Biomedical subjects

D Liu

Publications and source records attributed to D Liu.

At least 469 records · Page 26Linked to original sources

PubMed1997

Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration.

Physicochemical properties of the cationic liposomes, including structure of the cationic lipid-to-DNA ratio, liposome particle size, and inclusion of the helper lipids, were studied for their effect on the level, site, and duration time of gene expression in vivo by intravenous administration. Using a cytomegalovirus (CMV)-driven gene expression system containing either the luciferase or green fluorescence protein gene as a reporter and two cationic lipids [N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP)], we demonstrated in vivo by a single intravenous injection of DNA/liposome complexes into mice, that cationic liposomes are capable of transfecting cells in organs such as the lung, heart, liver, spleen, and kidney. Transfection efficiency is determined mainly by the structure of the cationic lipid and the ratio of cationic lipid to DNA. Although the presence of cholesterol in DOTAP liposomes did not affect transfection activity, inclusion of dioleoylphosphatidylethanolamine (DOPE) into either DOTAP or DOTMA liposomes significantly decreases liposome transfection activity in vivo. Results form time course show that gene expression in different organs is transient, with a peak level between 4 and 24 hr, dropping to less than 1% of the peak level by day 4. Experiments with repeated injections showed that the peak level of gene expression could be regained by subsequent injection.

Animals↗

PubMed1997

Identification of four novel human phosphoinositide 3-kinases defines a multi-isoform subfamily.

Phosphoinositide (PI) 3-kinases have critical roles in diverse cellular signalling processes and in protein trafficking. This suggests that like other intracellular signalling molecules, e.g., phospholipase C and protein kinase C, there might be a large family of PI 3-kinase isoforms with the individual members having discrete signalling roles. Reverse transcription-polymerase chain reaction methods, using degenerate oligonucleotide primers against the lipid kinase consensus region, revealed eight sequences from human cDNA containing a high degree of identity to the family of PI 3-kinases. The sequences obtained included the previously described p110 alpha, p110 beta, and p110 gamma isoforms and HsVps34. Additionally, we have identified four novel sequences which are related to PI 3-kinases. Three of the novel sequences would appear to form a distinct sub-family of PI 3-kinases. We report the expression of these novel PI 3-kinases in human tissues and in cells derived from normal breast.

Blotting, Northern↗

PubMed1997

Experimental spinal fusion with recombinant human bone morphogenetic protein-2 without decortication of osseous elements.

STUDY DESIGN: L4-L5 intertransverse process fusions were produced with 58 micrograms, 230 micrograms, or 920 micrograms of recombinant human bone morphogenetic protein-2 in 20 dogs. Eleven had traditional decortication of posterior elements before insertion of the implant. Nine were left undecorticated. All animals were evaluated 3 months after surgery. OBJECTIVES: To determine whether decortication is a prerequisite for successful fusion in the presence of osteoinductive proteins such as bone morphogenetic protein-2. SUMMARY OF BACKGROUND DATA: Recombinant osteoinductive proteins can induce de novo bone in ectopic soft-tissue sites in the absence of bone marrow elements. Traditional methods for achieving spinal fusion rely on exposure of bone marrow through decortication to facilitate osteogenesis. It is hypothesized that the presence of an implanted osteoinductive protein obviates the need for exposure and release of host inductive factors. METHODS: Recombinant human bone morphogenetic protein-2-induced intertransverse process fusions were performed with and without decortication. Fusion sites were evaluated by computed tomography imaging, high-resolution radiography, manual testing, mechanical testing, and histologic analysis. RESULTS: One hundred percent of decorticated spines and 89% of undecorticated spines were clinically fused by 3 months. Ninety-one percent of decorticated spines and 78% of undecorticated specimens exhibited bilateral transverse process osseous bridging. The only spines that failed to achieve solid bilateral arthrodesis were in the lowest dose group. With the higher two doses, there was histologic evidence of osseous continuity between the fusion mass and undecorticated transverse processes. CONCLUSIONS: There were no statistical differences in clinical and radiographic fusion rates between decorticated and undecorticated sites. With higher doses of recombinant human bone morphoganetic protein-2, there was little histologic distinction between fusions in decorticated versus undecorticated spines.

