varies with the cell cycle. Finally, dominant-negative mE2-C, created by changing a catalytic cysteine to serine, inhibits the in vitro ubiquitination and degradation of cyclin B in human HeLa cell extracts. These data suggest that part of the biologic effect of EWS/FLI1 could be to transcriptionally modulate genes involved in cell cycle regulation.

The kinase Eg2 is a component of the Xenopus oocyte progesterone-activated signaling pathway.

Quiescent Xenopus oocytes are activated by progesterone, which binds to an unidentified surface-associated receptor. Progesterone activates a poorly understood signaling pathway that results in the translational activation of mRNA encoding Mos, a MAP kinase kinase kinase necessary for the activation of MAP kinase and MPF, the resumption of meiosis, and maturation of the oocyte into the sperm-responsive egg. We have designed a screen to identify early signaling proteins based on the premise that some of these proteins would be phosphorylated or otherwise modified within minutes of progesterone addition. This screen has revealed Eg2, a Ser/Thr kinase. We find that Eg2 is phosphorylated soon after progesterone stimulation and provide evidence that it functions in the signaling pathway. Overexpression of Eg2 via mRNA microinjection shortens the time between progesterone stimulation and the appearance of new Mos protein, accelerates activation of MAP kinase and advances entry into the meiotic cell cycle. Finally, overexpression of Eg2 dramatically reduces the concentration of progesterone needed to trigger oocyte activation. These results argue that the kinase Eg2 is a component of the progesterone-activated signaling pathway that releases frog oocytes from cell cycle arrest.

Proteolytic ratchets that control progression through mitosis.

A key player in mitotic progression is a cell-cycle-regulated ubiquitin-protein ligase complex known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C is part of the machinery that promotes the metaphase-anaphase transition by mediating the ubiquitin-dependent destruction of anaphase inhibitors and initiates exit from mitosis by degrading mitotic cyclins. This review describes the known components and substrates of the mitotic ubiquitination machinery and discusses how a new subfamily of proteins that contain the WD40 repeat (the Fizzy/Cdc20p family) might activate the APC/C to allow temporal differences in substrate ubiquitination during progression through mitosis.

Functional analysis of the Saccharomyces cerevisiae UBC11 gene.

UBC11 is the Saccharomyces cerevisiae gene that is most similar in sequence to E2-C, a ubiquitin carrier protein required for the destruction of mitotic cyclins and proteins that maintain sister chromatid cohesion in animal cells and in Schizosaccharomyces pombe. We have disrupted the UBC11 gene and found it is not essential for yeast cell viability even when combined with deletion of UBC4, a gene that has also been implicated in mitotic cyclin destruction. Ubc11p does not ubiquitinate cyclin B in clam cell-free extracts in vitro and the destruction of Clb2p is not impaired in extracts prepared from delta ubc11 or delta ubc4 delta ubc11 cells. These results suggest Ubc4p and Ubc11p together are not essential for mitotic cyclin destruction in S. cerevisiae and we can find no evidence to suggest that Ubc11p is the true functional homologue of E2-C.

Components of the signaling pathway linking the 1-methyladenine receptor to MPF activation and maturation in starfish oocytes.

Starfish oocytes are arrested at the G2/M-phase border of meiosis I. Exposure to their natural mitogen, 1-methyladenine (1-MA), leads to the activation of MPF and MAP kinase, resumption of the meiotic cell cycle, and fertilization competency. The 1-MA receptor has not yet been identified, but it is known to be linked functionally to a pertussis toxin-sensitive G-protein. G beta gamma appears to be the major effector of the 1-MA receptor, since injection of G beta gamma, but not activated G alpha i, leads to the activation of MPF, entry into meiosis, and oocyte maturation. The components that connect G beta gamma to MPF and MAP kinase activation in oocytes are unknown. In mammalian cells, a novel phosphatidylinositol 3-kinase, PI-3 kinase-gamma, links G beta gamma to the MAP kinase activation pathway. Here we show that PI-3 kinase is required for starfish oocyte maturation. LY294002 and wortmannin, inhibitors of PI-3 kinase, block MPF and MAP kinase activation and entry into meiosis. Inhibition by LY294002 is reversible and limited to the hormone-dependent period. Neither inhibitor, however, blocks the earliest hormone-induced event, formation of actin spikes at the cell membrane. By contrast, pertussis toxin blocks both actin spiking and later events, arguing that PI-3 kinase functions downstream of G beta gamma. Finally, we show that unlike the well-studied case in Xenopus oocytes, where MAP kinase is an essential component of the MPF activation pathway, MAP kinase is not required for either MPF activation or subsequent oocyte maturation in starfish. Instead, its major role appears to be suppression of DNA synthesis in unfertilized, haploid eggs.

Combined trabeculectomy and phacoemulsification: a one-site vs a two-site approach.

