Biomedical subjects

N Morin

Publications and source records attributed to N Morin.

At least 55 records · Page 3Linked to original sources

PubMed1997

The roles of the human immunodeficiency virus type 1 Pol protein and the primer binding site in the placement of primer tRNA(3Lys) onto viral genomic RNA.

Factors that modulate the placement of primer tRNA(3Lys) onto the viral RNA genome in human immunodeficiency virus type 1 (HIV-1) were investigated through analysis of reverse-transcribed products that are extended from the tRNA(3Lys) primer. Mutations were introduced into the HIV-1 pol gene to result in the appearance of a stop codon in the open reading frame of the reverse transcriptase (RT) gene. These constructs, BH10-RT1 and BH10-RT2, yielded viruses with truncated Pol proteins. Alternatively, we altered the sequences involved in frameshifting by generating the construct BH10-FS. With each of these mutated viruses, we found that the primer tRNA(3Lys) that was placed onto viral genomic RNA was present in an unextended state. In contrast, as expected, tRNA(3Lys) in the case of wild-type BH10 virus had been extended by 2 bases. Furthermore, the amount of tRNA(3Lys) that was placed onto viral RNA in mutated viruses was significantly less than that placed in the wild-type virus. We also generated a mutant within the polymerase-active site of RT (D185H) (Asp-->His) that eliminated RT polymerase activity. We found that the placement of primer tRNA(3Lys) onto viral genomic RNA was independent of enzyme function; however, the tRNA(3Lys) that was placed was present in an unextended state due to the loss of RT activity. In contrast, the elimination of protease activity through a D25A (Asp-->Ala) point mutation in the protease-active site (construct BH10-PR) did cause a drop in the efficiency of tRNA(3Lys) placement. In this situation, a proportion of the placed tRNA(3Lys) was found to be extended by 2 bases, although not to the extent found with wild-type virus (BH10), due to a decrease in RT activity associated with unprocessed Gag-Pol protein that could not be cleaved because of the loss of protease activity. We also investigated the role of the primer binding site (PBS) in the placement of tRNA(3Lys) through a series of 2-, 4-, and 8-nucleotide (nt) deletions at the 3' end of the PBS, i.e., BH10-PBS2, BH10-PBS4, and BH10-PBS8, respectively. In mutated viruses BH10-PBS2 and BH10-PBS4, the 2-base-extended form of tRNA(3Lys) was still detected. However, less primer tRNA(3Lys) was placed onto viral genomic RNA as more nucleotides were deleted until the percentage of placement seen with wild-type BH10 virus dropped to only 4% in the virus with 8 nt deleted (BH10-PBS8). Consistently, these mutated viruses possessed decreased initial replication capacity compared with that of the wild-type virus, with the extent of incapacity corresponding to the size of the deletion. However, after several days, an increase in replication potential was accompanied by a reversion to a wild-type PBS.

Animals↗

PubMed1997

Fish nodavirus lytic cycle and semipermissive expression in mammalian and fish cell cultures.

In this study, Dicentrarchus labrax encephalitis virus (DlEV), which causes sea bass encephalitis, was propagated in cell culture, thus allowing study of its lytic cycle. DlEV infection of mammalian and fish cells induced different patterns of expression of capsid proteins, which were assembled as virus-like particles, accumulating in the cytoplasm either as diffuse masses or in vesicles, as shown by electron microscopy. These particles correspond to virions, as shown by their ability to induce secondary infection. Fish cells proved to be more permissive for DlEV than mammalian cells, although virus yield remained low. RNA analysis of infected sea bass cells revealed DlEV RNA3, in addition to genomic RNA1 and RNA2, and the presence of the RNA2 minus strand, thus demonstrating the replication of the DlEV genome. In addition, DlEV RNA-dependent RNA polymerase was associated with mature virions even after purification by a CsCl gradient, but it was dissociated when capsids were destabilized. In addition to providing more information about the relatedness of DlEV to the members of the family Nodaviridae, this study shows that fish nodaviruses may not be able to infect as wide a variety of cells as insect nodaviruses can.

Animals↗

PubMed1997

[Laser-assisted endoscopic resection: a new surgical technique for the treatment of benign prostatic hypertrophy. Preliminary results of a study involving 100 patients].

