Biomedical subjects

F Dong

Publications and source records attributed to F Dong.

At least 19 recordsLinked to original sources

PubMed1998

Rice (Oryza sativa) centromeric regions consist of complex DNA.

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.

Base Sequence↗

PubMed1998

Prolonged STAT1 activation related to the growth arrest of malignant lymphoma cells by interferon-alpha.

Multiple biologic effects of interferon-alpha (IFN-alpha), including cell growth inhibition and antiviral protection, are initiated by tyrosine phosphorylation of STAT proteins. Although this signal pathway has been intensively investigated, the relevance of STAT signal persistence has received scant attention. Using paired isogenic lymphoma cells (Daudi), which either are sensitive or resistant to growth inhibition by IFN-alpha, we found comparable initial tyrosine phosphorylation of multiple STAT proteins; however, the phosphorylation durations and associated DNA-binding activities diverged. Phosphorylation and DNA-binding capacity of STAT1 decreased after 4 to 8 hours in resistant cells, as compared with 24 to 32 hours in sensitive cells, whereas phosphorylation of STAT3 and STAT5b was briefer in both lines. Functional significance of the prolonged STAT1 signal, therefore, was explored by experimental interruption of tyrosine phosphorylation, either by premature withdrawal of the IFN-alpha or deferred addition of pharmacologically diverse antagonists: staurosporine (protein kinase inhibitor), phorbol 12-myristate 13-acetate (growth promoter), or aurintricarboxylic acid (ligand competitor). Results indicated that an approximately 18-hour period of continued STAT1 phosphorylation was associated with growth arrest, but that antiviral protection developed earlier. These differences provide novel evidence of a temporal dimension to IFN-alpha signal specificity and show that duration of STAT1 activation may be a critical variable in malignant cell responsiveness to antiproliferative therapy.

Antineoplastic Agents↗

PubMed1998

The role of cyclin D3-dependent kinase in the phosphorylation of p130 in mouse BALB/c 3T3 fibroblasts.

We have observed that cyclin D3-dependent kinase activity is increased in the late G1 phase in BALB/c 3T3 fibroblasts. The profile of cyclin D3-associated activity closely parallels that of cyclin D1, which is also induced after mitogenic stimulation of quiescent cells. These activities correlate with the appearance of hyperphosphorylated p130, an Rb family member important in regulating E2F-4 and E2F-5 activity in fibroblastic cells. We demonstrated, however, that only the cyclin D3 activity efficiently phosphorylated p130 in an in vitro kinase assay. This apparent specificity was further demonstrated by experiments which demonstrated that cyclin D3 was physically associated with p130 at the times when D3-dependent kinase activity and p130 hyperphosphorylation were observed. Examination of E2F by electrophoretic mobility shift assay revealed that E2F-4 DNA binding activity existed in a p130.E2F complex at times before D3-dependent kinase activity was apparent and in a free E2F-4 complex after D3 activity developed. Thus, our data suggest that cyclin D3 preferentially phosphorylates p130 and is thereby specifically targeted to overcoming growth-suppressive control mediated through p130 pathways.

3T3 Cells↗

PubMed1998

Molecular characterization of a cDNA that encodes six isoforms of a novel murine A kinase anchor protein.

We have cloned cDNA that encodes six novel A kinase anchor proteins (collectively named AKAP-KL). AKAP-KL diversity is generated by alternative mRNA splicing and utilization of two translation initiation codons. AKAP-KL polypeptides are evident in lung, kidney, and cerebellum, but are absent from many tissues. Different isoforms predominate in different tissues. Thus, AKAP-KL expression is differentially regulated in vivo. All AKAP-KL isoforms contain a 20-residue domain that avidly binds (Kd approximately 10 nM) regulatory subunits (RII) of protein kinase AII and is highly homologous with the RII tethering site in neuronal AKAP75. The distribution of AKAP-KL is strikingly asymmetric (polarized) in situ. Anchor protein accumulates near the inner, apical surface of highly polarized epithelium in tubules of nephrons. Both RII and AKAP-KL are enriched at an intracellular site that lies just below the plasma membrane of alveolar epithelial cells in lung. AKAP-KL interacts with and modulates the structure of the actin cytoskeleton in transfected cells. We also demonstrate that the tethering domain of AKAP-KL avidly ligates RII subunits in intact cells. AKAP-KL may be involved in (a) establishing polarity in signaling systems and (b) physically and functionally integrating PKAII isoforms with downstream effectors to capture, amplify, and precisely focus diffuse, trans-cellular signals carried by cAMP.

