Biomedical subjects

F Dong

Publications and source records attributed to F Dong.

At least 109 records · Page 6Linked to original sources

PubMed1996

A conserved repetitive DNA element located in the centromeres of cereal chromosomes.

Repetitive DNA sequences have been demonstrated to play an important role for centromere function of eukaryotic chromosomes, including those from fission yeast, Drosophila melanogaster, and humans. Here we report on the isolation of a repetitive DNA element located in the centromeric regions of cereal chromosomes. A 745-bp repetitive DNA clone pSau3A9, was isolated from sorghum (Sorghum bicolor). This DNA element is located in the centromeric regions of all sorghum chromosomes, as demonstrated by fluorescence in situ hybridization. Repetitive DNA sequences homologous to pSau3A9 also are present in the centromeric regions of chromosomes from other cereal species, including rice, maize, wheat, barley, rye, and oats. Probe pSau3A9 also hybridized to the centromeric region of B chromosomes from rye and maize. The repetitive nature and its conservation in distantly related plant species indicate that the pSau3A9 family may be associated with centromere function of cereal chromosomes. The absence of DNA sequences homologous to pSau3A9 in dicot species suggests a faster divergence of centromererelated sequences compared with the telomere-related sequences in plants.

Animals↗

PubMed1996

The ATP-activated hexameric helicase of bacteriophage T4 (gp41) forms a stable primosome with a single subunit of T4-coded primase (gp61).

We have examined the formation of the primosome subassembly of the bacteriophage T4-coded DNA replication (elongation) complex from its helicase, primase, and DNA components. Previously, we had shown that the T4 helicase (gp41) exists in solution in a stable monomer left and right arrow dimer equilibrium at physiological protein (and salt) concentrations and forms a hexamer upon activation by ATP (or GTP) binding (Dong, F., Gogol, E. P., and von Hippel, P. H.(1995) J. Biol. Chem. 270, 7462-7473). Here we report that the T4 primase (gp61) is a monomer in solution under the same conditions, and that the ATP-activated helicase binds to a single gp61 primase molecule on appropriate DNA templates to reconstitute a stable primosome. We show that: (i) the gp41 helicase alone does not form a stable complex with DNA templates, although this helicase by itself can carry out moderately processive ATP-driven translocation along single-stranded DNA (Young, M. C., Schultz, D. E., Ring, D., and von Hippel, P. H.(1994) J. Mol. Biol. 235, 1447-1458); (ii) the primase alone does form a stable complex with DNA; (iii) the helicase can bind to the primase-DNA complex in the presence of ATP or GTP to form a stable ternary complex; (iv) this complex consists of six helicase subunits and one primase subunit; and (v) the reconstituted primosome is stable for at least 10 to 20 min after NTP cleavage and dissociation of the hydrolysis products. These results strongly suggest that the functional T4 DNA replication primosome consists of an integrated 6:1 helicase-primase complex bound to DNA, and that the ATP-activated helicase hexamer remains intact throughout the processive DNA replication process.

Adenosine Diphosphate↗

PubMed1996

Differential modulation of G1 cyclins and the Cdk inhibitor p27kip1 by platelet-derived growth factor and plasma factors in density-arrested fibroblasts.

