Biomedical subjects

Feng Zhu

Publications and source records attributed to Feng Zhu.

At least 55 records · Page 3Linked to original sources

PubMed2004

The signal transduction networks required for phosphorylation of STAT1 at Ser727 in mouse epidermal JB6 cells in the UVB response and inhibitory mechanisms of tea polyphenols.

Signal transducers and activators of transcription (STATs) play a critical role in signal transduction pathways. STATs are a family of cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. Phosphorylation of STAT1 at Ser727 is essential for its activation and occurs in response to stress signals, inflammation or infection. We observed that UVB induced phosphorylation of STAT1 (Ser727) in mouse epidermal JB6 Cl41 cells. This stimulation was inhibited by PD98059 and UO126, wortmannin, LY294002, SB202190 and SP600125 and dominant negative mutants of ERK2 (DNM-ERK2), p38 (DNM-p38) and JNK1 (DNM-JNK1). The response was absent in Jnk1(-/-) or Jnk2(-/-) knockout cells, but was unaffected by a dominant negative mutant of the phosphatidylinositol-3 kinase (PI-3K) p85 subunit (DNM-Deltap85). STAT1 (Ser727) phosphorylation was also blocked in a Rsk2(-) cell line. In Pdk1(-/-) cells STAT1 was not activated by UVB stimulation compared with strong activation in Pdk1(+/+) cells. Our data indicate that phosphorylation of STAT1 (Ser727) occurs through PI-3K, ERKs, p38 kinase, JNKs, PDK1 and p90RSK2 in the cellular response to UVB. We also show an inhibitory effect of theaflavins and EGCG on UVB-induced STAT1 (Ser727), ERKs, JNKs, PDK1 and p90RSK2 phosphorylation.

Animals↗

PubMed2004

[Cell viability between anterior and posterior spinal growth plate in adolescent idiopathic scoliosis].

OBJECTIVE: To compare the cell viability of chondrocytes between the anterior and the posterior spinal growth plates in adolescent idiopathic scoliosis (AIS) by proliferation and apoptosis labelling. METHODS: Seventeen AIS patients (4 male and 13 female, mean age 13.6 years old, ranged from 10 to 17 years old) were recruited in this study. Growth plates were harvested during anterior and posterior surgery. PCNA (proliferating cell nuclear antigen) and TUNEL (terminal deoxynucleotide transferase-mediated nick end labeling) were used for proliferation and apoptosis labeling on chondrocytes respectively. RESULTS: In AIS, the distribution of the proliferating nests were denser and more parallel in anterior column than those in posterior under microscope observation. In the proliferative and hypertrophic zone the PCNA index and PCNA/TUNEL ratio were higher in the anterior column than those in the posterior column (P < 0.05 and P < 0.01 respectively). While in resting zone the differences were not so significant. CONCLUSION: In adolescent idiopathic scoliosis the growth viability of chondrocytes is more vigorous in anterior spinal column than in the posterior column.

Adolescent↗

PubMed2004

Roles of Helicobacter pylori infection and cyclooxygenase-2 expression in gastric carcinogenesis.

AIM: Cyclooxygenase (COX)-2 is over expressed in gastrointestinal neoplasm. Helicobacter pylori (H pylori) infection is causally linked to gastric cancer. However, the expression of COX-2 in various stages of H pylori-associated gastric carcinogenesis pathway has not been elucidated. Therefore, the aim of this study was to clarify the role of H pylori induced COX-2 expression during carcinogenesis in the stomach. METHODS: Gastric biopsies from 138 subjects (30 cases of chronic superficial gastritis (CSG), 28 cases of gastric glandular atrophy (GA), 45 cases of gastric mucosal intestinal metaplasia (IM), 12 cases of moderate gastric epithelial dysplasia and 23 cases of gastric cancer) were enrolled. H pylori infection was assessed by a rapid urease test and histological examination (modified Giemsa staining). The expression of COX-1 and COX-2 in human gastric mucosa was detected by immunohistochemical staining. RESULTS: H pylori infection rate was 64.3% in GA and 69.5% in gastric cancer, which was significantly higher than that (36.7%) in CSG (P<0.05). The positive expression rates of COX-2 were 10.0%, 35.7%, 37.8%, 41.7% and 69.5% in CSG, GA, IM, dysplasia and gastric cancer, respectively. From CSG to GA, IM, dysplasia and finally to gastric cancer, expression of COX-2 showed an ascending tendency, whereas COX-1 expression did not change significantly in the gastric mucosa. The level of COX-2 expression in IM and dysplasia was significantly higher in H pylori-positive than in H pylori-negative subjects (P<0.01). CONCLUSION: COX-2 expression induced by H pylori infection is a relatively early event during carcinogenesis in the stomach.