A Kinase Anchor Proteins↗

PubMed1998

Activation of the Jak2-Stat5 signaling pathway in Nb2 lymphoma cells by an anti-apoptotic agent, aurintricarboxylic acid.

Biological effects of many hormones and cytokines are mediated through receptor-associated Jak tyrosine kinases and cytoplasmic Stat transcription factors, including critical physiological processes such as immunity, reproduction, and cell growth and differentiation. Pharmaceuticals that control Jak-Stat pathways are therefore of considerable interest. Here we demonstrate that a single Jak-Stat pathway can be activated by aurintricarboxylic acid (ATA), a negatively charged triphenylmethane derivative (475 Da) with anti-apoptotic properties. In prolactin (PRL)-dependent Nb2 lymphocytes, ATA sustained cell growth in the absence of hormone and mimicked rapid PRL-induced tyrosine phosphorylation of Jak2 and activation of Stat5a and Stat5b with tyrosine phosphorylation, heterodimerization, DNA binding, and induction of the Stat5-regulated pim-1 protooncogene. ATA also mimicked PRL activation of serine kinases ERK1 and ERK2. However, unlike PRL, ATA did not regulate Stat1 or Stat3. ATA also did not affect Jak3, which is activated in these cells by interleukin-2 family cytokines. Although the mechanism and specificity by which ATA activates Jak2, Stat5, and ERKs in Nb2 cells are still unclear, the present study demonstrates that certain hormone or cytokine effects on Jak-Stat pathways can be discretely imitated by a low molecular weight, non-peptide pharmaceutical. The results are also consistent with Stat5 involvement in lymphocyte growth and survival.

Apoptosis↗

PubMed1998

Liquid or solid ultrasonically tissue-mimicking materials with very low scatter.

A new tissue-mimicking material for ultrasound, using evaporated milk as the primary absorption component, is described. It has very low backscatter but still exhibits the 1540 m s-1 propagation speed and proportionality of attenuation coefficient and frequency over the diagnostic frequency range. The material can be produced in solid or liquid form with attenuation coefficient slopes spanning the range 0.1-0.7 dB cm-1 MHz-1. The liquid form is useful in phantoms where detailed beam patterns are to be determined, either involving translation of measurement devices in the liquid or phantoms with fibers present for causing the only detectable echoes. In the latter case, the liquid quality allows removal of liquid with one attenuation coefficient slope and replacement with another. The solid form may be more useful than the liquid for two reasons. First, many simulated lesions (including ones that produce essentially no internal echoes) can lie in the scan slice with positions extending over the entire image area without enhancement or shadowing effects being of concern. Second, the lack of significant backscatter from the material in the absence of added scatterers allows the backscatter coefficient to be varied over a considerable range. A critical result is that intrinsic material contrast between targets and surroundings can be accurately predicted in terms of the concentrations of added scatterers and, assuming all scatterers are of the same type, the contrast will be completely independent of frequency. Use of the fungicide thimerosal eliminates deterioration, and ultrasonic properties have been shown to be stable over 2.5 years.

Animals↗

PubMed1998

Estimating the spatial autocorrelation function for ultrasound scatterers in isotropic media.

The autocorrelation function pertaining to spatial distributions of ultrasonic scatterers in soft tissue is believed to contain useful information related to tissue morphology. A simple processing method applied to radio-frequency echo signals estimates this function for a sample having isotropic scattering conditions. It utilizes backscattered echo signals from the sample and echo signals from a reference object having defined scattering properties. The ratio of the echo signal power spectrum from the sample to the echo signal power spectrum from the reference object is obtained, and corrected for attenuation differences between the two media. This yields a "form factor" for the sample, whose inverse Fourier transform is the autocorrelation function. The method was tested using tissue-mimicking samples for which spatial autocorrelation functions could be modeled from the dimensions of embedded scatterers. The shapes of the measured autocorrelation functions were in reasonable agreement with those estimated, although measured functions overestimated the function at small lag distances. Scatterer diameters estimated from the zeros of the autocorrelation function agreed to within 6% of expected values when the measurement system bandwidth satisfied minimal criteria.

Humans↗

PubMed1997

Phantoms and automated system for testing the resolution of ultrasound scanners.

