Effects of derivatives of sulfur dioxide on micronuclei formation in mouse bone marrow cells in vivo.
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Air Pollutants↗
Biomedical subjects
N Sang
Publications and source records attributed to N Sang.
RACK1 interacts with E1A and rescues E1A-induced yeast growth inhibition and mammalian cell apoptosis.
The adenoviral E1A proteins are able to promote proliferation and transformation, inhibit differentiation, induce apoptosis, and suppress tumor growth. The extreme N terminus and conserved region one of E1A, which are indispensable for transcriptional regulation and for binding to p300/CBP, TBP, and pCAF, play essential roles in these abilities. These observations strongly suggest an intrinsic link between E1A-mediated transcriptional regulation and other effects. In this report, we show that E1A inhibits the normal growth of Saccharomyces cerevisiae HF7c, and this inhibition also depends on the domains required for transcriptional regulation. We demonstrate that E1A associates with histone acetyltransferase activity and represses the transactivation activity of transcription factor in S. cerevisiae, suggesting that E1A may suppress the expression of genes required for normal growth. Based on yeast growth rescue, we present a genetic screening strategy that identified RACK1 as an E1A antagonizing factor. Expression of human RACK1 efficiently relieves E1A-mediated growth inhibition in HF7c and protects human tumor cells from E1A-induced apoptosis. Finally, we show that RACK1 decreases E1A-associated histone acetyltransferase activity in yeast and mammalian cells, and physically interacts with E1A. Our data demonstrate that RACK1 is a repressor of E1A, possibly by antagonizing the effects of E1A on host gene transcription.
Adenovirus E1A Proteins↗
Oxygen sensing and HIF-1 activation does not require an active mitochondrial respiratory chain electron-transfer pathway.
Hypoxia induces the stabilization and transcriptional activation of the hypoxia-inducible factor 1alpha (HIF-1alpha) protein, the regulatory member of the HIF-1 complex. The molecular mechanisms that are responsible for oxygen sensing and the downstream pathways utilized by the hypoxic signal are still poorly understood. One hypothesis for oxygen sensing has postulated that reactive oxygen species generated at mitochondrial complex III are the initiators of the hypoxic signal. Here we find that mitochondrial DNA-less (rho(o)) cells have a normal response to hypoxia, measured at the level of HIF-1alpha protein stabilization, nuclear translocation, and its transcriptional activation activity. Furthermore, overexpression of catalase, either in the mitochondria or in the cytosol, fails to modify the hypoxia response indicating that hydrogen peroxide is not a signaling molecule in the hypoxic signaling cascade that culminates with HIF-1 activation.
Cell Hypoxia↗
Multiple biological responses activated by nuclear protein kinase C.
Protein kinase C is a family of serine-threonine kinases that are physiologically activated by a number of lipid cofactors and are important transducers in many agonist-induced signaling cascades. To date, 12 different isozymes of this kinase have been identified and are believed to play distinct regulatory roles. Protein kinase C was thought to reside in the cytosol in an inactive conformation and translocate to the plasma membrane upon cell activation by different stimuli. Nevertheless, a growing body of evidence has illustrated that this family of isozymes is capable of translocating to other cellular sites, including the nucleus. Moreover, it seems that some protein kinase C isoforms are resident within the nucleus. A wealth of data is being accumulated, demonstrating that nuclear protein kinase C isoforms are involved in the regulation of several critical biological functions such as cell proliferation and differentiation, neoplastic transformation, and apoptosis. In this review, we will discuss the most significant findings concerning nuclear protein kinase C which have been published during the past 5 years.
Animals↗
Transduction of well-differentiated airway epithelium by recombinant adeno-associated virus is limited by vector entry.
The limitations of adeno-associated virus (AAV)-mediated vectors for lung-directed gene transfer were investigated by using differentiated human respiratory epithelium in air-liquid interface cultures. Transduction efficiency was high in undifferentiated cells and was enhanced in well-differentiated cells after basolateral application of the vector or after apical application following disruption of tight junctions or pretreatment of the cultures with glycosidases. These results indicate that transduction of airway epithelia by AAV vectors is limited by entry and reinforce the importance of a physical barrier on the airway surface.
