Biomedical subjects

D Liu

Publications and source records attributed to D Liu.

At least 37 records · Page 2Linked to original sources

PubMed2001

Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints.

During mitosis, a ras-related GTPase (Tem1) binds GTP and activates a signal transduction pathway to allow mitotic exit. During most of the cell cycle, Tem1 function is antagonized by a GTPase-activating protein complex, Bfa1/Bub2. How the Bfa1/Bub2 complex is regulated is not well understood. We find that Polo/Cdc5 kinase acts upstream of Bfa1/Bub2 in the mitotic exit network. Cdc5 phosphorylates Bfa1 and acts to antagonize Bfa1 function to promote mitotic exit. Bfa1 is regulated by multiple cell cycle checkpoints. The spindle assembly and spindle orientation checkpoints inhibit Bfa1 phosphorylation. DNA damage does not inhibit Bfa1 phosphorylation and instead causes a Rad53- and Dun1-dependent modification of Bfa1. Regulation of Bfa1 may therefore be a key step controlled by multiple checkpoint pathways to ensure a mitotic arrest.

Amino Acid Sequence↗

PubMed2001

Tumor necrosis factor-alpha induces the expression of DR6, a member of the TNF receptor family, through activation of NF-kappaB.

The tumor necrosis factor (TNF) receptor family are ligand-regulated transmembrane proteins that mediate apoptosis as well as activation of the transcription factor NF-kappaB. Exogenous expression of DR6, a recently identified member of the TNF receptor family, induced apoptosis in untransformed or tumor-derived cells and the apoptotic function of DR6 was inhibited by co-expression of Bcl-2, Bcl-x(L) or the inhibitor-of-apoptosis (IAP) family member, survivin. Expression of a dominant negative mutant of FADD failed to protect from DR6-mediated apoptosis indicating that unlike TNFR1 and Fas, DR6 induced apoptosis via a FADD-independent mechanism. Despite the ability of exogenous DR6 expression to induce apoptosis, DR6 mRNA and protein were found to be elevated in prostate tumor cell lines and in advanced stages of prostate cancer. Analysis of several anti-apoptotic proteins revealed that Bcl-x(L) levels and serine 32 phosphorylation of IkappaB, the natural inhibitor of NF-kappaB, were similarly elevated in cells expressing high levels of DR6, suggesting that NF-kappaB-regulated survival proteins may protect from DR6-induced apoptosis and that DR6 is a target of NF-kappaB regulation. Treatment of LnCAP cells with TNF-alpha resulted in increases in both DR6 mRNA and protein levels, and this induction was suppressed by inhibitors of NF-kappaB. Similarly, treatment of cells expressing high levels of DR6 with indomethacin and ibuprofen, compounds also known to perturb NF-kappaB function, resulted in a dose-dependent decrease in DR6 protein and mRNA levels. These results demonstrate that TNF-alpha signaling induces the expression of a member of its own receptor family through activation of NF-kappaB.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2001

Dendralene-type TTF vinylogs containing a 1,3-diselenole ring.

2-Diformylmethylene-1,3-diselenole was prepared and condensed with dithiolium phoshonium bromides and dithiolium phosphonates in the presence of base to give dendralene-type vinylogs of TTF bearing a 1,3-diselenole moiety. The electrochemistry of these dendrimers was studied. SEEPR measurements were also carried out. Calculations were carried out on the radical cations and correlated with the EPR values.

Journal Article↗

PubMed2001

The intratumoral expression of vascular endothelial growth factor and interleukin-8 associated with angiogenesis in nonsmall cell lung carcinoma patients.