This study was designed to assess the efficiency of 2 kind of laser prostatectomy devices in the treatment of Benign Prostatic Hyperplasia: a non contact technique versus a contact technique versus a contact one. From January 1994 to September 1994, 100 patients were included in a randomized comparison of 2 laser prostatectomy devices with right angle firing laser fibers: a non contact technique with Urolase fiber (Bard) (50 patients) versus a contact technique with Fibertom fiber (Dornier) (50 patients). The Urolase fiber was used at 60 Watts power setting for 60 seconds and administered to each lobe at 2, 4, 8 and 10 o'clock positions. The Fibertom fiber was used by dragging or the so called "painting" technique at 3 and 6 months with 3 parameters: Madsen symptom scores, peak urinary flow rates and post-void residual urine volumes. Operative morbidity rate was 9%. No difference in morbidity between both fibers. No blood transfusion was required in any case. Statistical analysis of the aforementioned parameters shows a p-value of < 0.001 for all parameters. Comparing the 2 different fibers, there was no statistical difference in outcome for any of these parameters. From this study we conclude that the preliminary results achieved, using the Urolase and the Fibertom fiber, are equivocal and interesting. However, a long term follow-up is necessary to evaluate the definitive efficiency of laser prostatectomy and to determine the optimal procedure.

Aged↗

PubMed1997

[The VLAP system (Visual Laser Ablation of the Prostate): comparative study of the contact versus non-contact techniques in a series of 100 cases].

OBJECTIVE: To compare two contact and non-contact techniques using the VLAP system (visual laser ablation of the prostate). PATIENTS AND METHOD: From January 1994 to April 1995, two groups of 50 patients were included in a randomized prospective study and were treated by the contact and non-contact laser technique, respectively. These patients presented with symptomatic BPH requiring transurethral electroresection. These patients were evaluated according to subjective (AUA score) and objective criteria (maximum flow rate, transrectal and suprapubic ultrasonography). ND:YAG laser energy was delivered by a Medilas 4100 source via a lateral beam fibre. Objective success criteria were an improvement of the AUA score by at least 50% and of the maximum flow rate by at least 50% with a difference of 4 mL/s. The failure criterion was the need to perform complementary electroresection during the first 6 postoperative months. RESULTS: The mortality was 2% (cardiovascular causes), and the overall morbidity (first postoperative month) was 29%, with 21% of a vesical irritation syndrome, 6% of urinary tract infection and 2% of bladder clots. The global result at 12 months showed a statistically significant postoperative improvement of the subjective and objective parameters studied preoperatively (p = 0.0001). In the contact group, the AUA score, mean maximum flow rate and residual urine, which had preoperative values of 19.1, 9.1 mL/s and 141 mL, respectively, were equal to 2.3, 15.6 mL/s and 45 mL at 12 months. In the non-contact group, the same parameters, which had preoperative values of 17.8, 9.2 mL/s and 87 mL, respectively, were equal to 3.8, 13.3 mL/s and 47 mL at 12 months. The results, evaluated as a function of objective success criteria, showed a 12-month success rate of 68% for the contact method and 61% for the non-contact method. The mean hospital stay was 6 days and the mean duration of bladder catheterization was 5 days with the two techniques. During follow-up, 7% of patients required revision by electroresection, regardless of the technique used. CONCLUSION: this study demonstrated identical results for the contact and non-contact techniques using the VLAP system.

Aged↗

PubMed1996

Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: role of calcium and protein kinase A and C.

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E2 (PGE2) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE2 provoked a 3.9 +/- 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca++ channel blockers, verapamil and nifedipine, and the Ca++/calmodulin inhibitor, W-7. The Ca+2 ionophore, ionomycin, mimicked the effects of PGE2 as did the phorbol ester PMA, which activates Ca++/-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE2 did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE2 secretagogue, IL-1 beta, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 +/- 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE2 does not mediate the effect of its secretagogue and that IL-1 beta signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2 receptor signalling pathways. Taken together, our results suggest that PGE2 modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism.

Calcium↗

PubMed1996

Cloning and expression of a novel neuropeptide Y receptor.

The neuropeptide Y family of peptides, which includes neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), are found in the central and peripheral nervous system and display a wide array of biological activities. These actions are believed to be mediated through pharmacologically distinct G protein-coupled receptors, and, to date, three members of the NPY receptor family have been cloned. In this study we describe the cloning and expression of a novel NPY receptor from mouse genomic DNA. This receptor, designated NPY Y5, shares 60% amino acid identity to the murine NPY Y1 receptor. The pharmacology of this novel receptor resembles that of the NPY Y1 receptor and is distinct from that described for the NPY Y2, Y3, and Y4 receptors. In situ hybridization of mouse brain sections reveals expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain.

