The appearance of dural sealants under MR imaging.

Dural sealants are an adjunct to obtain watertight closure after intradural procedures. This study aims to characterize the appearance on MR imaging of 3 commonly employed dural sealants: fibrin glue, PEGH, and BSAG. To this end, patients who underwent spinal intradural procedures that included the use of dural sealant during closure were identified retrospectively. Post-operative data on 15 patients, including complications such as pseudomeningocele formation and infection, were gathered. The appearance of dural sealants on follow-up MR imaging scans within 3 days of surgery was analyzed. Fifteen patients were identified (5 with fibrin glue, 5 with PEGH, and 5 with BSAG applied during closure) with appropriately timed post-operative MR imaging scans. All 3 substances were identifiable based on anatomic location and imaging characteristics on post-operative MR imaging in standard T1, T1 PGFS, and T2 FSE. Definite differentiation between CSF and fibrin glue or PEGH was not possible with the T1 or T1 PGFS, or with the T2 FSE. Differences in intensity between CSF and BSAG were also not significant on either T1 sequence, but they were statistically significant on the T2 FSE. All patients had an uneventful post-operative course, and no patients developed post-operative pseudomeningocele at 30 days. This study concludes that water-based dural sealants such as fibrin glue and PEGH are difficult to differentiate from CSF on standard T1, T1 PGFS and T2 FSE, while BSAG is easily recognized on the T2 FSE. Recognition of water-based sealants therefore requires communication between the neurosurgeon and the neuroradiologist to avoid post-operative misidentification.

Direct evidence for the role of centrosomally localized p53 in the regulation of centrosome duplication.

Abnormal amplification of centrosomes is the major cause of mitotic defects and chromosome instability in cancer cells. Centrosomes duplicate once in each cell cycle, and abrogation of the regulatory mechanism underlying centrosome duplication leads to centrosome amplification. p53 tumor suppressor protein is involved in the regulation of centrosome duplication: loss of p53 as well as expression of certain p53 mutants result in deregulated centrosome duplication and centrosome amplification. p53 at least in part depends on its transactivation function to control centrosome duplication, primarily via upregulation of p21 cyclin-dependent kinase (CDK) inhibitor, which prevents untimely activation of CDK2/cyclin E, a key initiator of centrosome duplication. However, numerous studies have shown the presence of p53 at centrosomes, yet the role of the centrosomally localized p53 in the regulation of centrosome duplication had been enigmatic. Here, we comparatively examined wild-type p53 and p53 mutants that are transactivation(+)/centrosome-binding(-), transactivation(-)/centrosome-binding(+) and transactivation(-)/centrosome-binding(-) for their abilities to control centrosome duplication. We found that the transactivation(+)/centrosome-binding(-) and transactivation(-)/centrosome-binding(+) mutants suppress centrosome duplication only partially compared with wild-type p53. Moreover, the transactivation(-)/centrosome-binding(-) mutant almost completely lost the ability to suppress centrosome duplication. These observations provide direct evidence for the centrosomally localized p53 to participate in the regulation of centrosome duplication in a manner independent of its transactivation function in addition to its transactivation-dependent regulation of centrosome duplication.

Overexpression of calbindin D(28k) in dentate gyrus granule cells alters mossy fiber presynaptic function and impairs hippocampal-dependent memory.

Calcium is a key signaling ion for induction of synaptic plasticity processes that are believed to influence cognition. Mechanisms regulating activity-induced increases in neuronal calcium and related synaptic modifications are not fully understood. Moreover, involvement of specific synapses in discrete aspects of spatial learning remains to be elucidated. We used herpes simplex amplicons to overexpress calbindin D(28k) (CaBP) selectively in dentate gyrus (DG) granule cells. We then examined the effects on hippocampal network activity by recording evoked synaptic responses in vivo and in vitro and analyzing hippocampal-dependent behavior. Relative to Lac-Z- and sham-infected controls, CaBP overexpression increased mossy fiber (MF-CA3) excitatory postsynaptic potentials and reduced paired-pulse facilitation (PPF), suggesting an increase in presynaptic strength. Additionally, CaBP overexpression reduced long-term potentiation (LTP), caused a frequency-dependent inhibition of post-tetanic potentiation (PTP), and impaired spatial navigation. Thus, increasing CaBP levels selectively in the DG disrupts MF-CA3 presynaptic function and impairs spatial cognition. The results demonstrate the power of gene delivery in the study of the neural substrates of learning and memory and suggest that mossy fiber synaptic plasticity is critical for long-term spatial memory.

Difference in the centrosome duplication regulatory activity among p53 'hot spot' mutants: potential role of Ser 315 phosphorylation-dependent centrosome binding of p53.