BACKGROUND: Angiogenesis has important effects on tumor growth and metastasis. It is regulated by a variety of angiogenic and angiostatic factors. METHODS: To evaluate the effects of tumor cell-derived angiogenic factors, we performed an immunohistochemic study to evaluate the intratumoral expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in relation to intratumoral microvessel density (IMD) in tumors from 104 nonsmall cell lung carcinoma (NSCLC)patients. RESULTS: Fifty-four carcinomas were VEGF-positive, 47 carcinomas were IL-8-positive, and 53 carcinomas were hypervascular tumors. There was no significant correlation between the percentages of positive VEGF-staining and positive IL-8-staining in NSCLCs (rho = 0.174, P = 0.080). The IMD of VEGF-positive carcinomas was significantly greater than that of VEGF-negative carcinomas (P = 0.023). In addition, the IMD of IL-8-positive carcinomas was significantly greater than that of IL-8-negative carcinomas (P =0.013). The overall survival rate of patients with hypervascular tumors was significantly lower than that of patients with hypovascular tumors (41.0% versus 67.0%, P = 0.004). Cox proportional-hazards regression model also demonstrated that angiogenesis was one of the significant factors in predicting the survival of NSCLC patients (relative risk = 1.944, P = 0.041). CONCLUSIONS: Intratumoral expression of VEGF and IL-8 was associated with angiogenesis in NSCLCs. Tumor angiogenesis significantly affected the prognosis of NSCLC patients.

Adult↗

PubMed2001

TGF-beta inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of skeletal muscle differentiation, but the molecular mechanism and signaling events that lead to this inhibition are poorly characterized. Here we show that the TGF-beta intracellular effector Smad3, but not Smad2, mediates the inhibition of myogenic differentiation in MyoD-expressing C3H10T1/2 cells and C2C12 myoblasts by repressing the activity of the MyoD family of transcriptional factors. The Smad3-mediated repression was directed at the E-box sequence motif within muscle gene enhancers and the bHLH region of MyoD, the domain required for its association with E-protein partners such as E12 and E47. The repression could be overcome by supplying an excess of E12, and covalent tethering of E47 to MyoD rendered the E-box-dependent transcriptional activity refractory to the effects of Smad3 and TGF-beta. Smad3 physically interacted with the HLH domain of MyoD, and this interaction correlated with the ability of Smad3 to interfere with MyoD/E protein heterodimerization and binding of MyoD complexes to oligomerized E-box sites. Together, these results reveal a model for how TGF-beta, through Smad3-mediated transcriptional repression, inhibits myogenic differentiation.

Animals↗

PubMed2001

The plasticity of dendritic cell responses to pathogens and their components.

Dendritic cells are involved in the initiation of both innate and adaptive immunity. To systematically explore how dendritic cells modulate the immune system in response to different pathogens, we used oligonucleotide microarrays to measure gene expression profiles of dendritic cells in response to Escherichia coli, Candida albicans, and influenza virus as well as to their molecular components. Both a shared core response and pathogen-specific programs of gene expression were observed upon exposure to each of these pathogens. These results reveal that dendritic cells sense diverse pathogens and elicit tailored pathogen-specific immune responses.

Antigen Presentation↗

PubMed2001

Antibody response to Epstein-Barr virus Rta protein in patients with nasopharyngeal carcinoma: a new serologic parameter for diagnosis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is associated closely with Epstein-Barr virus (EBV). The authors previously reported that an EBV immediate-early gene, BRLF1, was expressed frequently in NPC tumors, and a significant elevation in immunoglobulin G (IgG) antibodies directed against BRLF1 gene product Rta was detected in NPC sera by a radioactive immunoprecipitation assay. To simplify and to make the detection more quantitative, an enzyme-linked immnunosorbent assay (ELISA) was developed in this study. METHODS: Antigen domains of Rta were identified further using an immunoprecipitation assay. Two glutathione-S-transferase (GST) recombinant Rta fragments (R150-GST and R185-GST) were prepared subsequently and were used as antigens in the ELISA. Serum samples derived from 51 patients with NPC patients, 115 non-NPC ENT patients, and 47 healthy volunteers were examined for the presence of antibodies directed against Rta. RESULTS: Among the patients with NPC, 74.5% showed a positive IgG response to R150-GST, and 62.7% showed a positive IgG response to R185-GST, with 80.4% positive for either fragment. In contrast, the reactions were positive in only 8.5% of healthy volunteers and 13.0% of control patients. When using a mixture of the two recombinant Rta proteins as coating antigens, the IgG positive responses were 82.3%, 10.6%, and 14.8%, respectively, in patients with NPC, healthy volunteers, and control patients. It is noteworthy that 51.0% of the NPC sera showed a positive immunoglobulin A (IgA) response, with none of the control patients showing obvious reactivity. Both the IgG response and the IgA response to Rta protein in patients with NPC were correlated with the IgA response to EBV early antigens and virus capsid antigens, the classic serologic markers used to diagnose NPC. CONCLUSIONS: The ELISA method described for the detection of IgG antibodies directed against recombinant Rta proteins is simple and reliable and may be useful as a serologic parameter for the screening and diagnosis of patients with NPC.

