Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

The Greatwall kinase: a new pathway in the control of the cell cycle.

New data have recently established that protein phosphorylation during mitosis is the result of a controlled balance between kinase and phosphatase activities and that, as for mitotic kinases, phosphatases are also regulated during cell division. This regulation is at least in part induced by the activation of the Greatwall (Gwl) kinase at mitotic entry. Activated Gwl phosphorylates its substrates cAMP-regulated phospho protein 19 (Arpp19) and α-endosulfine (ENSA), promoting their binding to and the inhibition of PP2A. Interestingly, besides the role of the Gwl-Arpp19/ENSA in the control of mitotic division, new data in yeast support the involvement of this pathway in mRNA stabilization during G(0) program initiation, although in this case the phosphatase PP2A appears not to be implicated. Finally, Gwl activity has been shown to be required for DNA checkpoint recovery. These new findings support the view that Gwl, Arpp19 and ENSA could function as the core of a new signalization pathway that, by targeting different final substrates, could participate in a variety of physiological functions.

RSK2 is a kinetochore-associated protein that participates in the spindle assembly checkpoint.

The spindle assembly checkpoint (SAC) prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The MAP kinase (MAPK) is an important player in this pathway, however its exact role is not fully understood. One major target of MAPK is the p90 ribosomal protein S6 kinase (RSKs) family. In this study, we analyse whether Rsk2 could participate in the activation of the SAC. Our data indicate that this protein is localized at the kinetochores under checkpoint conditions. Moreover, it is essential for the SAC activity in Xenopus egg extracts as its depletion prevents metaphase arrest as well as the kinetochore localization of the other SAC components. We also show that this kinase might also participate in the maintenance of the SAC in mammalian cells as Rsk2 knockdown in these cells prevents the kinetochore localization of Mad1, Mad2 and CENP-E under checkpoint conditions.

Chfr interacts and colocalizes with TCTP to the mitotic spindle.

Chfr is a checkpoint protein that plays an important function in cell cycle progression and tumor suppression, although its exact role and regulation are unclear. Previous studies have utilized overexpression of Chfr to determine the signaling pathway of this protein in vivo. In this study, we demonstrate, by using three different antibodies against Chfr, that the endogenous and highly overexpressed ectopic Chfr protein is localized and regulated differently in cells. Endogenous and lowly expressed ectopic Chfr are cytoplasmic and localize to the spindle during mitosis. Higher expression of ectopic Chfr correlates with a shift in the localization of this protein to the nucleus/PML bodies, and with a block of cell proliferation. In addition, endogenous and lowly expressed ectopic Chfr is stable throughout the cell cycle, whereas when highly expressed, ectopic Chfr is actively degraded during S-G2/M phases in an autoubiquitination and proteasome-dependent manner. A two-hybrid screen identified TCTP as a possible Chfr-interacting partner. Biochemical analysis with the endogenous proteins confirmed this interaction and identified beta-tubulin as an additional partner for Chfr, supporting the mitotic spindle localization of Chfr. The Chfr-TCTP interaction was stable throughout the cell cycle, but it could be diminished by the complete depolymerization of the microtubules, providing a possible mechanism where Chfr could be the sensor that detects microtubule disruption and then activates the prophase checkpoint.

The zebrafish bcl-2 homologue Nrz controls development during somitogenesis and gastrulation via apoptosis-dependent and -independent mechanisms.

Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.

Penetration of surface-inoculated bacteria as a result of electrically generated hydrodynamic shock wave treatment of boneless skinless chicken breasts.

The top surface of boneless skinless chicken breasts was inoculated with either green fluorescent protein (GFP)-labeled Escherichia coli (E. coli-GFP) or rifampicin-resistant E. coli (E. coli-Rif) and subjected to electrically generated hydrodynamic shock wave treatment (HVADH). Cryostat sampling in concert with laser scanning confocal microscopy or plating onto antibiotic selective agar was used to determine if HVADH treatment resulted in the movement of the inoculated bacteria from the outer inoculated surface to the interior of intact boneless skinless chicken breasts. In HVADH-treated boneless skinless chicken breasts, marker bacteria were detected within the first 200 microm below the inoculated surface, 50 to 100 microm beyond the depth of untreated surface inoculated boneless skinless chicken breasts. The exact depth at which the marker bacteria were found was dependent on the cryostat sampling distance used. These results suggest that HVADH treatments affect the movement of surface bacteria.