Cell Differentiation↗
Binding of CDK9 to TRAF2.
CDK9 has been recently shown to have increased kinase activity in differentiated cells in culture and a differentiated tissue-specific expression in the developing mouse. In order to identify factors that contribute to CDK9's differentiation-specific function, we screened a mouse embryonic library in the yeast two-hybrid system and found a tumor necrosis factor signal transducer, TRAF2, to be an interacting protein. CDK9 interacts with a conserved domain in the TRAF-C region of TRAF2, a motif that is known to bind other kinases involved in TRAF-mediated signaling. Endogenous interaction between the two proteins appears to be specific to differentiated tissue. TRAF2-mediated signaling may incorporate additional kinases to signal cell survival in myotubes, a cell type that is severely affected in TRAF2 knockout mice.
Animals↗
[The pharmacokinetic study of desflurane, sevoflurane, isoflurane and enflurane in general anesthesia].
OBJECTIVE: To compare pharmacokinetics of desflurane, sevoflurane, isoflurane and enflurane in general anesthesia. METHODS: 40 patients scheduled for abdominal hysterectomy under general anesthesia were randomly divided into desflurane(D), sevoflurane(S), isofluane(I) and enflurane(E) groups. After induction of anesthesia and endotracheal intubation, desired fraction (Fd) of desflurane(6%), sevoflurane(2%), isoflurane (1.15%) and enflurane(1.7%) in oxygen and nitrous oxide(1:2) were inhaled in D, S, I and E groups, respectively. The fractional end tidal alveolar concentration (Fa) was adjusted to 1MAC during the maintenance of anesthsia. Fa and the fractional inspired concentration of inhaled anesthetics (Fi) were monitored continuously. During operation, fentanyl was infused continuously and pancuronium was injected intermittently. RESULTS: After the beginning of inhalational anesthesia, the time required for Fa/Fi = 1:2 and Fa = 1MAC in D and S groups was significantly shorter than that in E and I groups. The rates of Fa/Fi in D and S groups were significantly higher than those in E and I groups during the maintenance of anesthesia, so were those of Fa/Fd. After cessation of inhalational anesthesia, the time required for Fa equaled to 50% of Fa0(the last Fa during stoping administration of the inhalational anesthesia) in D group was significantly faster than that in the other three groups. CONCLUSIONS: The rates of desflurane wash-in and wash-out are faster than those of other inhaled anesthetics. The depth of anesthesia is easy to control when desflurane is used in general anesthesia.
Anesthesia, General↗
Roles of p300, pocket proteins, and hTBP in E1A-mediated transcriptional regulation and inhibition of p53 transactivation activity.
The conserved region 1 and the extreme N-terminus of adenoviral oncoprotein E1A are essential for transforming activity. They also play roles in the interaction of E1A with p300/CBP and pRb and are involved in both transactivation and repression of host gene expression. It was reported recently that p53-mediated transactivation is specifically repressed by E1A and that p53-induced apoptosis can be protected by pRb. In this report, we investigated the roles of pRb and p300 in the N-terminus of E1A-mediated transcriptional regulation. We demonstrate here that p300 and pRb have no effect on DBD.1-70 transactivation and that overexpression of p300 or pRb failed to relieve the repression by E1A. Repression of p53 transactivation requires both the extreme amino terminus and CR1 but not CR2. This repressive activity of E1A specifically correlates with E1A's ability to bind p300 and TBP. On the other hand, E1A inhibited the transactivation activity of a fusion construct containing the DNA binding domain of yeast Gal4 and the transactivation domain of p53. When p53 was contransfected with E1A, similar inhibition was found in Saos-2 cells that lack endogenous pRb and p53 activity. Introduction of pRb into Saos-2 cells did not affect p53 transcription activity. E1A-mediated repression can be relieved be overexpression of either p300, hTBP, or-TFIIB but cannot be released by overexpression of pocket proteins. Our data suggest that p300/CBP and TBP but not the pocket proteins, pRb, p107, and pRb2/p130 are functional targets of E1A in transcriptional regulation and that p53 transactivation requires the function of the p300/TBP/TFIIB complex, thus delineating a new pathway by which E1A may exert its transforming activity.