Antibodies, Viral↗

PubMed2001

[Triptolide inhibits vascular endothelial growth factor expression and production by endothelial cells].

OBJECTIVE: To investigate the effect of triptolide on vascular endothelial growth factor (VEGF) expression and secretion by endothelial cells, and explore the mechanism of anti-proteinuric effect of triptolide on glomerular nephritis. METHOD: A human umbilical endothelium derived cell line (ECV-304) from American Type Culture Collection(ATCC) was used in this study. The effects of triptolide on VEGF mRNA expression, and intracellular protein production and secretion induced by PMA were measured by RT-PCR, flow cytometry and enzyme linked immunosorbent assay (ELISA). The effects of triptolide on endothelial c-jun/c-fos mRNA expression were investigated by RT-PCR. RESULTS: 100 ng/ml PMA significantly increased VEGF mRNA expression, intracellular production and secretion of VEGF in endothelial cells, while triptolide inhibited the effects of PMA in a dose-dependent manner. Moreover, triptolide dose-dependently inhibited endothelial c-jun/c-fos mRNA expression. CONCLUSION: Triptolide is a potent inhibitor of VEGF expression and production, suggesting that the inhibitory influence of triptolide on VEGF expression and production may be one of the mechanisms underlying its anti-proteinuric effect on glomerular nephritis. Triptolide might inhibit VEGF expression and production by interfering with transcription factor AP-1 formation.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2001

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies.

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.

Aspartic Acid↗

PubMed2001

Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides.

PKB/Akt and serum and glucocorticoid-regulated kinase (SGK) family kinases are important downstream targets of phosphatidylinositol 3 (PI-3) kinase and have been shown to mediate a variety of cellular processes, including cell growth and survival. Although regulation of Akt can be achieved through several mechanisms, including its phosphoinositide-binding Pleckstrin homology (PH) domain, how SGK kinases are targeted and regulated remains to be elucidated. Unlike Akt, cytokine-independent survival kinase (CISK)/SGK3 contains a Phox homology (PX) domain. PX domains have been implicated in several cellular events involving membrane trafficking. However, their precise function remains unknown. We demonstrate here that the PX domain of CISK interacts with phosphatidylinositol (PtdIns)(3,5)P2, PtdIns(3,4,5)P3, and to a lesser extent PtdIns(4,5)P2. The CISK PX domain is required for targeting CISK to the endosomal compartment. Mutation in the PX domain that abolished its phospholipid binding ability not only disrupted CISK localization, but also resulted in a decrease in CISK activity in vivo. These results suggest that the PX domain regulates CISK localization and function through its direct interaction with phosphoinositides. Therefore, CISK and Akt have evolved to utilize different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling.

Amino Acid Sequence↗

PubMed2001

Effects of N-(4-hydroxyphenyl)retinamide on hTERT expression in the bronchial epithelium of cigarette smokers.