Tissue-mimicking phantoms and an automated system have been developed for testing the resolution performance of ultrasound scanners by determining detectability of low to higher contrast spherical lesions over the entire depth of field. Axial, lateral and elevational resolutions are accounted for simultaneously and equally. Tissue-mimicking spherical simulated lesions are either 3 or 4 mm in diameter and have one of four different intrinsic material contrasts. For each diameter and contrast, there is a set of 109 lesions in a regular array with coplanar centers extending from 0.5-15.5 cm in depth. With the scan slice superimposed on the spheres, the image is frame-grabbed for automated analysis. A diameter-dependent lesion signal-to-noise ratio is computed for each pixel position in the image, excluding a 5-mm boundary. Two universal thresholds, resulting from maximization of agreement between the automated system and human observers, give rise to a depth range, or "resolution zone", over which detection exists for each type lesion.

Image Enhancement↗

PubMed1997

Mutations in the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia.

Previously, nonsense mutations in the gene encoding the granulocyte colony-stimulating factor receptor (G-CSF-R) have been described in three patients with severe congenital neutropenia (SCN) (Proc Natl Acad Sci USA 1994; 91: 4480; New Engl J Med 1995; 333: 487). The mutations resulted in the truncation of the carboxy-terminal region of G-CSF-R essential for transduction of maturation signals. Two of these patients developed acute myeloblastic leukemia (AML). We present the results of a search among 20 additional cases of congenital neutropenia (CN) and SCN for the presence of mutations in the cytoplasmic domain of G-CSF-R. This series includes patients with familial and nonfamilial forms of CN and SCN. Mutations in the G-CSF-R gene were found in two new SCN cases. These mutations were nonsense mutations, located in the same cytoplasmic region of G-CSF-R as those found earlier, resulting in the truncation of the C-terminus. Both of these patients developed AML. None of the other patients showed clinical symptoms or cytogenetic features indicative of AML or progression to leukemia. The analysis in this extended series of patients thus has revealed five SCN cases with G-CSF-R mutations, four of whom developed AML. These results add support to the notion that mutations in the G-CSF-R gene, affecting the maturation signaling function of the receptor, define a distinct subgroup of SCN with increased susceptibilty to AML.

Adolescent↗

PubMed1997

Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway.

Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways.

Animals↗

PubMed1997

Attenuation and backscatter estimation using video signal analysis applied to B-mode images.

Most methods for in vivo quantitation of ultrasound attenuation and backscatter are not available clinically because they rely upon acquiring and analyzing radio frequency (rf) echo signals. This paper describes a technique to estimate ultrasound attenuation and backscatter from B-mode image data. The video signal analysis (VSA) technique utilizes images of a reference phantom, taken using the same instrument settings used to record images from the patient or sample, to account for effects of the transducer beam, system gain and signal processing on image data. A 'gray-scale look-up table' is derived to convert image pixel value data within a region of interest to echo signal amplitudes relative to echo signals from the same depth in the reference phantom. These relative echo levels enable estimates of attenuation and backscatter in the region of interest. VSA was used to quantify acoustic properties of test phantoms using 3 different clinical scanners and various transducers. The level of agreement between results obtained with different ultrasound imaging systems was very good. VSA attenuation and backscatter levels also compare favorably with attenuation coefficients and backscatter coefficients obtained using rf analysis.

Humans↗

PubMed1997

Model of complications of NIDDM. I. Model construction and assumptions.

OBJECTIVE: To develop a model of NIDDM for analyzing prevention strategies for NIDDM. RESEARCH DESIGN AND METHODS: A Markov type model with Monte Carlo techniques was used. Age, sex, and ethnicity of cohort was based on U.S. data. Incidence rates of complications were also based on community and population studies. RESULTS: Nonproliferative retinopathy, proliferative retinopathy, and macular edema are predicted in 79, 19, and 52%, respectively, of people with NIDDM; 19% are predicted to develop legal blindness. Microalbuminuria, gross proteinuria, and end-stage renal disease related to diabetes are predicted in 53, 40, and 17%, respectively. Symptomatic sensorimotor neuropathy and lower-extremity amputation are predicted in 31 and 17%, respectively. Cardiovascular disease is predicted in 39%. Higher rates of complications (1.1-3.0x) are predicted in minority populations. Predicted average life expectancy is 17 years after diagnosis. CONCLUSIONS: A probabilistic model of NIDDM predicts the vascular complications of NIDDM in a cohort representative of the incident cases of diabetes in the U.S. before age 75 years. Predictions of complications and mortality are consistent with the known epidemiology of NIDDM. The model is suitable for evaluating the effect of preventive interventions on the natural history of NIDDM.