Adult↗

PubMed2004

A caspase-6 and anti-human epidermal growth factor receptor-2 (HER2) antibody chimeric molecule suppresses the growth of HER2-overexpressing tumors.

Clinical studies have suggested that human epidermal growth factor receptor-2 (HER2) provide a useful target for antitumor therapy. We previously described the generation of a chimeric HER2-targeted immunocasp-3 protein. In this study, we extend the repertoire of chimeric proapoptotic proteins with immunocasp-6, a construct that comprises a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A, and an active caspase-6, which can directly cleave lamin A leading to nucleus damage and inducing programmed cell death. We demonstrate that the secreted immunocasp-6 molecule selectively recognizes and induces apoptosis in HER2-overexpressing tumor cells in vitro, but not in cells with undetectable HER2. The immunocasp-6 gene was next transferred into BALB/c athymic mice bearing human breast SK-BR-3 tumors by i.m. injection of liposome-encapsulated vectors, by intratumor injection of adenoviral vectors, or by i.v. injection of PBMC modified by retroviral infection. Regardless of the method used, expression of immunocasp-6 suppressed tumor growth and prolonged animal survival significantly. Our data show that the chimeric immunocasp-6 molecule can recognize HER2-positive tumor cells, promptly attack their nucleus, and induce their apoptotic death, suggesting the potential of this strategy for the treatment of human cancers that overexpress HER2.

ADP Ribose Transferases↗

PubMed2004

Involvement of ERKs and mitogen- and stress-activated protein kinase in UVC-induced phosphorylation of ATF2 in JB6 cells.

Activating transcription factor 2 (ATF2) has been shown to regulate gene expression in the cellular response to environmental stresses such as ultraviolet (UV) irradiation. However, the signal transduction mechanism of ATF2 activation by UV is not as yet completely understood. In the present study, we provide evidence showing that UVC-stimulated phosphorylation of ATF2 (Thr71) was to varying degrees prevented by a dominant negative mutant of p38beta kinase, c-Jun N-terminal kinase 1 (JNK1) or extracellular signal-regulated kinase 2 (ERK2). The phosphorylation was also suppressed by PD98059, an MEK inhibitor, or H89, a potent inhibitor of mitogen- and stress-activated protein kinase 1 (MSK1), and a C- or N-terminal 'kinase-dead' mutant of MSK1 (MSK1-Cd or MSK1-Nd). Furthermore, co- immunoprecipitation experiments revealed a potential intracellular signaling complex consisting of ATF2 and ERKs and/or MSK1. In vitro kinase assays revealed that ERK1, ERK2 and MSK1, like p38 kinase and JNK2, directly phosphorylate ATF2 at Thr71, but addition of RSK2 or Akt1 had almost no effect. Active kinase immunoprecipitated by an MSK1, ERKs or p38 antibody from an extract of JB6 cells irradiated by UVC can directly phosphorylate ATF2 at Thr71, suggesting UVC induces a direct phosphorylation of ATF2 by ERKs or MSK1. Overall, our results reveal that MSK1 and ERKs, like p38 kinase and JNKs, are required for ATF2 phosphorylation (Thr71) in the UVC response.