BACKGROUND: Telomerase activation plays a critical role in tumorigenesis. To determine the role of telomerase in early lung carcinogenesis and as a potential biomarker in chemoprevention trials, we analyzed the expression of the human telomerase reverse transcriptase catalytic subunit (hTERT) in bronchial biopsy specimens from cigarette smokers who were enrolled in a randomized, double-blinded, placebo-controlled chemoprevention trial of N-(4-hydroxyphenyl)retinamide (4-HPR). METHODS: We obtained biopsy specimens from six predetermined sites in the bronchial tree from the 57 participants, before treatment and 6 months after treatment with 4-HPR or placebo. We used in situ hybridization to examine hTERT messenger RNA (mRNA) expression in 266 pretreatment (baseline) and post-treatment site-paired biopsy specimens from 27 patients in the 4-HPR-treated group and from 30 patients in the placebo-treated group. All statistical tests were two-sided. RESULTS: At baseline, 62.4% (95% confidence interval [CI] = 53.9% to 71%) of the biopsy specimens obtained from the group treated with 4-HPR and 65.2% (95% CI = 57.4% to 73.1%) of the biopsy specimens obtained from the placebo-treated group expressed hTERT mRNA. After 6 months, 45.6% (95% CI = 36.9% to 54.3%) of the biopsy specimens obtained from the 4-HPR-treated group and 68.1% (95% CI = 60.4% to 75.8%) of the biopsy specimens obtained from the placebo-treated group expressed hTERT mRNA. The reduction in hTERT expression observed between the two treatment groups over time was statistically significant (P =.01) when we used the biopsy site as the unit of analysis, but not when we used the individual as the unit of analysis (P =.37). CONCLUSIONS: Telomerase is frequently reactivated in the lungs of cigarette smokers. The modulation of hTERT expression in 4-HPR-treated smokers suggests that a novel molecular mechanism underlies the potential chemopreventive properties of 4-HPR. hTERT expression is a promising potential biomarker for risk assessment and for the evaluation of the efficacy of chemopreventive agents in lung carcinogenesis.

Adult↗

PubMed2001

De novo design, synthesis, and characterization of antimicrobial beta-peptides.

beta-Peptides are a class of polyamides that have been demonstrated to adopt a variety of helical conformations. Recently, a series of amphiphilic L(+2) helical beta-peptides were designed, which were intended to mimic the overall physicochemical properties of a class of membrane-active antimicrobial peptides, including magainin and cecropin. Although these peptides showed potent antimicrobial activity, they also showed significant activity against human erythrocytes. Operating under the assumption that their lack of specificity arose from excessive hydrophobicity, two additional beta-peptides H-(beta(3)-HAla-beta(3)-HLys-beta(3)-HVal)(n)-NH(2) (n = 4, 5) were designed and synthesized. Both have high antimicrobial activities, but very low hemolytic potencies. The peptides bind in an L(+2) conformation to phospholipid vesicles, inducing leakage of entrapped small molecules. The peptides have a low affinity for membranes consisting of neutral phosphatidylcholine lipids, but bind avidly to vesicles containing 10 mol % of acidic phosphatidylserine lipids. Differences in vesicle leakage kinetics for the two peptides suggest that chain length could affect their mechanisms of disrupting cell membranes. Thus, insights gained from the study of variants of natural alpha-peptides have provided a useful guide for the design of nonnatural antimicrobial beta-peptides.

Anti-Infective Agents↗

PubMed2001

Long-term impact of smoking on lung epithelial proliferation in current and former smokers.

BACKGROUND: Lung cancer risk remains elevated for many years after quitting smoking. To assess using proliferation indices in bronchial tissues as an intermediate endpoint biomarker in lung cancer chemoprevention trials, we determined the relationship between the extent, intensity, and cessation of tobacco smoking and proliferative changes in bronchial epithelial biopsy specimens. METHODS: Bronchial biopsy specimens were obtained from up to six epithelial sites in 120 current smokers (median pack-years, 42) and 207 former smokers (median pack-years, 40; median quit-years, 8.1). Sections from the paraffin-embedded specimens were stained with hematoxylin--eosin to determine the metaplasia index and with an antibody to Ki-67 to determine the proliferative (labeling) index for the basal and parabasal (Ki-67 PLI) layers. All statistical tests were two-sided. RESULTS: Biopsy sites with metaplasia had statistically significantly higher Ki-67-labeling indices than those without metaplasia (P<.001) in both current and former smokers. Increased proliferation was observed in multiple biopsy sites, with the average Ki-67 PLI of the subject strongly correlating with the metaplasia index (r =.72 for current smokers; P<.001), even in sites without metaplasia (r =.23 for current smokers; P<.001). In current smokers, the Ki-67 PLI was associated with the number of packs smoked/day (P =.02) but not with smoking years or pack-years. In subjects who had quit smoking, the Ki-67 PLI dropped statistically significantly within 1 year (P =.008) but remained detectable for more than 20 years, even in the absence of squamous metaplasia. CONCLUSION: Smoking appears to elicit a dose-related proliferative response in the bronchial epithelia of active smokers. Although the proliferative response decreased gradually in former smokers, a subset of individuals had detectable proliferation for many years and may benefit from targeted chemoprevention. Bronchial epithelial proliferation, measured by Ki-67, may provide a useful biomarker in the assessment of lung cancer risk and in the response to chemopreventive interventions.