Stimulation of quiescent Balb/c 3T3 fibroblasts into S phase requires the synergistic action of platelet-derived growth factor (PDGF) and progression factors found in platelet-poor plasma (PPP). Traverse of the G1/S phase boundary and the initiation of DNA replication require functional cyclin E-cyclin-dependent kinase (Cdk) 2 and cyclin A-Cdk2 complexes; however, the mechanisms by which PDGF and PPP regulate Cdk2 activation are not known. Density-arrested fibroblasts contain low levels of cyclins E and A, and high levels of the Cdk inhibitor p27kip1. Exposure of PDGF, which stimulates cell cycle entry but not progression through G1, induces the formation of cyclin D1-Cdk4 complexes that bind p27kip1 and titrate the pool of Kip1 available to inhibit Cdk2. In addition, PDGF stimulates a moderate transient reduction in the abundance of p27kip1 protein. However, limited expression of cyclin E and cyclin A is observed after PDGF treatment, and in the absence of PPP, p27 levels are sufficient to bind and inactivate existing cyclin-Cdk complexes. Although plasma does not significantly increase the proportion of Kip1 bound to cyclin D1-Cdk4, stimulation of PDGF-treated cells with plasma does overcome the threshold inhibition of p27kip1 by further increasing the expression of cyclins E and A and decreasing the amount of Kip1 over a prolonged time period. Our results indicate that the distinct mitogenic activities of PDGF and PPP differentially influence the activation of cyclin E- and cyclin A-associated kinases that ultimately regulate entry into S phase.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G-CSF-mediated activation of signaling complexes of the p21ras route.

Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G-CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G-CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.

Animals↗

PubMed1996

The membrane-distal cytoplasmic region of human granulocyte colony-stimulating factor receptor is required for STAT3 but not STAT1 homodimer formation.

Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) involves the activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway. G-CSF induces tyrosine phosphorylation of Jak1, Jak2, STAT1, and STAT3. The membrane-proximal region of G-CSF-R is sufficient for activation of Jaks. It is still unclear how STAT proteins are activated by G-CSF-R. We investigated the possible involvement of the C-terminal region of G-CSF-R in the recruitment of STAT proteins using BAF3 cell transfectants expressing wild type (WT) G-CSF-R, C-terminal deletion mutants and tyrosine-to-phenylalanine substitution mutants. Electrophoretic mobility shift assays with STAT-binding oligonucleotides (m67) showed that activation of WT G-CSF-R induces three distinct STAT complexes, namely STAT3 homodimers, STAT1-STAT3 heterodimers, and STAT1 homodimers. However, STAT1 homodimers and STAT1-STAT3 heterodimers were predominantly formed after activation of a C-terminal deletion mutant d685, which lacks all four conserved cytoplasmic tyrosine residues, located at positions 704, 729, 744, and 764. Antiphosphotyrosine immunoblots of STAT3 immunoprecipitates showed that activation of WT G-CSF-R induced phosphorylation of STAT3. In contrast, no phosphorylation of STAT3 was observed after activation of deletion mutant d685. These findings establish that the C-terminal region of G-CSF-R plays a major role in the activation of STAT3. By using tyrosine-to-phenylalanine substitution mutants of G-CSF-R, we further show that tyrosine 704, present in a YXXQ consensus sequence shown to be essential for STAT3 binding to gp130, is not exclusively involved in the activation of STAT3 by G-CSF-R.

Amino Acid Sequence↗

PubMed1996

Specific involvement of tyrosine 764 of human granulocyte colony-stimulating factor receptor in signal transduction mediated by p145/Shc/GRB2 or p90/GRB2 complexes.

Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) occurs via multiple pathways, one of which involves activation of p21Ras and mitogen-activated protein kinase. The SH2 domain-containing proteins Shc and GRB2 have been implicated in this latter signaling route. We studied the role of these proteins in signal transduction from wild type (WT) G-CSF-R, C-terminal deletion mutants, and tyrosine-to-phenylalanine substitution mutants in transfectants of the mouse pro-B cell line, BAF3. G-CSF stimulation of BAF3 cells expressing WT G-CSF-R induced tyrosine phosphorylation of Shc. Anti-Shc antibodies co-immunoprecipitated tyrosine-phosphorylated 145-kD proteins (p145), whereas GRB2 immunoprecipitates contained phosphorylated Shc, Syp, and proteins of 145 and 90 kD (p90). Neither of these complexes were detected after activation of a C-terminal deletion mutant of G-CSF-R that lacked all four conserved cytoplasmic tyrosine residues. G-CSF induced formation of Syp/GRB2 complexes in all the tyrosine-substitution mutants, suggesting that this association did not depend on the presence of single specific tyrosine residues in G-CSF-R. In contrast, tyrosine 764 of G-CSF-R appeared to be exclusively required for tyrosine phosphorylation of Shc and its association with p145 and GRB2. In addition, tyrosine 764 also specifically mediated binding of GRB2 to p90 without the involvement of Shc. These findings indicate that tyrosine 764 of G-CSF-R has a prominent role in G-CSF signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Severe congenital neutropenia terminating in acute myeloid leukemia: disease progression associated with mutations in the granulocyte-colony stimulating factor receptor gene.