Activating Transcription Factor 2↗

PubMed2004

Response of REV3 promoter to N-methyl-N'-nitro-N-nitrosoguanidine.

Previously, we have shown that low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) led to the upregulation of REV3 gene at transcriptional level in cultured human amnion FL cells. In this study, using bioinformatic analysis the putative binding sites for different transcription factors were found to exist in REV3 gene promoter region. A 2570-bp fragment of the 5' flanking region of REV3 gene was amplified by PCR from PAC clone RP3-415N12 and inserted into the pGL3-Basic reporter vector. Dual-luciferase reporter assay demonstrated that the reconstructed plasmid did respond to MNNG exposure in transfected FL cells. Several variants of the reporter plasmids with different deletions of the REV3 promoter region were also constructed and their promoter strength was analyzed. It was found that the MNNG response element might locate at the REV3 gene promoter region -404 to -102 between two Sma1 sites. The shortest responsive fragment containing the putative binding sites for transcription factors CREBP, AP-2, NF-kappaB, and SP1 was also identified.

Base Sequence↗

PubMed2004

Hydrolytic degradation and cracks in resin-modified glass-ionomer cements.

Water-absorption affects the basic properties of resin-modified glass-ionomer cements (RMGICs). Fick's law is usually invoked to explain the absorption process. The purpose of this study is to show that the absorption in accordance with the Fickian model cannot be extended to the whole of the specimen, and that microcrack formation is the main degradation mechanism for specimens cured in a closed environment. For this purpose, flat disk-shaped paste specimens 1.5 mm thick (aspect ratio 4), irradiated in closed conditions between two glass slides and stored in water for approximately 20 months, were analyzed periodically gravimetrically and under confocal fluorescence microscopy, with absorbed eosin used as the fluorescent probe. At pH 7.0, the specimen surface (10-20 micrometers in depth) absorbed water rapidly, swelled, and disintegrated in 20-40 days. Long-term storage produced isolated cracks and grains, no progress in the swelling, and a slow weight decrease. A lower pH (pH 3.5) produced a significant increase of the number of microcracks. The decrease in the irradiation time (30 s or less) enhanced the erosion process, producing very broad cracks. It was concluded that the prevalent mechanism of long-term hydrolytic degradation was based on the slow formation of cracks, whereas only in the early stage of storage did absorption occur quickly in accordance with the Fickian diffusion.

Composite Resins↗

PubMed2004

Evidence of STAT1 phosphorylation modulated by MAPKs, MEK1 and MSK1.

Phosphorylation at Ser727 in signal transducer and activator of transcription 1 (STAT1) is essential for its activation and signal transduction. However, the upstream kinases responsible for phosphorylating Ser727 are still elusive. Here, we provide evidence showing that UVA-induced mitogen-activated protein kinase (MAPK) signaling pathways lead to STAT1 Ser727 phosphorylation. Our experimental results show that UVA-induced Ser727 phosphorylation of STAT1 was, to different degrees, diminished by PD98059 and U0126, two specific inhibitors of MEKs, and SB202190 and PD169316, inhibitors of p38 kinase and c-Jun N-terminal kinases (JNKs), respectively. STAT1 phosphorylation was also blocked by a dominant negative mutant of p38beta kinase or JNK1, JNK1- or JNK2-deficiency, or an N-terminal or C-terminal kinase-dead mutant of mitogen- and stress-activated protein kinase 1 (MSK1), a downstream kinase closer to p38 kinase and extracellular signal-regulated kinases (ERKs). In vitro kinase assays using the combined STAT1 proteins as substrates from immunoprecipitation and glutathione S-transferase pull down show that active ERK1, JNK1, p38 kinase, MEK1 and MSK1 stimulated phosphorylation of STAT1 (Ser727) indirectly through an unidentified factor or a downstream kinase. Overall, our data indicate that phosphorylation of STAT1 at Ser727 occurs through diverse MAPK cascades including MEK1, ERKs, p38 kinase, JNKs and MSK1 in the cellular response to UVA.