Animals↗

PubMed1997

Raf-1 kinase and exoenzyme S interact with 14-3-3zeta through a common site involving lysine 49.

14-3-3 proteins are a family of conserved dimeric molecules that bind to a range of cellular proteins involved in signal transduction and oncogenesis. Our solution of the crystal structure of 14-3-3zeta revealed a conserved amphipathic groove that may allow the association of 14-3-3 with diverse ligands (Liu, D., Bienkowska, J., Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191-194). Here, the contributions of three positively charged residues (Lys-49, Arg-56, and Arg-60) that lie in this Raf-binding groove were investigated. Two of the charge-reversal mutations greatly (K49E) or partially (R56E) decreased the interaction of 14-3-3zeta with Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 14-3-3zeta with another target protein, exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3zeta required for ExoS activation increased by approximately 110-, 5-, and 2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic reduction of 14-3-3zeta/ligand affinity by the K49E mutation is due to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteolysis and circular dichroism analysis. This work identifies the first point mutation (K49E) that dramatically disrupts 14-3-3zeta/ligand interactions. The parallel effects of this single point mutation on both Raf-1 binding and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3zeta.

14-3-3 Proteins↗

PubMed1997

ERF: genomic organization, chromosomal localization and promoter analysis of the human and mouse genes.

ERF (Ets2 Repressor Factor) is a ubiquitously expressed ets-domain protein that exhibits strong transcriptional repressor activity, has been shown to suppress ets-induced transformation and has been suggested to be regulated by MAPK phosphorylation. We report here the sequence of the mouse gene, the genomic organization of the human and the mouse genes, their chromosomal position and the analysis of the promoter region. Genomic clones encompassing either the human ERF or the mouse Erf gene were isolated and utilized to define their molecular organization. The gene in both species consists of 4 exons over a 10 kb region. Utilizing FISH, somatic cell hybrids and linkage analysis, we identified the chromosomal position of ERF on human chromosome 19q13.1 and on its syntenic region in the mouse, on chromosome 7. Sequence analysis of the mouse gene indicated a 90% identity to the human gene within the coding and promoter regions. The predicted Erf protein is 98% identical to the human protein and all of the identifiable motifs are conserved between the two proteins. However, the mouse protein is three amino acids longer (551 versus 548 aa). The area surrounding the region that is homologous to the 5' end of the human cDNA can serve as a promoter in transfection into eukaryotic cells. This region is highly conserved between the mouse and the human genes. A number of conserved transcription factor binding sites can be identified in the region including an ets binding site (EBS). Interestingly, removal of a small segment that includes the EBS, seriously hampers promoter function, suggesting the ERF transcription may be regulated by ets-domain proteins.

Amino Acid Sequence↗

PubMed1997

A sampling artifact in the microdialysis study of changes in extracellular [Mg2+] upon spinal cord injury.

Whether depletion of a substance from the extracellular fluid by removal through the dialysis probe can affect the measured efflux of that substance into the extracellular fluid upon injury is examined. When Mg(2+)-free artificial cerebrospinal fluid was used to perfuse the microdialysis fiber, a significant increase in collected Mg2+ was observed immediately following injury. However, when [Mg2+] inside the fiber was equal to the basal concentration in the extracellular fluid, no increase in [Mg2+] was detected. The difference between the two sets of results is attributed to efflux down an increased intracellular/extracellular [Mg2+] gradient that occurs when the basal extracellular concentration is not balanced within the fiber, but not otherwise. This is a previously unreported problem with microdialysis.

Amino Acids↗

PubMed1997

A magnetic colloid system for isolation of rare cells from blood for fish analysis.