Adult↗

PubMed1997

Model of complications of NIDDM. II. Analysis of the health benefits and cost-effectiveness of treating NIDDM with the goal of normoglycemia.

OBJECTIVE: To analyze the health benefits and economics of treating NIDDM with the goal of normoglycemia. RESEARCH DESIGN AND METHODS: Incidence-based simulation model of NIDDM was used. Hazard rates for complications were adjusted for glycemia using risk gradients from the Diabetes Control and Complications Trial. Treatment costs were estimated from national survey data and clinical trials. Incremental costs and benefits were expressed in present value dollars (3% discount rate). Life-years were adjusted for quality of life, yielding quality-adjusted life-years (QALYs). RESULTS: Comprehensive treatment of NIDDM that maintains an HbA1c value of 7.2% is predicted to reduce the cumulative incidence of blindness, end-stage renal disease, and lower-extremity amputation by 72, 87, and 67%, respectively. Cardiovascular disease risk increased by 3% (no effect of treating glycemia is assumed). Life expectancy increased 1.39 years. The cost of treating hyperglycemia increased by almost twofold, which is partially offset by reductions in the cost of complications. The estimated incremental cost/QALY gained is $16,002. Treatment is more cost-effective for those with longer glycemic exposure (earlier onset of diabetes), minorities, and those with higher HbA1c under standard care. CONCLUSIONS: The incremental effectiveness of treating NIDDM with the goal of normoglycemia is estimated to be approximately $16,000/QALY gained, which is in the range of interventions that are generally considered cost-effective.

Adult↗

PubMed1996

A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis.

We have developed a coupled helicase-polymerase DNA unwinding assay and have used it to monitor the rate of double-stranded DNA unwinding catalyzed by the phage T4 DNA replication helicase (gp41). This procedure can be used to follow helicase activity in subpopulations in systems in which the unwinding-synthesis reaction is not synchronized on all the substrate-template molecules. We show that T4 replication helicase (gp41) and polymerase (gp43) can be assembled onto a loading site located near the end of a long double-stranded DNA template in the presence of a macro-molecular crowding agent, and that this coupled "two-protein" system can carry out ATP-dependent strand displacement DNA synthesis at physiological rates (400 to 500 bp per sec) and with high processivity in the absence of other T4 DNA replication proteins. These results suggest that a direct helicase-polymerase interaction may be central to fast and processive double-stranded DNA replication, and lead us to reconsider the roles of the other replication proteins in processivity control.

Bacteriophage T4↗

PubMed1996

Fluorescence monitoring of T4 polymerase holoenzyme accessory protein interactions during loading of the sliding clamp onto the template-primer junction.

Assembly of the T4 polymerase holoenzyme requires coordinated interactions among the core polymerase and the clamp loader (gp44/62) and sliding clamp (gp45) accessory proteins. Here we describe the creation of a mutant of gp45 that can be uniquely modified by fluorescent probes within each protein monomer at a site-specific cysteine residue. The fluorescently labeled gp45 was shown to have the same biological activity as the wild-type protein. These strike "labeled" gp45 adducts were then used in steady-state and fluorescence polarization studies to monitor the interaction of gp45 with the gp44/62 clamp-loading complex and template-primer DNA in the presence and absence of ATP, and of the non-hydrolyzable analog, adenosine 5'-O-(3-thiotriphosphate). We find that a complex of ATP-activated gp44/62 with appropriately labeled gp45 shows significant fluorescent enhancement (and an increase in fluorescent anisotropy), that can be partially reversed by interaction with template-primer DNA. Fluorescence-monitored binding curves between gp45 and ATP-activated gp44/62 reveal that the two protein complexes bind with a 1:1 stoichiometry. Analysis shows that these methods can be used to follow the ATP-driven loading of gp45 onto the template-primer by the gp44/62 clamp-loading complex, and in combination with the kinetic data presented in the companion article, provide insight into the rate-limiting steps during clamp assembly on template-primer DNA. A reaction pathway for this processivity clamp-loading process is proposed.

Adenosine Triphosphatases↗

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↗