Animals↗

PubMed2004

Analysis of etiology and drug resistance of biliary infections.

The bile was collected from fro patients with biliary infections, with the bacterium isolated to study the sensitivity of each kind of the bacterium to several antibiotics in common use. Except G- bacterium, we also found some kinds of G+ bacterium in infection bile. G- bacterium were not sensitive to Clindamycin, G+ bacterium were sensitive to Ciprofloxacin. Escherichia coli, Xanthomonas maltophilia, Enterobacter cloacae, Pseudomonas aeruginosa were sensitive to Ampicillin. G+ bacterium were not sensitive to Azactam. Enterococcus faecalis, Enterococcus faecium, Enterobacter cloacae were not sensitive to Ceftazidime. Enterococcus faecalis, Staphylococcus coagulase negative, Staphylococcus epidermidis, Pseudomonas aeruginosa were not sensitive to Ceftriaxone Sodium. We didn't found any bacterium resistance Imipenem. The possibility of the existence of G+ bacterium as well as drug resistance should be considered n patients with biliary infections. The value of susceptibility test should be respected to avoid drug abuse of antibiotics.

Adult↗

PubMed2004

Protease domain of human ADAM33 produced by Drosophila S2 cells.

Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in several insect cells. The pro-CAT was cloned into baculovirus under the regulation of the polyhedron promoter and using either the honeybee mellitin or ADAM33 signal sequence. Sf9 or Hi5 cells infected with these recombinant viruses expressed the majority of the protein unprocessed and as inclusion bodies ( approximately 10 mg/L). On the other hand, similar constructs could be expressed, processed, and secreted by Drosophila S2 cells using a variety of constitutive (actin, pAc5.1) or inducible (metallothionein, PMT) promoters and leader sequences (e.g., native and BiP). Higher expression level of 10-fold was observed for the inducible system resulting in an average yield of 20 mg/L after purification. The majority of the catalytic domain purified from the Drosophila conditioned media remained associated with the pro-domain after several chromatography steps. An induction cocktail containing cadmium chloride and zinc chloride was subsequently developed for the PMT system as an alternative to using cupric sulfate or cadmium chloride as single inducers. The novel induction cocktail resulted in an increased ratio of secreted catalytic to pro-domain, and yielded milligram amounts of highly purified protease. The availability of this modified expression system facilitated purification of the wild type and several glycosylation mutants, one of which (N231Q) crystallized recently for X-ray structure determination [J. Mol. Biol. 335 (2003) 129].

ADAM Proteins↗

PubMed2004

Lymphotactin enhances the in-vitro immune efficacy of dendritoma formed by dendritic cells and mouse hepatocellular carcinoma cells.

OBJECTIVE: To investigate the in-vitro antitumor immune responses of dendritoma formed by mouse hepatocellular carcinoma (HCC) cells and lymphotactin (Lptn) gene modified dendritic cells (DCs). METHOD: DCs prepared from mouse bone marrow were genetically modified by lymphotactin adenovirus, and fused with H22 cells by polyethylene glycol (PEG). RT-PCR and ELISA were employed to identify lymphotactin expression at mRNA and protein level. Cell phenotypes and fusion efficiency was detected by FACS. The stimulatory effect of DC on T cells was detected by mixed lymphocyte reaction. The cytotoxicity activity against H22 cells was assayed by LDH method. RESULTS: Lymphotactin could be efficiently expressed by DCLptn/H22 hybridoma. DCLptn/H22 cells could induce potent T cell proliferation effect and generate strong cytotoxic T lymphocyte (CTL) reaction against allogenic H22 cells. CONCLUSION: Lymphotactin genetic modification could enhance the in vitro immune activity of the dendritoma.

Animals↗

PubMed2004

[Computer-assisted surgical navigation technique].