Adenovirus E1A Proteins↗
p300 is required for MyoD-dependent cell cycle arrest and muscle-specific gene transcription.
The nuclear phosphoprotein p300 is a new member of a family of 'co-activators' (which also includes the CREB binding protein CBP), that directly modulate transcription by interacting with components of the basal transcriptional machinery. Both p300 and CBP are targeted by the adenovirus E1A protein, and binding to p300 is required for E1A to inhibit terminal differentiation in both keratinocytes and myoblasts. Here we demonstrate that, in differentiating skeletal muscle cells, p300 physically interacts with the myogenic basic helix-loop-helix (bHLH) regulatory protein MyoD at its DNA binding sites. During muscle differentiation, MyoD plays a dual role: besides activating muscle-specific transcription, it induces permanent cell cycle arrest by up-regulating the cyclin-dependent kinase inhibitor p21. We show that p300 is involved in both these activities. Indeed, E1A mutants lacking the ability to bind p300 are greatly impaired in the repression of E-box-driven transcription, and p300 overexpression rescues the wild-type E1A-mediated repression. Moreover, p300 potentiates MyoD- and myogenin-dependent activation of transcription from E-box-containing reporter genes. We also provide evidence, obtained by microinjection of anti-p300 antibodies, that p300 is required for MyoD-dependent cell cycle arrest in either myogenic cells induced to differentiate or in MyoD-converted C3H10T1/2 fibroblasts, but is dispensable for maintenance of the postmitotic state of myotubes.
Animals↗
Extreme N terminus of E1A oncoprotein specifically associates with a new set of cellular proteins.
By interacting with key regulatory proteins such as the pRb family, cyclins, cyclin-dependent kinases and p300/CBP of host cells, adenoviral E1A interferes with various cellular processes to provide a suitable environment for the replication of viruses. E1A may promote DNA synthesis and cell cycle progression, immortalize rodent cells in culture and transform cultured cells in cooperation with E1B, Ras, or other oncoproteins. Both extreme N terminus and conserved region 1 of E1A are required for the immortalization and the transformation of rodent cells, transcriptional repression and specific induction of the expression of cellular genes such as the proliferating cell nuclear antigen (PCNA) and heat shock protein 70 (HSP70). Although the molecular mechanisms of these functions of E1A are not fully understood, it is believed that protein-protein interactions may play essential roles. In this communication, we report that a new set of cellular proteins with apparent molecular weight of 200, 90, 45, 30, and 28 specifically associate with the extreme N terminus of E1A. Further analysis demonstrate that these associations do not depend on E1A's association with p300 or pRB. Neither the 30 kDa nor the 28 kDa polypeptide is identical to Cdc2 or Cdk2. The region of E1A required for the protein interaction is also required for the recently identified N-terminal transactivation activity of E1A. Our observations suggest that in addition to p300/CBP, the new set of cellular proteins may be involved in the functional complexity of the N terminus of E1A, thus predicting a p300/CBP independent pathway.
3T3 Cells↗
Transforming region of 243R E1A contains two overlapping but distinct transactivation domains.
Conserved regions 1 and 2 as well as the amino terminus of E1A are required for the transforming activity of the E1A oncoprotein. We show here that the amino terminus of 243R E1A has transactivation activity when brought to a promoter in yeast. Recruitment to a specific promoter is essential. Mutagenesis studies correlated the transactivation function with the extreme amino terminus and the conserved region 1 of E1A. Cotransfection assays in rodent cells confirmed that two overlapping but distinguishable domains, amino acids 1-65 and 37-80, can transactivate independently when targeted to a promoter. We also observed that when recruited to the proliferating cell nuclear antigen (PCNA) promoter, the amino-terminal region was sufficient to transactivate the PCNA promoter. On the other hand, deletion of the amino terminus of E1A resulted in failure to induce PCNA expression. Fusion of VP16 with the amino-terminal-deleted E1A mutant was able to restore the ability to induce the PCNA promoter. We further show that the amino-terminal region also is required for 243R E1A to repress the transactivation mediated by a universal transactivator DBD.VP16 and DBD.E1A. This repression could be specifically relieved by overexpression of TBP but not TFIIB. In addition, we show that the amino terminus of E1A is involved in in vitro interaction with the TATA binding protein (TBP). Thus the amino-terminal transforming region of E1A may regulate cellular gene expression in species that are distant in evolution via a common mechanism, functionally targeting TBP.