Penetration of surface-inoculated bacteria as a result of hydrodynamic shock wave treatment of beef steaks.

The top surface of the raw eye of round steaks was inoculated with either green fluorescent protein (GFP)-labeled Escherichia coli (E. coli-GFP) or rifampin-resistant E. coli (E. coli-rif). Cryostat sampling in concert with laser scanning confocal microscopy (LSCM) or plating onto antibiotic selective agar was used to determine if hydrodynamic shock wave (HSW) treatment resulted in the movement of the inoculated bacteria from the outer inoculated surface to the interior of intact beef steaks. HSW treatment induced the movement of both marker bacteria into the steaks to a maximum depth of 300 microm (0.3 mm). Because popular steak-cooking techniques involve the application of heat from the exterior surface of the steak to achieve internal temperatures ranging from 55 to 82 degrees C, the extent of bacterial penetration observed in HSW-treated steaks does not appear to pose a safety hazard to consumers.

Levels of Vibrio vulnificus and organoleptic quality of raw shellstock oysters (Crassostrea virginica) maintained at different storage temperatures.

Temperature abuse during raw oyster harvesting and storage may allow for the multiplication of natural spoilage flora as well as microbial pathogens, thus posing a potential health threat to susceptible consumers and compromising product quality. The objective of this study was to provide a scientific basis for determining whether different refrigeration and abuse temperatures for raw oysters would result in a spoiled product before it became unsafe. Raw shellstock oysters (Crassostrea virginica) purchased from a commercial Virginia processor were subjected to different temperature abuse conditions (7, 13, and 21 degrees C) over a 10-day storage period. Salinity, pH, halophilic plate count (HPC), total culturable Vibrio counts, and culturable Vibrio vulnificus counts were determined at each abuse condition. V. vulnificus isolates were confirmed by a specific enzyme-linked immunosorbent assay. Olfactory analysis was performed to determine consumer acceptability of the oysters at each abuse stage. The pH of the oysters decreased over time in each storage condition. The HPC increased 2 to 4 logs for all storage conditions, while olfactory acceptance decreased over time. V. vulnificus levels increased over time, reaching 10(5) to 10(6) CFU/g by day 6. The length of storage had a greater effect on the bacterial counts and olfactory acceptance of the oysters (P < 0.05) over time than did the storage temperature (P < 0.05).

c-Mos and cyclin B/cdc2 connections during Xenopus oocyte maturation.

Fully-grown G2 arrested Xenopus oocytes can be induced to enter and progress into meiotic cell cycle by progesterone stimulation. This process is termed oocyte maturation. An early response to progesterone is the synthesis of the onco-protein c-Mos, defined as the candidate initiator of Xenopus oocyte maturation, which triggers the MAPK cascade, MPF activation and promotes CSF activity. Here we review our current knowledge on the synthesis, activation and functions of c-Mos in connection with MPF activation during maturation. We also discuss our recent results concerning the dispensability of cyclin B degradation in meiosis I-meiosis II transition and the stabilization of c-Mos through its direct phosphorylation by cyclin B/cdc2.

Interaction between cyclin T1 and SCF(SKP2) targets CDK9 for ubiquitination and degradation by the proteasome.

CDK9 paired with cyclin T1 forms the human P-TEFb complex and stimulates productive transcription through phosphorylation of the RNA polymerase II C-terminal domain. Here we report that CDK9 is ubiquitinated and degraded by the proteasome whereas cyclin T1 is stable. SCF(SKP2) was recruited to CDK9/cyclin T1 via cyclin T1 in an interaction requiring its PEST domain. CDK9 ubiquitination was modulated by cyclin T1 and p45(SKP2). CDK9 accumulated in p45(SKP2-/-) cells, and its expression during the cell cycle was periodic. The transcriptional activity of CDK9/cyclin T1 on the class II major histocompatibility complex promoter could be regulated by CDK9 degradation in vivo. We propose a novel mechanism whereby recruitment of SCF(SKP2) is mediated by cyclin T1 while ubiquitination occurs exclusively on CDK9.

[When chromosomal dynamics control cell division]. / Quand la dynamique chromosomique contrôle la division cellulaire.