Amino Acid Sequence↗

PubMed1996

MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts.

Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.

Animals↗

PubMed1996

[Mechanisms of reduction of CD4 receptor expression on the surface of HIV-1 infected cells].

Specific interactions between the cell surface CD4 receptor and the HIV-1 envelope glycoprotein gp120 are responsible for the entry of HIV into host cells. Following infection, a down-modulation of CD4 at the cell surface is commonly observed. This may render cells resistant to subsequent infection by HIV as well as other viruses that also use CD4 as a portal of entry. This phenomenon is termed retroviral interference. CD4 down-modulation is complex and involves at least 3 viral gene products which include the envelope precursor gp160 and 2 auxilliary proteins Nef and Vpu. CD4 down-modulation has been observed in each of primary CD4+ T-lymphocytes and monocyte-derived macrophages, as well as both T and monocytic cell lines. CD4 down-regulation may occur at different levels. Specific binding of soluble gp120 may lead to internalization of CD4. The HIV-1 nef gene product which is expressed prior to HIV-1 structural proteins also causes the internalization of CD4 followed by its lysosomal degradation. During the late phase of viral gene expression i.e. viral structural protein synthesis, CD4-gp160 complexes forming in the ER represent another important factor leading to CD4 down-modulation. Finally, CD4 which is retained by gp160 in the ER, is specifically degraded in the presence of Vpu. Thus, it appears that CD4 down-regulation is of central importance to the life cycle of HIV-1.

Animals↗

PubMed1995

MAT1 ('menage à trois') a new RING finger protein subunit stabilizing cyclin H-cdk7 complexes in starfish and Xenopus CAK.

The kinase responsible for Thr161-Thr160 phosphorylation and activation of cdc2/cdk2 (CAK:cdk-activating kinase) has been shown previously to comprise at least two subunits, cdk7 and cyclin H. An additional protein co-purified with CAK in starfish oocytes, but its sequencing did not reveal any similarity with any known protein. In the present work, a cDNA encoding this protein is cloned and sequenced in both starfish and Xenopus oocytes. It is shown to encode a new member of the RING finger family of proteins with a characteristic C3HC4 motif located in the N-terminal domain. We demonstrate that the RING finger protein (MAT1: 'menage à trois') is a new subunit of CAK in both vertebrate and invertebrates. However, CAK may also exist in oocytes as heterodimeric complexes between cyclin H and cdk7 only. Stable heterotrimeric CAK complexes were generated in reticulocyte lysates programmed with mRNAs encoding Xenopus cdk7, cyclin H and MAT1. In contrast, no heterodimeric cyclin H-cdk7 complex could be immunoprecipitated from reticulocyte lysates programmed with cdk7 and cyclin H mRNAs only. Stabilization of CAK complexes by MAT1 does not involve phosphorylation of Thr176, as the Thr176-->Ala mutant of Xenopus cdk7 could engage as efficiently as wild-type cdk7 in ternary complexes. Even though starfish MAT1 is almost identical to Xenopus MAT1 in the RING finger domain, the starfish subunit could not replace the Xenopus subunit and stabilize cyclin H-cdk7 in reticulocyte lysate, suggesting that the MAT1 subunit does not (or not only) interact with cyclin H-cdk7 through the RING finger domain.

Amino Acid Sequence↗

PubMed1995

Sequence analysis and transcriptional mapping of the orf-2 gene of Autographa californica nuclear polyhedrosis virus.

Sequencing of the dnapol promoter region of Autographa californica nuclear polyhedrosis virus (AcNPV) revealed an overlapping open reading frame (ORF) in an antisense orientation, referred to as ORF-2. Analysis of the ORF-2 deduced amino-acid sequence revealed two short regions of homology with a similar ORF from Lymantria dispar nuclear polyhedrosis virus (LdNPV). Two 3' processing signals of this gene, expressed late during infection, were shown to be located on the orf-2 stop codon and 162 nucleotides further downstream.

Amino Acid Sequence↗

PubMed1995

Onset of chromosome segregation at the metaphase to anaphase transition of the cell cycle.