Severe congenital neutropenia (SCN) is a heterogeneous disease condition with a variable family history and a propensity to progress towards myelodysplastic syndrome (MDS) and acute myeloblastic leukemia (AML). In a subgroup of patients, point mutations in the G-CSF-R gene have been found. These nonsense mutations result in the truncation of the C-terminal cytoplasmic region, a subdomain that is crucial for G-CSF induced maturation. SCN patients with mutations in the G-CSF-R gene appear to be predisposed to develop AML. Here, we recapitulate our view of how defective G-CSF-R may contribute to neutropenia and leukemogenesis.

Acute Disease↗

PubMed1996

Hematopoietic growth factors in leukemia.

The responses of leukemic cells to recombinant hematopoietic growth factors (HGFs) have been extensively studied, both in vitro and in vivo. Here, we summarize the current knowledge of the role that HGFs and growth modulatory cytokines play in the growth of leukemia. Particular attention is paid to the proliferation and maturation abilities of acute myeloblastic leukemia (AML), and the role HGFs play in these processes. The HGF responses of leukemic cells are discussed in the context of autocrine growth mechanisms, (cyto-)genetic abnormalities and defective function of HGF receptors. These studies have contributed considerably to our insight into the heterogeneous pathophysiology of leukemia.

Hematopoietic Cell Growth Factors↗

PubMed1996

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells.

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.

3T3 Cells↗

PubMed1996

The C-terminal cytoplasmic region of the granulocyte colony-stimulating factor receptor mediates apoptosis in maturation-incompetent murine myeloid cells.

Granulocyte colony-stimulating factor (G-CSF) promotes the survival and proliferation of myeloid progenitors and induces maturation of these cells toward terminally differentiated neutrophils. Using transfectants of the murine IL-3-dependent myeloid cell line 32D that express the human G-CSF receptor (32D/WT cells), we show here that G-CSF can also exert adverse effects on myeloid cell survival. Although initially enhancing IL-3-driven proliferation of 32D/WT cells, G-CSF strongly inhibited cell survival at later stages of culture. The loss of viability of 32D/WT cells following sustained G-CSF stimulation was not accompanied by progressive neutrophilic maturation. Instead, 32D/WT cells exhibited features characteristic of apoptosis. The apoptosis-inducing effect of G-CSF was seen at concentrations of IL-3 that could support long-term proliferation and survival of 32D/WT cells in the absence of G-CSF. Experiments with 32D cells expressing mutant forms of the G-CSF receptor revealed that the death signals were mediated exclusively through the membrane-distal cytoplasmic part of the G-CSF receptor, a region also involved in maturation signaling.

Animals↗

PubMed1996

[Expression of c-erbB-2 oncogene and mutation of p53 gene in myoepithelioma and myoepithelial carcinoma].

Expression of c-erbB-2 oncogene and mutant p53 protein were detected immunohistochemically in 14 cases of myoepithelioma and 6 cases of myoepithelial carcinoma of the salivary gland. Five of the 6 myoepithelial carcinomas and nine of the 14 myoepitheliomas were overexpression of c-erbB-2 oncoprotein. The salivary ductal epithelial cells near tumors were overexpression of c-erbB-2 oncoprotein. These results indicate that c-erbB-2 may be an initial oncogene during the histogenesis of myoepithelial tumors. The p53 protein was positive in five cases of the 6 myoepithelial carcinoma, whereas all myoepitheliomas were negative. The results indicate that the mutant p53 gene may play an important role in the carcinogenesis of myoepithelial carcinoma.