The p53 tumor suppressor protein regulates centrosome duplication through multiple pathways, and p21(Waf1/Cip1) (Waf1), a major target of p53's transactivation function, has been shown to be one of the effectors. However, it had been unclear whether the p53's Waf1-independent centrosome duplication regulatory pathways require its transactivation function. In human cancers, specific residues of p53 are mutated at a high frequency. These 'hot spot' mutations abrogate p53's transactivation function. If p53 regulates centrosome duplication in a transactivation-independent manner, different 'hot spot' mutants may regulate centrosome duplication differently. To test this, we examined the effect of two 'hot spot' mutants (R175H and R249S) for their centrosome duplication regulatory activities. We found that R175H lost the ability to regulate centrosome duplication, while R249S partially retained it. Moreover, R249S associates with both unduplicated and duplicated centrosomes similar to wild-type p53, while R175H only associates with duplicated, but not unduplicated centrosomes. Since cyclin-dependent kinase 2 (CDK2) triggers initiation of centrosome duplication, and p53 is phosphorylated on Ser 315 by CDK2, we examined the p53 mutants with a replacement of Ser 315 to Ala (A) and Asp (D), both of which retain the transactivation function. We found that S315D retained a complete centrosome duplication activity, while S315A only partially retained it. Moreover, S315D associates with both unduplicated and duplicated centrosomes, while S315A associates with only duplicated, but not unduplicated centrosomes. Thus, p53 controls the centrosome duplication cycle both in transactivation-dependent and transactivation-independent manners, and the ability to bind to unduplicated centrosomes, which is controlled by phosphorylation on Ser 315, may be important for the overall p53-mediated regulation of centrosome duplication.

Direct regulation of the centrosome duplication cycle by the p53-p21Waf1/Cip1 pathway.

The function of the centrosomes to direct mitotic spindles is critical for accurate chromosome transmission to daughter cells. Since each daughter cell inherits one centrosome, each centrosome must duplicate prior to the next mitosis, and do so only once. Thus, there are control mechanism(s) that ensure the coordinated progression of centrosome duplication and other cell cycle events (i.e. DNA synthesis), and limit centrosome duplication to once per cell cycle. Deregulation of the centrosome duplication cycle results in abnormal amplification of centrosomes, leading to aberrant mitoses and increased chromosome transmission errors. This has been found to be the case for cells lacking functional p53 tumor suppressor protein. However, it had remained to be determined whether the deregulation of the centrosome duplication cycle is the direct or indirect effect of loss/mutational inactivation of p53. Here, we found that the normal centrosome duplication cycle is almost completely restored in p53(-/-) cells by re-introduction of wild-type p53 at a physiologically relevant level, demonstrating that p53 is directly involved in the regulation of centrosome duplication. Since cyclin dependent kinase 2 (CDK2)/cyclin E triggers DNA synthesis as well as centrosome duplication, we tested whether Waf1, a CDK inhibitor and a major target of p53's transactivation function, is an effector of p53-mediated regulation of centrosome duplication. We found that induced expression of Waf1 in p53(-/-) cells only partially restored the centrosome duplication control, suggesting that Waf1 comprises one of the multiple effector pathways of the p53-mediated regulation of the centrosome duplication cycle.

Specific phosphorylation of nucleophosmin on Thr(199) by cyclin-dependent kinase 2-cyclin E and its role in centrosome duplication.

The kinase activity of cyclin-dependent kinase 2 (CDK2)-cyclin E is required for centrosomes to initiate duplication. We have recently found that nucleophosmin (NPM/B23), a phosphoprotein primarily found in nucleolus, associates with unduplicated centrosomes and is a direct substrate of CDK2-cyclin E in centrosome duplication. Upon phosphorylation by CDK2-cyclin E, NPM/B23 dissociates from centrosomes, which is a prerequisite step for centrosomes to initiate duplication. Here, we identified that threonine 199 (Thr(199)) of NPM/B23 is the major phosphorylation target site of CDK2-cyclin E in vitro, and the same site is phosphorylated in vivo. NPM/T199A, a nonphosphorylatable NPM/B23 substitution mutant (Thr(199) --> Ala) acts as dominant negative when expressed in cells, resulting in specific inhibition of centrosome duplication. As expected, NPM/T199A remains associated with the centrosomes. These observations provide direct evidence that the CDK2-cyclin E-mediated phosphorylation on Thr(199) determines association and dissociation of NPM/B23 to the centrosomes, which is a critical control for the centrosome to initiate duplication.

Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication.

In animal cells, duplication of centrosomes and DNA is coordinated. Since CDK2/cyclin E triggers initiation of both events, activation of CDK2/cyclin E is thought to link these two events. We identified nucleophosmin (NPM/B23) as a substrate of CDK2/cyclin E in centrosome duplication. NPM/B23 associates specifically with unduplicated centrosomes, and NPM/B23 dissociates from centrosomes by CDK2/cyclin E-mediated phosphorylation. An anti-NPM/B23 antibody, which blocks this phosphorylation, suppresses the initiation of centrosome duplication in vivo. Moreover, expression of a nonphosphorylatable mutant NPM/ B23 in cells effectively blocks centrosome duplication. Thus, NPM/B23 is a target of CDK2/cyclin E in the initiation of centrosome duplication.

Synergistic induction of centrosome hyperamplification by loss of p53 and cyclin E overexpression.