3T3 Cells↗
Generation of site-directed mutagenesis by extralong, high-fidelity polymerase chain reaction.
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Adenovirus E1A Proteins↗
Chromosomal mapping of members of the cdc2 family of protein kinases, cdk3, cdk6, PISSLRE, and PITALRE, and a cdk inhibitor, p27Kip1, to regions involved in human cancer.
Orderly progression through the cell cycle requires sequential activation and inactivation of cyclin-dependent kinases (cdks). This is achieved in part through the association of cdks with positive regulators called cyclins and inactivation of cyclin-cdk complexes by a rapidly growing number of cyclin-cdk inhibitors. Recently, the role of cell cycle control proteins both as primary effectors and as mediators of tumorigenesis has become a subject of increased interest. Here we report the chromosomal mapping of two cdks, cdk3 and cdk6, two putative cdks, PISSLRE and PITALRE, and one cyclin-dependent kinase inhibitor, p27, to chromosomal regions which may be altered in human tumors and examine their possible involvement in some of these malignancies. In particular, two of the kinases, cdk3 and PISSLRE and PITALRE, the cdc2-related kinases recently cloned by us, map to regions previously shown to exhibit loss of heterozygosity in breast and other tumors.
Base Sequence↗
Cyclins, cyclin-dependent kinases and cdk inhibitors: implications in cell cycle control and cancer.
A significant portion of cell scientific literature published is dedicated to describing the cloning, the link to cancer, or the characterization of proteins involved in the progression of the cell cycle. With this abundance of information, the cascading pathways of molecular events that occur in the cell cycle are proving to be exceedingly complicated. Originally, the sole regulator of the fission yeast cells division cycle, cdc2, was thought to also regulate mammalian cell cycles in the same manner. However, mammalian cdc2 has now been joined by seven well-characterized relatives acting at distinct points in the cell cycle. These kinases are activated by larger proteins called cyclins, named with respect to their cyclical expression and degradation. Therefore, the catalytic subunits of these complexes are named cyclin-dependent kinases (cdks). In the event that the cell must stop normal cycling behavior, a number of cdk inhibitors, which have only begun to be characterized, function in inhibiting the kinase ability of cdks, among other nonproliferative acts. The external environment manipulates cellular proliferation and differentiation by stimulating or inhibiting certain signal transduction pathways. However, each component of the cell cycle machinery, as they are the final executors in cell division, has the potential to elicit or to contribute to a neoplastic phenotype. This review focuses on the characterization of each member of the cell cycle protein family and also addresses the potential role each plays in cancer.
Animals↗
PITALRE, a nuclear CDC2-related protein kinase that phosphorylates the retinoblastoma protein in vitro.
Members of the cell division cycle 2 (CDC2) family of kinases play a pivotal role in the regulation of the eukaryotic cell cycle. In this communication, we report the isolation of a cDNA that encodes a CDC2-related human protein kinase temporarily designated PITALRE for the characteristic Pro-Ile-Thr-Ala-Leu-Arg-Glu motif. Its deduced amino acid sequence is 47% identical to that of the human cholinesterase-related cell division controller (CHED) kinase, which is required during hematopoiesis, and 42% identical to the Saccharomyces cerevisiae SGV1 gene product, a putative kinase involved in the response to pheromone via its guanine nucleotide-binding protein alpha subunit. PITALRE expression is ubiquitous, but its expression levels are different in various human tissues. PITALRE is an approximately 43-kDa protein that associates with three cellular polypeptides of 80, 95, and 155 kDa. PITALRE is localized primarily to the nucleus. In addition, we have identified a retinoblastoma protein kinase activity associated with PITALRE immunocomplexes that cannot phosphorylate histone H1, suggesting that the target phosphorylation site of PITALRE differs from that of CDC2 kinase. Interestingly, the retinoblastoma kinase activity associated with PITALRE does not oscillate during the cell cycle.