Adult↗

PubMed2001

[Expression and significance of CD44 and angiogenic growth factor in nasal NK/T cells lymphoma].

OBJECTIVE: To investigate the molecular basis of the high aggressiveness of nasal NK/T cells lymphoma. METHODS: Immunohistochemical study was used on 17 cases of nasal NK/T cells lymphoma to examine CD20, CD43/CD45RO, CD3, CD56, TIA-1, and CD44. In situ hybridization was used to examine Epstein-Barr virus (EBV) infection, VEGF, and bFGF. RESULTS: The positive rates of CD20, CD43/CD45RO, CD3, CD56, TIA-1, and CD44 were 0%, 88.2%, 70.6%, 88.2%, 100%, and 41.2%. Fifteen cases (88.2%) were positive in EBER1 (EBV infection). VEGF was expressed in 11 cases (64.7%). VEGF expression was negative in 2 out of 4 cases with angioinvasion, and negative in 4 cases out of 7 cases who died of tumor. bFGF was weakly expressed in 3 cases (17.6%) and was not expressed in 2 out of 4 cases with angioinvasion and 6 out of 7 cases who died of tumor. CD44 expression was negative in all 3 cases with dysplasia, and in 4 out of the 7 cases who died of tumor. CONCLUSION: CD44 can not predict the potentiality of NK/T cells lymphoma in invasion, and does not correlate with prognosis. The expression of VEGF is far more frequent than that of bFGF in NK/T cells lymphoma. The significance of VEGF and bFGF expression remains to be investigated.

Angiogenesis Inducing Agents↗

PubMed2001

Combined interferon-alfa, 13-cis-retinoic acid, and alpha-tocopherol in locally advanced head and neck squamous cell carcinoma: novel bioadjuvant phase II trial.

PURPOSE: Retinoids and interferons (IFNs) have single-agent and synergistic combined effects in modulating cell proliferation, differentiation, and apoptosis in vitro and clinical activity in vivo in the head and neck and other sites. Alpha-tocopherol has chemopreventive activity in the head and neck and may decrease 13-cis-retinoic acid (13-cRA) toxicity. We designed the present phase II adjuvant trial to prevent recurrence or second primary tumors (SPTs) using 13-cRA, IFN-alpha, and alpha-tocopherol in locally advanced-stage head and neck cancer. PATIENTS AND METHODS: After definitive local treatment with surgery, radiotherapy, or both, patients with locally advanced SCCHN were treated with 13-cRA (50 mg/m(2)/d, orally, daily), IFN-alpha (3 x 10(6) IU/m(2), subcutaneous injection, three times a week), and alpha-tocopherol (1,200 IU/d, orally, daily) for 12 months, with a dose modification. Screening for recurrence or SPTs was performed every 3 months. RESULTS: Tumors of 11 (24%) of the 45 treated patients were stage III, and 34 (76%) were stage IV. Thirty-eight (86%) of 44 patients completed the full 12-month treatment (doses modified as needed). Toxicity generally was consistent with previous IFN and 13-cRA reports and included mild to moderate mucocutaneous and flu-like symptoms; occasional significant fatigue (grade 3 in 7% of patients), mild to moderate hypertriglyceridemia in 30% of patients who continued treatment along with antilipid therapy, and mild hematologic side effects. Six patients did not complete the planned treatment because of intolerable toxicity or social problems. At a median 24-months of follow-up, our clinical end point rates were 9% for local/regional recurrence (four patients), 5% for local/regional recurrence and distant metastases (two patients), and 2% for SPT (one patient), which was acute promyelocytic leukemia (ie, not of the upper aerodigestive tract). Median 1- and 2-year rates of overall survival were 98% and 91%, respectively, and of disease-free survival were 91% and 84%, respectively. CONCLUSION: The novel biologic agent combination of IFN-alpha, 13-cRA, and alpha-tocopherol was generally well tolerated and promising as adjuvant therapy for locally advanced squamous cell carcinoma of the head and neck. We are currently conducting a phase III randomized study of this combination (v no treatment) to confirm these phase II study results.