Chromosome segregation is one of the most important acts in the life of the cell. Unequal inheritance of chromosomes (aneuploidy) is a cause of a number of disorders, particularly in humans, even though eukaryotic cells can arrest or delay the transition from metaphase to anaphase if an event critical to the completion of metaphase is impaired. In this report, we review recent advances in our knowledge of how the complex process of chromosome segregation is coupled with cell cycle progression, and starts at onset of anaphase with sister chromatids separation of the replicated chromosomes.

Anaphase↗

PubMed1995

Sequence requirements for encapsidation of deletion mutants and chimeras of human immunodeficiency virus type 1 Gag precursor into retrovirus-like particles.

Interacting domains in human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55gag) expressed in recombinant baculovirus-infected cells were investigated by three different methods: (i) trans rescue and coencapsidation of C-terminal deletion (amber) Gag mutants and Gag chimeras into retrovirus-like particles in complementation experiments with HIV-1 wild-type (WT) Pr55gag, (ii) Gag-Gag interactions in vitro in Gag ligand affinity blotting assays, and (iii) quantitative immunoelectron microscopy of retrovirus-like Gag particles, using a panel of monoclonal antibodies to probe the epitope accessibility of encapsidated HIV-1 WT Pr55gag. Four discrete regions, within residues 210 to 241, 277 to 306 (major homology region), and 307 to 333 in the capsid (CA) protein and residues 358 to 374 at the CA-spacer peptide 2 (sp2) junction, were found to have a significant influence on Gag trans-packaging efficiency. A fifth region, within residues 375 to 426, overlapping the sp2-nucleocapsid (NC) protein junction and most of the NC, seemed to be essential for stable inter-Gag binding in vitro. The coincidence of the two regions from 358 to 374 and 375 to 426 with an immunologically silent domain in WT Gag particles suggested that they could participate in direct Gag interactions.

Animals↗

PubMed1995

Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase.

1,3-beta-D-Glucan is a major structural polymer of yeast and fungal cell walls and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-beta-D-glucan synthase. Previous work has shown that the FKS1 gene encodes a 215-kDa integral membrane protein (Fks1p) which mediates sensitivity to the echinocandin class of antifungal glucan synthase inhibitors and is a subunit of this enzyme. We have cloned and sequenced FKS2, a homolog of FKS1 encoding a 217-kDa integral membrane protein (Fks2p) which is 88% identical to Fks1p. The residual glucan synthase activity present in strains with deletions of fks1 is (i) immunodepleted by antibodies prepared against FKS2 peptides, demonstrating that Fks2p is also a component of the enzyme, and (ii) more sensitive to the echinocandin L-733,560, explaining the increased sensitivity of fks1 null mutants to this drug. Simultaneous disruption of FKS1 and FKS2 is lethal, suggesting that Fks1p and Fks2p are alternative subunits with essential overlapping function. Analysis of FKS1 and FKS2 expression reveals that transcription of FKS1 is regulated in the cell cycle and predominates during growth on glucose, while FKS2 is expressed in the absence of glucose. FKS2 is essential for sporulation, a process which occurs during nutritional starvation. FKS2 is induced by the addition of Ca2+ to the growth medium, and this induction is completely dependent on the Ca2+/calmodulin-dependent phosphoprotein phosphatase calcineurin. We have previously shown that growth of fks1 null mutants is highly sensitive to the calcineurin inhibitors FK506 and cyclosporin A. Expression of FKS2 from the heterologous ADH1 promoter results in FK506-resistant growth. Thus, the sensitivity of fks1 mutants to these drugs can be explained by the calcineurin-dependent transcription of FKS2. Moreover, FKS2 is also highly induced in response to pheromone in a calcineurin-dependent manner, suggesting that FKS2 may also play a role in the remodeling of the cell wall during the mating process.

Amino Acid Sequence↗

PubMed1995

[Molecular basis and clinical significance of HIV-1 resistance to nucleoside compounds].