Amino Acid Sequence↗
PISSLRE, a human novel CDC2-related protein kinase.
The cell division cycle have been shown to be regulated by a closely-related family of protein kinases named CDKs (by cyclin-dependent kinases). Using a PCR-based cloning technique, we have isolated cDNAs encoding a human CDC2-related protein kinase. The full-length cDNA accommodates an open reading frame that does not contain any ATG initiation codon upstream of the sequence encoding the catalytic domain of this putative kinase. Three putative non-ATG initiation codons have been detected. Starting at the most 5' non-ATG initiation site, the encoded product is 316 amino acids long with a predicted molecular weight of 35.8 kDa. Analysis of the deduced amino acid sequence showed it to contain the XI subdomains present in all known protein kinases and a PSTAIRE-like motive, PISSLRE, which temporarily names this kinase. PISSLRE is most related to p58/GTA (55% identity in the catalytic domain), the galactosyl transferase associated protein, which has been shown to inhibit entry into S-phase when over-expressed in CHO cells. PISSLRE shares 38-45% identity with all CDKs and contains the regulatory Tyr and Thr residues present in most of the members of the CDK family of protein kinases, which suggests similar modes of regulation. PISSLRE is expressed in all human tissues tested, including those which contain high proportion of terminally differentiated cells. However, the levels of the PISSLRE transcripts are dissimilar among different tissues.
Amino Acid Sequence↗
Cloning of a new member of the retinoblastoma gene family (pRb2) which binds to the E1A transforming domain.
The product of the retinoblastoma tumor suppressor gene (pRb) and p107 share a high degree of structural homology in the pocket region, which is thought to play a primary role in the function of these proteins. It is conceivable that there exists a larger family of cellular proteins containing this pocket region. In this communication, we report cloning of a new human cDNA encoding a polypeptide that shows a high level of identity with pRb and p107 and possesses a pocket region. We have named it pRb2. From the deduced amino acid sequence, pRb2 has a predicted molecular weight of approximately 120 kD and its in vitro translated product binds to the adenovirus E1A protein. Due to its size, pRb2 may correspond to p130, which has previously been shown by us to interact with the transforming region of E1A in in vivo studies. Interestingly, pRb2 fails to bind an E1A mutant in the transforming domain 2 suggesting that pRb2 is involved in the transforming capacity of E1A.
Adenovirus E1A Proteins↗
Clinical significance of flow cytometric deoxyribonucleic acid measurements of deparaffinized specimens in bladder tumors.
A retrospective study of flow cytometric measurements on paraffin-embedded tumor specimens from 188 patients with bladder tumor was conducted. Results were analyzed in combination with the morphological variation in bladder tumors. It was found that the deoxyribonucleic acid (DNA) ploidy pattern, the degree of infiltration and the multiplicity of bladder tumor were closely related with tumor recurrence, among which the DNA ploidy pattern was most significant. In aneuploid bladder tumors, the recurrence rate and the mean annual recurrence frequency were 76.7% and 1.46, respectively, and in the diploid bladder tumors, they were 18.7% and 0.33, respectively. Aneuploidy was the most indicative parameter of recurrence in bladder tumors. In addition, according to the DNA content and the ploidy level of the aneuploid cell lines, the aneuploid tumors in our group were divided into four types, namely tetraploid tumors, noneuploid tumors with an DNA index (DI) less than or equal to 1.5, hypotetraploid tumors and aneuploid tumors with several cell lines. The results showed that the recurrence rate of tetraploid tumors was relatively lower, and it became higher and higher in the following order: noneuploid tumors with DI less than or equal to 1.5, noneuploid tumors with DI greater than 1.5, and two-aneuploid tumors. This indicates that there are different biological behaviors in tumors with different ploidy patterns. Finally, the correlation between the DNA ploidy pattern and tumor metastases was also discussed.
Carcinoma, Transitional Cell↗