Surgical navigation system is a system that processes medical images by computer graphics and image processing technique, reconstructs 2D or 3D medical image models. It builds a real-time loop among the eyes of doctor, the surgical device and the head of patient by several of space localization technique, so that to realize displaying the localization of surgical devices real-time. The history and research status of surgical navigation system is summarized and the system configuration and the key techniques are expatiated emphatically in this paper. The key techniques include localization in space, image processing and displaying technique, system registration technique and head location technique. At last the progress direction of navigation systems is given.

Computer Graphics↗

PubMed2004

[nhibitory effect of adenosine analogues on invasion of human ovarian cancer cell line HO-8910PM].

BACKGROUND & OBJECTIVE: Adenosine plays an essential role in cells signal transduction, including inhibiting the activity of phosphatidylinositol 4-kinase, up-regulating the activity of adenylate cyclase and the concentration of cAMP, and inhibiting platelet actin polymerization. Adenosine analogues have the same bioactivities as adenosine, but are slowly deaminated by adenosine deaminase. This study was to observe the effects of 2-chloro-adenosine(2-ClAdo), 2-chloro-2'-deoxyadenosine (2-CldAdo), and 2'-deoxyadenosine (2'-dAdo) on invasion of human ovarian cancer cell line HO-8910PM. METHODS: HO-8910PM cells were treated with 2-ClAdo, 2-CldAdo, and 2'-dAdo, Trypan blue dye exclusion assay was used to examine cytotoxities of these 3 adenosine analogues to HO-8910PM cells, 5-fluorouracil (5-FU) was used as sensitive control. Reconstituted basement membrane invasion assay was used to evaluate invasive, adhesive, and kinetic activities of the cells. PAGE substrate zymography was used to analyze secretion,and activity of matrix metalloproteinase-9 (MMP-9). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyze mRNA levels of mta1 and nm23H1. RESULTS: The 50% inhibition concentrations (IC(50)) of 2-ClAdo, and 2-CldAdo at 72 h were 5.80, and 2.61 micromol/L; while inhibitory effect of 2'-dAdo on the cells was not significant, its IC(50) was more than 100 micromol/L. Inhibition rates of 6.0 micromol/L of 2-ClAdo, and 2-CldAdo on cells invasion were (42.5+/-1.5)%, and (9.9+/-0.5)%; those on cells migration were (36.9+/-2.1)%,and (17.1+/-0.4)%; and those on cells adhesion were (32.2+/-2.3)%, and (19.5+/-3.1)%. All these inhibition rates were significantly higher than those of control (P< 0.05). While 2'-dAdo had no significant effect on cells invasion. All 3 chemicals could not inhibit the production and activity of MMP-9 in HO-8910PM cells. 2-ClAdo down-regulated mRNA level of mta1, but neither 2-ClAdo nor 2-CldAdo affected mRNA level of nm23H1. CONCLUSION: Of these 3 adenosine analogues, 2-ClAdo might be a potential chemical inhibiting tumor invasion and metastasis because of its weaker cytotoxity but stronger inhibitive effect on invasion of tumor cells.

2-Chloroadenosine↗

PubMed2003

Catalytic activity of human ADAM33.

ADAM33 (a disintegrin and metalloproteinase) is an asthma susceptibility gene recently identified through a genetic study of asthmatic families (van Eerdewegh et al. (2002) Nature 418, 426-430). In order to characterize the catalytic properties of ADAM33, the metalloproteinase domain of human ADAM33 was expressed in Drosophila S2 cells and purified. The N-terminal sequence of the purified metalloproteinase was exclusively (204)EARR, indicating utilization of one of three furin recognition sites. Of many synthetic peptides tested as potential substrates, four peptides derived from beta-amyloid precursor protein (APP), Kit-ligand-1 (KL-1), tumor necrosis factor-related activation-induced cytokine, and insulin B chain were cleaved by ADAM33; mutation at the catalytic site, E346A, inactivated catalytic activity. Cleavage of APP occurred at His(14)/Gln(15), not at the alpha-secretase site and was inefficient (k(cat)/K(m) (1.6 +/- 0.3) x 10(2) m(-1) s(-1)). Cleavage of a juxtamembrane KL-1 peptide occurred at a site used physiologically with a similar efficiency. Mutagenesis of KL-1 peptide substrate indicated that the P3, P2, P1, and P3' residues were critical for activity. In a transfected cell-based sheddase assay, ADAM33 functioned as a negative regulator of APP shedding and mediated some constitutive shedding of KL-1, which was not regulated by phorbol 12-myristate 13-acetate activation. ADAM33 activity was sensitive to several hydroxamate inhibitors (IK682, K(i) = 23 +/- 7 nm) and to tissue inhibitors of metalloproteinase (TIMPs). Activity was inhibited moderately by TIMP-3 and TIMP-4 and weakly inhibited by TIMP-2 but not by TIMP-1, a profile distinct from other ADAMs. The identification of ADAM33 peptide substrates, cellular activity, and a distinct inhibitor profile provide the basis for further functional studies of ADAM33.