In most tumor cells a chromosomal instability leads to an abnormal chromosome number (aneuploidy). The mitotic checkpoint is essential for ensuring accurate chromosome segregation by allowing mitotic delay in response to a spindle defect. This checkpoint delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. When unattached kinetochores are present, the metaphase/anaphase transition is not allowed and the time available for chromosome-microtubule capture increases. Genes required for this delay were first identified in Saccharomyces cerevisiae (the MAD, BUB and MPS1 genes) and subsequently, homologs have been identified in higher eucaryotes showing that the spindle checkpoint pathway is highly conserved. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC) from ubiquitinylating proteins whose destruction is required for anaphase onset.

Cyclin B/cdc2 induces c-Mos stability by direct phosphorylation in Xenopus oocytes.

The c-Mos proto-oncogene product plays an essential role during meiotic divisions in vertebrate eggs. In Xenopus, it is required for progression of oocyte maturation and meiotic arrest of unfertilized eggs. Its degradation after fertilization is essential to early embryogenesis. In this study we investigated the mechanisms involved in c-Mos degradation. We present in vivo evidence for ubiquitin-dependent degradation of c-Mos in activated eggs. We found that c-Mos degradation is not directly dependent on the anaphase-promoting factor activator Fizzy/cdc20 but requires cyclin degradation. We demonstrate that cyclin B/cdc2 controls in vivo c-Mos phosphorylation and stabilization. Moreover, we show that cyclin B/cdc2 is capable of directly phosphorylating c-Mos in vitro, inducing a similar mobility shift to the one observed in vivo. Tryptic phosphopeptide analysis revealed a practically identical in vivo and in vitro phosphopeptide map and allowed identification of serine-3 as the largely preferential phosphorylation site as previously described (Freeman et al., 1992). Altogether, these results demonstrate that, in vivo, stability of c-Mos is directly regulated by cyclin B/cdc2 kinase activity.

Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint.

The mitotic checkpoint acts to inhibit entry into anaphase until all chromosomes have successfully attached to spindle microtubules. Unattached kinetochores are believed to release an activated form of Mad2 that inhibits APC/C-dependent ubiquitination and subsequent proteolysis of components needed for anaphase onset. Using Xenopus egg extracts, a vertebrate homolog of yeast Mps1p is shown here to be a kinetochore-associated kinase, whose activity is necessary to establish and maintain the checkpoint. Since high levels of Mad2 overcome checkpoint loss in Mps1-depleted extracts, Mps1 acts upstream of Mad2-mediated inhibition of APC/C. Mps1 is essential for the checkpoint because it is required for recruitment and retention of active CENP-E at kinetochores, which in turn is necessary for kinetochore association of Mad1 and Mad2.

The APC is dispensable for first meiotic anaphase in Xenopus oocytes.

Here we show that segregation of homologous chromosomes and that of sister chromatids are differentially regulated in Xenopus and possibly in other higher eukaryotes. Upon hormonal stimulation, Xenopus oocytes microinjected with antibodies against the anaphase-promoting complex (APC) activator Fizzy or the APC core subunit Cdc27, or with the checkpoint protein Mad2, a destruction-box peptide or methylated ubiquitin, readily progress through the first meiotic cell cycle and arrest at second meiotic metaphase. However, they fail to segregate sister chromatids and remain arrested at second meiotic metaphase when electrically stimulated or when treated with ionophore A34187, two treatments that mimic fertilization and readily induce chromatid segregation in control oocytes. Thus, APC is required for second meiotic anaphase but not for first meiotic anaphase.

Growth and histamine formation of Morganella morganii in determining the safety and quality of inoculated and uninoculated bluefish (Pomatomus saltatrix).

The objective of this study was to determine the effect of normal microflora and Morganella morganii on histamine formation and olfactory acceptability in raw bluefish under controlled storage conditions. Fillets inoculated with and without M. morganii were stored at 5, 10, and 15 degrees C for 7 days. Microbial isolates from surface swabs were identified and screened for histidine decarboxylase activity. Olfactory acceptance was performed by an informal sensory panel. Histamine levels were quantified using high-performance liquid chromatography and fluorescence detection. While olfactory acceptance decreased, histamine concentration and bacterial counts increased. Storage temperature had a significant effect on histamine levels, bacterial counts, and olfactory acceptance of the bluefish. Inoculation with M. morganii had a positive significant effect on histamine formation for bluefish held at 10 and 15 degrees C (P < 0.0001). The results of the study will serve in supporting U.S. Food and Drug Administration (FDA) regulations regarding guidance and hazard levels of histamine in fresh bluefish.