Carcinoma↗

PubMed1996

[Clinical application and observation of silicone oil in complicated retinal detachment].

Nine eyes with complicated retinal detachment underwent vitrectomy and silicone oil tamponate among whom there were seven eyes with PVRC3-PVRD2, one eye with giant retina tear and PVRD3 and one eye with giant retina tear. All the eyes were followed-up for an average of 12.6 months with a range from 2 to 23 months. Retina were completely reattached in there eyes. Vision were improved in six eyes. Retina was redetached in one eye after removal of silicone oil. Silicone oil tamponate can be used in complicated retinal detachment that fails to respond to conventional method. However, silicone oil tamponate may cause a number of complications, so its indications should be properly taken care of.

Adolescent↗

PubMed1996

Surgical management of macular epiretinal membranes.

PURPOSE: To evaluate the effects of surgical treatment on macular epiretinal membranes. METHODS: Vitrectomy and membrane removal were undergone for idopathic secondary macular epiretinal membrane. RESULTS: Fourteen eyes of 15 patients (93%) had vision improvement after operation in which 8 eyes (57%) increased 3 or more Senellen lines. Only one case suffered from paracentral scotoma. No other complications were noted. CONCLUSION: Surgical management of macular epiretinal membrane is safe and effective with good visual outcome and few complications.

Adult↗

PubMed1995

Mutations in the gene for the granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia.

BACKGROUND: In severe congenital neutropenia the maturation of myeloid progenitor cells is arrested. The myelodysplastic syndrome and acute myeloid leukemia develop in some patients with severe congenital neutropenia. Abnormalities in the signal-transduction pathways for granulocyte colony-stimulating factor (G-CSF) may play a part in the progression to acute myeloid leukemia. METHODS: We isolated genomic DNA and RNA from hematopoietic cells obtained from two patients with acute myeloid leukemia and histories of severe congenital neutropenia. The nucleotide sequences encoding the cytoplasmic domain of the G-CSF receptor were amplified by means of the polymerase chain reaction and sequenced. Murine myeloid 32D.C10 cells were transfected with complementary DNA encoding the wild-type or mutant G-CSF receptors and tested for their responses to G-CSF. RESULTS: Point mutations in the gene for the G-CSF receptor were identified in both patients. The mutations, a substitution of thymine for cytosine at the codon for glutamine at position 718 (Gln718) in one patient and at the codon for glutamine at position 731(Gln731) in the other, caused a truncation of the C-terminal cytoplasmic region of the receptor. Both mutant and wild-type genes for the G-CSF receptor were present in leukemic cells from the two patients. In one patient, the mutation was also found in the neutropenic stage, before the progression to acute myeloid leukemia. The 32D.C10 cells expressing mutant receptors had abnormally high proliferative responses but failed to mature when cultured in G-CSF. The mutant G-CSF receptors also interfered with terminal maturation mediated by the wild-type G-CSF receptor in the 32D.C10 cells that coexpressed the wild-type and mutant receptors. CONCLUSIONS: Mutations in the gene for the G-CSF receptor that interrupt signals required for the maturation of myeloid cells are involved in the pathogenesis of severe congenital neutropenia and associated with the progression to acute myeloid leukemia.

Acute Disease↗

PubMed1995

Functional assay of plasma antithrombin using polyethylene glycol (PEG) defibrinated plasma.