PURPOSE: To compare the results of combined trabeculectomy and phacoemulsification surgery with intraocular lens implant by means of a one-site vs a two-site approach. METHODS: Glaucomatous patients with a coexisting cataract were randomly assigned to undergo either a one-site or two-site combined procedure. One-site surgery was performed with a limbus-based conjunctival flap and scleral tunnel at the 12-o'clock position. Two-site surgery was performed with a limbus-based conjunctival flap for the trabeculectomy in the superior nasal quadrant and a temporal clear cornea incision for phacoemulsification. Mitomycin C (0.4 mg/ml for 2 minutes) was applied to the scleral surface at the trabeculectomy site for both approaches. All patients received intraocular lens implants at the time of combined surgery. RESULTS: Thirty-three eyes of 33 patients were included in this study. Preoperative intraocular pressure and number of glaucoma medications were similar in the two groups. Corrected visual acuity improved similarly in both groups. Intraocular pressure decreased in both groups at last follow-up but was not significantly different (P = .129) between the one-site and two-site groups. At last follow-up, the one-site group required significantly more (P = .030) medications than did the two-site group. CONCLUSIONS: Combined trabeculectomy and phacoemulsification surgery in which one-site and two-site techniques were used yielded similar improvements in corrected visual acuity and intraocular pressure reduction. However, the one-site group required more medication to maintain intraocular pressure control than did the two-site group.

Combination of systemic acetazolamide and topical dorzolamide in reducing intraocular pressure and aqueous humor formation.

OBJECTIVE: The study aimed to determine whether topical dorzolamide and systemic acetazolamide have an additive effect on intraocular pressure (IOP) and aqueous humor formation (AHF). DESIGN: This was a prospective, open-label, two-protocol clinical study. PARTICIPANTS: Sixteen patients with ocular hypertension or with primary open-angle glaucoma were studied. INTERVENTION: Baseline AHF was measured by computerized fluorophotometry and IOP by pneumatonometry without antiglaucoma therapy. In the first protocol, dorzolamide was randomized to one eye (N = 10) and IOP and AHF measurements were repeated. One week later, having used dorzolamide in one eye three times daily, patients had measurements performed before and after the single administration of oral acetazolamide 250 mg. In the second protocol, having used acetazolamide 250 mg four times daily for 4 to 7 days (N = 6), patients had measurements performed before and after a single drop of dorzolamide was instilled randomly into one eye. The patient continued acetazolamide and unilateral dorzolamide for 4 to 7 more days and returned for IOP and AHF measurements. MAIN OUTCOME MEASURES: Intraocular pressure and AHF were measured in treated and contralateral control eyes. RESULTS: In the first protocol, IOP (mmHg +/- standard deviation) was significantly (P = 0.02) lower in the dorzolamide (16.3 +/- 2.6) than in the contralateral control (19.9 +/- 2.9) eyes. Aqueous humor formation (microliter/minute +/- standard deviation) also was lower (P = 0.02) in dorzolamide eyes (1.79 +/- 0.4 vs. 2.33 +/- 0.5). After oral acetazolamide 250 mg, IOP was unchanged in dorzolamide eyes (17.6 +/- 2.0 preacetazolamide vs. 17.9 +/- 2.0 postacetazolamide), whereas it was reduced (P = 0.003) in control eyes (20.5 +/- 2.2 preacetazolamide vs. 16.9 +/- 2.3 postacetazolamide). Aqueous humor formation was reduced in control eyes (2.31 +/- 0.8 preacetazolamide vs. 1.73 +/- 0.6 postacetazolamide; P = 0.005) but not in dorzolamide-treated eyes (1.56 +/- 0.45 preacetazolamide vs. 1.77 +/- 0.39 postacetazolamide). In the second protocol, acetazolamide 250 mg four times daily symmetrically reduced IOP and AHF in both eyes. After single-drop dorzolamide in one eye, IOP and AHF did not change significantly. After 4 to 7 days of acetazolamide and unilateral dorzolamide, IOP and AHF remained reduced to a similar level in dorzolamide and control eyes not receiving topical therapy. CONCLUSION: Topical dorzolamide and oral acetazolamide, in the concentrations and doses used in this study, are not additive. Either drug alone results in maximum reduction in IOP and AHF. Concomitant glaucoma therapy of a topical and systemic carbonic anhydrase inhibitor is not warranted.

Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase.

Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis.

Cognitive and functional assessments of stroke patients: an analysis of their relation.

OBJECTIVES: To improve the assessment of stroke patients for the purpose of designing rehabilitation treatments and predicting rehabilitation outcomes. Specific objectives included the evaluation of the power of functional scales to properly assess both physical and cognitive disabilities, and the evaluation of the relations between functional, neurological, physical, and cognitive assessments. The hypothesis was that the relations between different assessment types (eg, functional, neurological, etc) can be assessed by the relations between the results of these assessments when administered to stroke patients. DESIGN: Sixty-six stroke patients were administered a series of tests including functional assessments (Functional Independence Measure, barthel Index, Rankin Functional Scale), neurological assessments (Canadian Neurological Scale, National Institute of Health Stroke Scale), physical assessments (Stages of Motor Recovery, Clinical Outcome Variables Scale), and cognitive assessments (Stroke Unit Mental Status Examination, Mini Mental State, Raven Matrices, Boston Naming Test). RESULTS: Analysis of correlation coefficients revealed that the stronger relationships were observed between functional assessments and physical assessments, and between functional assessments and neurological assessments. Cognitive tests did not correlate highly with any of the functional tests used in this study. Three factors were extracted using factor analysis. They were interpreted as being a physical disability factor (50% of the variance), a cognitive disability factor (23% of the variance), and a dementia factor (12% of variance). Functional scales obtained higher loads on the physical disability factor only. CONCLUSIONS: Considering that cognitive functions are frequently affected in stroke patients, cognitive impairment needs to be more seriously considered when describing and/or predicting a patient's level of independence. In brain injured patients, such as stroke patients, we suggest that the total score provided by standardized functional scales should be interpreted with care. We believe that rehabilitation outcome could be better predicted if the results of functional assessment were coupled with in-depth cognitive assessment.