ADAM Proteins↗

PubMed2003

Ataxia telangiectasia mutated proteins, MAPKs, and RSK2 are involved in the phosphorylation of STAT3.

Phosphorylation at Ser(727) is known to be required for complete activation of STAT3 by diverse stimuli including UV irradiation, but the kinase(s) responsible for phosphorylating STAT3 (Ser(727)) is still not well discerned. In the present study, we observed that activation of ATM is required for a UVA-stimulated increase in Ser(727) phosphorylation of STAT3 as well as in activation and phosphorylation of p90 ribosomal protein S6 kinases (RSKs). Moreover, UVA-stimulated activation of upstream kinases, such as c-Jun N-terminal kinases (JNKs) and ERKs, involved in mediating phosphorylation of RSKs and STAT3 was defective or delayed in ATM-deficient cells. Furthermore, we provide evidence that RSK2-deficient cells were defective for UV-induced Ser(727) phosphorylation of STAT3, and the defect was restored after ectopic expression of transfected full-length RSK2. In vitro experiments showed that active RSK2 and JNK1 induce the phosphorylation of STAT3 precipitates from immunoprecipitation but not from glutathione S-transferase (GST) pull-down. Interestingly, the GST fusion STAT3 proteins mixed together with STAT3 immunoprecipitates can be phosphorylated by JNK. However, the in vitro phosphorylation of STAT3 was reduced by the GST-STAT3 beta protein, a dominant negative form of STAT3. Taken together, our results demonstrate that the STAT3 phosphorylation at Ser(727) is triggered by active RSK2 or JNK1 in the presence of a downstream kinase or a cofactor, and thereby the intracellular phosphorylation process is stimulated through a signaling pathway involving ATM, MAPKs, RSK2, and an as yet unidentified kinase or cofactor. Additionally, RSK2-mediated phosphorylation of STAT3 (Ser(727)) was further determined to be required for basal and UVA-stimulated STAT3 transcriptional activities.

Acute-Phase Proteins↗

PubMed2003

Response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine and its role in mutagenesis.

AIM: To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis. METHODS: The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRT locus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FL-REV3(-) by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3(-) and FL-REV3(-). The blockage effect of REV3 was measured by combination of reverse transcription-polymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3 protein level. RESULTS: The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27 fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P<0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P<0.05). In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein. MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P<0.05) and 5.8 fold (P<0.01) respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3 was blocked by antisense RNA, and almost recovered to their spontaneous mutation levels. The spontaneous HPRT mutation was disappeared in REV3-disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66 x 10(-6) in FL cells to 0.14 x 10(-6) in transgenic cells as well (P<0.01). CONCLUSION: The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.

Base Sequence↗

PubMed2003

DNA polymerase zeta: new insight into eukaryotic mutagenesis and mammalian embryonic development.