Adult↗

PubMed2001

Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice.

Encephalitogenic T cells that mediate experimental autoimmune encephalomyelitis (EAE) are commonly assumed to be exclusively CD4+, but formal proof is still lacking. In this study, we report that synthetic peptides 35-55 from myelin oligodendrocyte glycoprotein (pMOG(35-55)) consistently activate a high proportion of CD8+ alphabetaTCR+ T cells that are encephalitogenic in C57BL/6 (B6) mice. The encephalitogenic potential of CD8+ MOG-specific T cells was established by adoptive transfer of CD8-enriched MOG-specific T cells. These cells induced a much more severe and permanent disease than disease actively induced by immunization with pMOG(35-55). CNS lesions in pMOG(35-55) CD8+ T cell-induced EAE were progressive and more destructive. The CD8+ T cells were strongly pathogenic in syngeneic B6 and RAG-1(-/-) mice, but not in isogeneic beta2-microglobulin-deficient mice. MOG-specific CD8+ T cells could be repeatedly reisolated for up to 287 days from recipient B6 or RAG-1(-/-) mice in which disease was induced adoptively with <1 x 10(6) T cells sensitized to pMOG(35-55). It is postulated that MOG induces a relapsing and/or progressive pattern of EAE by eliciting a T cell response dominated by CD8+ autoreactive T cells. Such cells appear to have an enhanced tissue-damaging effect and persist in the animal for long periods.

Adoptive Transfer↗

PubMed2001

VIP gene transcription is regulated by far upstream enhancer and repressor elements.

SK-N-SH human neuroblastoma subclones differ widely in basal and second messenger induction of the gene encoding the neuropeptide vasoactive intestinal peptide (VIP). These differences were recapitulated by a chimeric gene which consisted of 5.2 kb of the human VIP gene 5' flanking sequence fused to a reporter. Subsequent gene deletion experiments revealed several regulatory regions on the gene, including a 645-bp sequence located approximately 4.0 upstream from the transcription start site. Here we examined this upstream region in detail. Inhibitory sequences were found to be present on each end of the 645-bp fragment. When removed, basal transcription increased more than 50-fold. Subsequent deletion/mutation analysis showed that the 213-bp fragment contained at least two enhancer elements. One of these was localized to an AT-rich 42-bp sequence shown by others to bind Oct proteins in neuroblastoma cells, while the other corresponded to a composite AP-1/ets element. In addition to these enhancers, a 28-bp sequence on the 213-bp fragment with no apparent homology to known silencers inhibited transcription. The studies provide molecular details of a complex regulatory region on the VIP gene that is likely to be used to finely tune the level of gene transcription in vivo.

5' Untranslated Regions↗

PubMed2001

Pds1 phosphorylation in response to DNA damage is essential for its DNA damage checkpoint function.

In Saccharomyces cerevisiae, Pds1 is an anaphase inhibitor and plays an essential role in DNA damage and spindle checkpoint pathways. Pds1 is phosphorylated in response to DNA damage but not spindle disruption, indicating distinct mechanisms delaying anaphase entry. Phosphorylation of Pds1 is Mec1 and Chk1 dependent in vivo. Here, we show that Pds1 is phosphorylated at multiple sites in vivo in response to DNA damage by Chk1. Mutation of the Chk1 phosphorylation sites on Pds1 abolished most of its DNA damage-inducible phosphorylation and its checkpoint function, whereas its anaphase inhibitor functions and spindle checkpoint functions remain intact. Loss of Pds1 phosphorylation correlates with APC-dependent Pds1 destruction in response to DNA damage. We also show that APC(Cdc20) is active in preanaphase arrested cells after DNA damage. This suggests that Pds1 is stabilized by phosphorylation in response to DNA damage, but APC(Cdc20) activity is not altered. Our results indicate that phosphorylation of Pds1 by Chk1 is the key function of Chk1 required to prevent anaphase entry.

Amino Acid Sequence↗