Polyethylene glycol(PEG) was used to precipitate fibrinogen to prepare defibrinated plasma in the two stage clotting assay of antithrombin activity. Five percent PEG-8000 precipitated fibrinogen from plasma without loss of antithrombin activity in the defibrinated plasma. Fibrin degradation products(FDP) as high as 640 ug/ml did not interfere the two stage clotting assay using PEG defibrinated plasma possibly because part of FDP was precipitated by PEG in the process of plasma defibrination. The two stage clotting assay was very sensitive to the changes of antithrombin activity in the range of 60%-100% of normal level. The assay was reproducible and correlated with chromogenic assay. The decrease of plasma antithrombin activity in a baboon septic shock model was demonstrated with this assay.

Animals↗

PubMed1995

The phage T4-coded DNA replication helicase (gp41) forms a hexamer upon activation by nucleoside triphosphate.

Sedimentation and high performance liquid chromatography studies show that the functional DNA replication helicase of bacteriophage T4 (gp41) exists primarily as a dimer at physiological protein concentrations, assembling from gp41 monomers with an association constant of approximately 10(6) M-1. Cryoelectron microscopy, analytical ultracentrifugation, and protein-protein cross-linking studies demonstrate that the binding of ATP or GTP drives the assembly of these dimers into monodisperse hexameric complexes, which redissociate following depletion of the purine nucleotide triphosphatase (PuTP) substrates by the DNA-stimulated PuTPase activity of the helicase. The hexameric state of gp41 can be stabilized for detailed study by the addition of the nonhydrolyzable PuTP analogs ATP gamma S and GTP gamma S and is not significantly affected by the presence of ADP, GDP, or single-stranded or forked DNA template constructs, although some structural details of the hexameric complex may be altered by DNA binding. Our results also indicate that the active gp41 helicase exists as a hexagonal trimer of asymmetric dimers, and that the hexamer is probably characterized by D3 symmetry. The assembly pathway of the gp41 helicase has been analyzed, and its structure and properties compared with those of other helicases involved in a variety of cellular processes. Functional implications of such structural organization are also considered.

Adenosine Triphosphate↗

PubMed1995

A point mutation in the granulocyte colony-stimulating factor receptor (G-CSF-R) gene in a case of acute myeloid leukemia results in the overexpression of a novel G-CSF-R isoform.

A novel human granulocyte colony-stimulating factor (G-CSF) receptor isoform, designated SD, has been identified in which the distal C-terminal cytoplasmic region, previously shown to be essential for maturation signalling, is substituted by an altered C-terminus. The SD receptor has a high affinity for G-CSF and retains the membrane-proximal cytoplasmic region known to be sufficient for proliferative signalling. Nonetheless, the SD isoform lacks the ability to transduce growth signals in murine BAF3 cells and, in contrast to the wild-type G-CSF receptor, is scarcely capable of activating JAK2 kinase. Expression of SD receptor was found to be low in normal granulocytes, but was significantly increased in a patient with acute myeloid leukemia (AML). The leukemic cells of this patient harbour a point mutation in the SD splice donor site of the G-CSF receptor gene. These findings provide the first evidence that mutations in the G-CSF receptor gene can occur in certain cases of clinical de novo AML. The possible contribution of defective G-CSF receptor signalling to leukemogenesis is discussed.

Acute Disease↗

PubMed1995

A method for estimating an overlying layer correction in quantitative ultrasound imaging.

A new method is tested to compensate for attenuation losses through the intervening layers in quantitative ultrasound imaging. The method subtracts the echo signal power spectrum acquired from a uniform region beneath the overlying layers from the signal power spectrum obtained from a reference phantom using the same instrumentation system settings. Changes in spectral components with frequency are then used to estimate the attenuation of the overlying layers. Several phantoms were used to test the method, among which was a phantom having three windows, one with no overlying layers and the other two with fat and muscle mimicking layers of different degrees of irregularity. Attenuation losses through the windows were compensated for using the technique, producing backscatter estimator images of a simulated tumor inside the phantom. After applying the method, consistent results for the backscatter estimator of the tumor, as well as the backscatter coefficient of the background material, were obtained from the various windows.

Humans↗