Information about the mechanisms that generate mutations in eukaryotes is likely to be useful for understanding human health concerns, such as genotoxicity and cancer. Eukaryotic mutagenesis is largely the outcome of attacks by endogenous and environmental agents. Except for DNA repair, cell cycle checkpoints and DNA damage avoidance, cells have also evolved DNA damage tolerance mechanism, by which lesion-targeted mutation might occur in the genome during replication by specific DNA polymerases to bypass the lesions (translesion DNA synthesis, TLS), or mutation on undamaged DNA templates (untargeted mutation) might be induced. DNA polymerase zeta (pol zeta), which was found firstly in budding yeast Saccharomyces cerevisiae and consists of catalytic subunit scRev3 and stimulating subunit scRev7, has received more attention in recent years. Pol zeta is a member of DNA polymerase eta subfamily, which belongs to DNA polymerase B family, and exists in almost all eukaryotes. Human homolog of the scRev3 gene is located in chromosome region 6q21, and the mouse equivalent maps to chromosome 10, distal to the c-myb gene and close to the Macs gene. Alternative splicing, upstream out-of frame ATG can be found in yeast scRev3, mouse and human homologs. Furthermore, the sequence from 253-323 immediate upstream of the AUG initiator codon has the potential to form a stem-loop hairpin secondary structure in REV3 mRNA, suggesting that human REV3 protein may be expressed at low levels in human cells under normal growth conditions. The functional domain analysis showed that yeast Rev3-980 tyrosine in conserved region II is at the polymerase active site. Human REV3 amino acid residues 1 776-2 195 provide a REV7 binding domain, and REV7 amino acid residues 1-211 provide a bind domain for REV1, REV3 and REV7 itself. More interestingly, REV7 interacts with hMAD2 and therefore might function in the cell cycle control by affecting the activation of APC (anaphase promoting complex). Currently it has been known that pol zeta is involved in most spontaneous mutation, lesion-targeted mutation via TLS, chemical carcinogen induced untargeted mutation and somatic hypermutation of antibody genes in mammalian. In TLS pathway, pol zeta acts as a "mismatch extender" with combination of other DNA polymerases, such as pol iota. Unlike in yeast, it was found that pol zeta also functioned in mouse embryonic development more recently. It was hypothesized that the roles of pol zeta in TLS and cell cycle control might contribute to mouse embryonic lethality.

Animals↗

PubMed2003

Isolation of a novel member of small G protein superfamily and its expression in colon cancer.

AIM: APMCF1 is a novel human gene whose transcripts are up-regulated in apoptotic MCF-7 cells. In order to learn more about this gene's function in other tumors, we cloned its full length cDNA and prepared its polyclonal antibody to investigate its expression in colon cancers with immunohistochemistry. METHODS: With the method of 5' rapid amplification of cDNA end (RACE) and EST assembled in GenBank, we extended the length of APMCF1 at 5' end. Then the sequence encoding the APMCF1 protein was amplified by RT-PCR from the total RNA of apoptotic MCF-7 cells and cloned into the prokaryotic expression vector pGEX-KG to construct recombinant expression vector pGEX-APMCF1. The GST-APMCF1 fusion protein was expressed in E. coli and used to immunize rabbits to get the rabbit anti-APMCF1 serum. The specificity of polyclonal anti-APMCF1 antibody was determined by Western blot. Then we investigated the expression of Apmcf1 in colon cancers and normal colonic mucosa with immunohistochemistry. RESULTS: A cDNA fragment with a length of 1 745 bp was obtained. APMCF1 was mapped to chromosome 3q22.2 and spanned at least 14.8 kb of genomic DNA with seven exons and six introns contained. Bioinformatic analysis showed the protein encoded by APMCF1 contained a small GTP-binding protein (G proteins) domain and was homologous to mouse signal recognition particle receptor beta(SRbeta). A coding region covering 816 bp was cloned and polyclonal anti-APMCF1 antibody was prepared successfully. The immunohistochemistry study showed that APMCF1 had a strong expression in colon cancer. CONCLUSION: APMCF1 may be the gene coding human signal recognition particle receptor beta and belongs to the small-G protein superfamily. Its strong expression pattern in colon cancer suggests it may play a role in colon cancer development.

Amino Acid Sequence↗