Part of Xenopus translin is localized in the centrosomes during mitosis.

During oogenesis, maternal mRNAs are synthesised and stored in a translationally dormant form due to the presence of regulatory elements at the 3' untranslated regions (3'UTR). In Xenopus oocytes, several studies have described the presence of RNA-binding proteins capable to repress maternal-mRNA translation. The testis-brain RNA-binding protein (TB-RBP/Translin) is a single-stranded DNA- and RNA-binding protein which can bind the 3' UTR regions (Y and H elements) of stored mRNAs and can suppress in vitro translation of the mRNAs that contain these sequences. Here we report the cloning of the Xenopus homologue of the TB-RBP/Translin protein (X-translin) as well as its expression, its localisation, and its biochemical association with the protein named Translin associated factor X (Trax) in Xenopus oocytes. The fact that this protein is highly present in the cytoplasm from stage VI oocytes until 48 h embryos and that it has been described as capable to inhibit paternal mRNA translation, indicates that it could play an important role in maternal mRNA translation control during Xenopus oogenesis and embryogenesis. Moreover, we investigated X-translin localisation during cell cycle in XTC cells. In interphase, although a weak and diffuse nuclear staining was observed, X-translin was mostly present in the cytoplasm where it exhibited a prominent granular staining. Interestingly, part of X-translin underwent a remarkable redistribution throughout mitosis and associated with centrosomes, which may suggest a new unknown role for this protein in cell cycle.

The polo-like kinase Plx1 prevents premature inactivation of the APC(Fizzy)-dependent pathway in the early Xenopus cell cycle.

Members of the polo-like family of protein kinases have been involved in the control of APC (anaphase-promoting complex) during the cell cycle, yet how they activate APC is not understood in any detail. In Xenopus oocytes, Ca2+-dependent degradation of cyclin B associated with release from arrest at second meiotic metaphase was demonstrated to require the polo-like kinase Plx1. The aim of the present study was to examine, beyond Ca2+-dependent resumption of meiosis, the possible role of Plx1 in the control of cyclin degradation during the early mitotic cell cycle. Plx1 was found to be dispensable for MPF to turn on the cyclin degradation machinery. However, it is required to prevent premature inactivation of the APC-dependent proteolytic pathway. Microcystin suppresses the requirement for Plx1 in both Ca2+-dependent exit from meiosis, associated with degradation of both cyclin B and A downstream of CaMK2 activation, and prevention of premature APC(Fizzy) inactivation in the early mitotic cell cycle. These results are consistent with the view that Plx1 antagonizes an unidentified microcystin-sensitive phosphatase that inactivates APC(Fizzy).

The Xenopus XMAP215 and its human homologue TOG proteins interact with cyclin B1 to target p34cdc2 to microtubules during mitosis.

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2-cyclinB1 complex). It has previously been demonstrated that the p34cdc2-cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215-cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.

Biogenic amine survey and organoleptic changes in fresh, stored, and temperature-abused bluefish (Pomatomus saltatrix).

Changes in histamine, putrescine, and cadaverine concentrations in bluefish filets (Pomatomus saltatrix) stored at 5, 10, and 15 degrees C were determined using high-performance liquid chromatography. An organoleptic assessment was conducted simultaneously with the biogenic amine analyses. The histamine levels found in fresh bluefish obtained from wholesale seafood distributors ranged between <1 ppm and 99 with an average of 39 ppm. Putrescine and cadaverine were not found in fresh bluefish. Fish fillets stored at each of the three temperatures developed histamine. The greatest accumulation of histamine was observed in fish stored at 15 degrees C, which developed histamine levels as high as 2,200 ppm. Putrescine levels increased at each temperature during storage. Cadaverine was present only in uninoculated bluefish stored at 15 degrees C. Histamine achieved higher levels in bluefish pieces inoculated with Morganella morganii, which demonstrates that bluefish support bacterial histamine formation. Histamine levels at each temperature exceeded the 50-ppm advisory level established by the Food and Drug Administration before 100% sensory rejection. Standard plate counts increased during storage of fish at all temperatures, but the correlation between histamine levels and standard plate count was not significant.