The prolonged use of anti-viral nucleosides (ZDV, ddI, ddC) in HIV-infected patients has given rise to the isolation of viral variants that display resistance against these compounds. Tissue culture selection experiments, involving increasing concentrations of anti-viral compounds, have likewise been shown to select for drug-resistant strains of HIV. Cloning, sequencing and site-directed mutagenesis have shown that a series of point mutations in the viral reverse transcriptase (RT) are responsible for this phenomenon. A different series of mutations in RT are responsible for resistance against non-nucleoside inhibitors of this enzyme. These mutations are due to the error-prone nature of RT during viral replication. Mutated forms of recombinant RT, that derive from drug-resistant viruses, have reduced affinity for relevant triphosphorylated nucleosides.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Heterodimeric retinoic acid receptor-beta and retinoid X receptor-alpha complexes stimulate expression of the intercellular adhesion molecule-1 gene.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the leukocyte-function associated antigen-1 and for Mac-1, plays an important role in immune responses. ICAM-1 expression is regulated by various proinflammatory cytokines, phorbol myristate acetate, and retinoic acid. In this study, we investigated the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by retinoic acid in Cos-1 cells. Deletion mutant analysis provided evidence that a region located between -393 and -176 from the translational start site is critical to retinoic acid stimulation of luciferase activity. This region harbors the consensus sequence for a retinoic acid-responsive element (RARE) 5'-GGGTCATCGCCCTGCCA-3'. The Smal(-270)/Smal (-178) fragment containing this element conferred appropriate retinoic acid responsiveness to an enhancerless SV40 promoter. Cotransfection of expression vectors encoding the retinoic acid receptor alpha, beta, or gamma and retinoid X receptor alpha with reporter plasmids harboring the putative RARE demonstrated that the ICAM-1 gene is regulated by retinoic acid in a retinoic acid receptor beta/retinoid X receptor alpha-dependent fashion. Electrophoretic mobility shift assays showed that ICAM-1 and ADH3 RARE, a well-characterized RARE, display the same band shift pattern, bind retinoic acid receptor beta and retinoid X receptor alpha, and are mutually competitive.

Base Sequence↗

PubMed1994

The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.

In Saccharomyces cerevisiae, mutations in FKS1 confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETG1 confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-beta-D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z. The FKS1 and ETG1 genes were cloned by complementation of these phenotypes and were found to be identical. Disruption of the gene results in (i) a pronounced slow-growth phenotype, (ii) hypersensitivity to FK506 and cyclosporin A, (iii) a slight increase in sensitivity to echinocandin, and (iv) a significant reduction in 1,3-beta-D-glucan synthase activity in vitro. The nucleotide sequence encodes a 215-kDa polypeptide predicted to be an integral membrane protein with 16 transmembrane helices, consistent with previous observations that the etg1-1 mutation results in echinocandin-resistant glucan synthase activity associated with the nonextractable membrane fraction of the enzyme. These results suggest that FKS1 encodes a subunit of 1,3-beta-D-glucan synthase. The residual activity present in the disruption mutant, the nonessential nature of the gene, and results of Southern blot hybridization analysis point to the existence of a glucan synthase isozyme.

Base Sequence↗

PubMed1994

p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes.

p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified kinase) as features of p40MO15 that are required for the generation of active CAK.

Amino Acid Sequence↗

PubMed1994

The proteolysis-dependent metaphase to anaphase transition: calcium/calmodulin-dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs.

It has been shown, using spindles assembled in vitro in extracts containing CSF (the cytostatic factor responsible for arresting unfertilized vertebrate eggs at metaphase), that onset of anaphase requires Ca(2+)-dependent activation of the ubiquitin-dependent proteolytic pathway that destroys both mitotic cyclins and an unknown protein responsible for metaphase arrest (Holloway et al., 1993, Cell, 73, 1382-1402). We showed recently that Ca2+/calmodulin-dependent protein kinase II (CaM KII) activates the ubiquitin-dependent cyclin degradation pathway in CSF extracts (Lorca et al., 1993, Nature, 366, 270-273), but did not investigate its possible effect on sister chromatid segregation. In this work we identify CaM KII as the only target of Ca2+ in inducing anaphase in CSF extracts, and further show that transition to anaphase does not require the direct phosphorylation of metaphase spindle components by CaM KII. A possible interpretation of the above results could have been that the ubiquitin-dependent degradation pathway is required for onset of anaphase only when spindles are clamped at metaphase due to CSF activity, and not in the regular cell cycle that occurs in the absence of CSF activity. We ruled out this possibility by showing that competitive inhibition of the ubiquitin-dependent degradation pathway still prevents the onset of anaphase in cycling extracts that lack CSF and do not require Ca2+ for sister chromatid separation.

Anaphase↗