Centrosome hyperamplification and the consequential mitotic defects contribute to chromosome instability in cancers. Loss or mutational inactivation of p53 has been shown to induce chromosome instability through centrosome hyperamplification. It has recently been found that Cdk2-cyclin E is involved in the initiation of centrosome duplication, and that constitutive activation of Cdk2-cyclin E results in the uncoupling of the centrosome duplication cycle and the DNA replication cycle. Cyclin E overexpression and p53 mutations occur frequently in tumors. Here, we show that cyclin E overexpression and loss of p53 synergistically increase the frequency of centrosome hyperamplification in cultured cells as well as in tumors developed in p53-null, heterozygous, and wildtype mice. Through examination of cells derived from Waf1-null mice, we further found that Waf1, a potent inhibitor of Cdk2-cyclin E and a major target of p53's transactivation function, is involved in coordinating the initiation of centrosome duplication and DNA replication, suggesting that Waf1 may act as a molecular link between p53 and Cdk2-cyclin E in the control of the centrosome duplication cycle.

p53 mutation and mitotic infidelity.

Chromosome instability (a high frequency of chromosomal loss and gain and genome doubling, often referred to as karyotypic instability) is one of the major characteristics of cancer cells. It facilitates carcinogenesis by increasing the chance of specific mutations responsible for malignant phenotypes. Chromosome instability in most cases reflects the occurrence of defective mitosis, including unequal distribution of chromosomes to daughter cells and failure to undergo cytokinesis, which leads to generation of aneuploid cells. Both in vivo and in vitro, chromosome instability has been shown to correlate with loss or mutation of the p53 tumor suppressor protein, the product of one of the most frequently mutated genes in cancer. The major function of p53 is to prevent cells from proceeding through the cell cycle when cells experience stress, insults, or errors that disturb the preprogrammed cell cycle progression. During the last several years, significant advances have been made in understanding how p53 is involved in the regulation of mitosis and how loss or mutation of p53 affects mitotic fidelity, which will be the subject of this review.

Centrosome hyperamplification in human cancer: chromosome instability induced by p53 mutation and/or Mdm2 overexpression.

We have previously reported that loss of p53 tumor suppressor protein results in centrosome hyperamplification, which leads to aberrant mitosis and chromosome instability. Since p53 is either deleted or mutated in human cancers at a high frequency, we investigated whether human cancers showed centrosome hyperamplification. Screening of advanced stage breast ductal carcinomas and squamous cell carcinomas of the head and neck (SCCHN) revealed that centrosome hyperamplification is frequent in both tumor types. Moreover, through the analyses of p53 in SCCHN samples by direct sequencing and by loss-of-heterozygosity test, we found that p53 mutations correlated with occurrence of centrosome hyperamplification. However, in some cases, we observed centrosome hyperamplification in tumors that retained wild-type p53. These tumors contained high levels of Mdm2. Since Mdm2 can inactivate p53 through physical association, we investigated whether Mdm2 overexpression induced centrosome hyperamplification. We found that Mdm2 overexpression, like loss of p53, induced centrosome hyperamplification and chromosome instability in cultured cells.

DNA binding and transcriptional activation by the Ski oncoprotein mediated by interaction with NFI.

The Ski oncoprotein has been found to bind non-specifically to DNA in association with unindentified nuclear factors. In addition, Ski has been shown to activate transcription of muscle-specific and viral promoters/enhancers. The present study was undertaken to identify Ski's DNA binding and transcriptional activation partners by identifying specific DNA binding sites. We used nuclear extracts from a v-Ski-transduced mouse L-cell line and selected Ski-bound sequences from a pool of degenerate oligonucleotides with anti-Ski monoclonal antibodies. Two sequences were identified by this technique. The first (TGGC/ANNNNNT/GCCAA) is the previously identified binding site of the nuclear factor I (NFI) family of transcription factors. The second (TCCCNNGGGA) is the binding site of Olf-1/EBF. By electophoretic mobility shift assays we find that Ski is a component of one or more NFI complexes but we fail to detect Ski in Olf-1/EBF complexes. We show that Ski binds NFI proteins and activates transcription of NFI reporters, but only in the presence of NFI. We also find that homodimerization of Ski is essential for co-activation with NFI. However, the C-terminal dimerization domain of c-Ski, which is missing in v-Ski, can be substituted by the leucine zipper domain of GCN4.

Use of a simulation model in occupational dose assessment at a high-level radioactive waste repository.

Occupational radiation dose assessment at a nuclear facility can benefit from a systems approach that recognizes the complex dynamics of the facility. A simulation model can be a useful tool for accomplishing this goal. This technique can quantify the uncertainty in estimates of occupational dose equivalent and illustrate the effects of system interactions. A simulation model of operations at a high-level radioactive waste repository together with a radiological dose assessment model were used to illustrate this technique. Annual individual and collective occupational dose equivalents were estimated in the truck cask receiving and handling stations at a repository. The uncertainty of these estimates is shown through the use of complementary cumulative distribution functions. The effects of changing parameters in cask handling operations are examined.