To improve cell recovery of trophoblast and nucleated red blood cells from maternal blood for diagnosis of chromosomal abnormalities, we have investigated the use of a magnetic sorting system that utilizes a ferrofluid. The main advantage of this system is that the beads used are small enough to allow visualization of chromosome-specific sequences by fluorescence in situ hybridization (FISH). The ferrofluid was validated using MAb340, a trophoblast-specific antibody, and anti-CD71, used to sort for nucleated red blood cells. Antigen-positive cells could be efficiently sorted from a 1000-fold excess of antigen-negative cells and easily stained by FISH. We are currently evaluating its use on maternal blood samples.

Blood Cells↗

PubMed1997

The CARET asbestos-exposed cohort: baseline characteristics and comparison to other asbestos-exposed cohorts.

The Carotene and Retinol Efficacy Trial (CARET) was a double-blind, placebo-controlled trial of the daily administration of 25,000 IU vitamin A and 30 mg beta-carotene for the prevention of lung cancer. Of close to 18,500 participants, more than 4,000 were asbestos-exposed men recruited from shipyard and construction trades at five study centers in the United States. While the primary endpoint of the trial was the incidence of lung cancer, a number of questions about the natural history of asbestos-related disease will also be addressed. The mean age at entry into the trial was 57 years and the mean duration of follow-up on active intervention was 4 years. With the exception of 133 never-smoker pilot participants (3%), all subjects recruited were by intention current (38%) or ex-smokers (58%), with a mean cumulative smoking exposure at entry of 43 pack-years. Mean years from first asbestos exposure were 35, and mean duration of asbestos exposure in a high-risk trade was 19 years. The distribution of radiographic abnormalities was as follows: normal, 34%; parenchymal opacities (ILO profusion score > 1/0) alone, 18%; pleural thickening alone, 27%; both parenchymal opacities and pleural thickening, 21%. The CARET cohort, when compared to previously reported asbestos-exposed cohorts, is characterized by substantial asbestos exposure and high proportion of asbestos-related radiographic findings. The active intervention was halted in 1996, after a mean duration of 40 years. Passive follow-up of the cohort will continue until the year 2000.

Asbestos↗

PubMed1997

Secondary and tertiary structural changes in gamma delta resolvase: comparison of the wild-type enzyme, the I110R mutant, and the C-terminal DNA binding domain in solution.

gamma delta Resolvase is a site-specific DNA recombinase (M(r) 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low-salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten tertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I110R resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I110R mutant contains a folded catalytic core of approximately 60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I110R resolvase. No increase in the helicity of the linker region of I110R resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.

Ammonium Sulfate↗

PubMed1997

Redistribution of platelet membrane glycoprotein IV and release of intracellular alpha-granule thrombospondin in patients with chronic myelogenous leukemia.

The redistribution of platelet membrane glycoprotein IV (GPIV) and the release of intracellular alpha-granule thrombospondin (TSP) were examined and the inhibition of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) in patients with chronic myelogenous leukemia (CML) was observed and quantitation of beta-TG and PF4 in sera was conducted. GPIV in inactive platelet from CML was 36080 +/- 17010 molecules/platelet as compared with 13190 +/- 4810 from the controls (P < 0.01). No abnormality was found in the distribution of platelet membrane GPIb and GPIIb/IIIa (P > 0.05). The GPIV redistribution on active platelet membrane induced thrombin (IU/ml) from CML and healthy donors was 44320 +/- 32310 and 22800 +/- 12700 molecules/platelet respectively (P < 0.01). The difference in the release of intracellular alpha-granule TSP between CML and the control group was not found (P > 0.05). There was no direct correlation between GPIV expression and TSP binding after platelet activation. The high levels of beta-TG and PF4 in sera inhibited release of intracellular alpha-granule TSP in vitro. These results indicate that the abnormality of platelet membrane GPIV is a common marker in CML, therefore the specific increase of platelet GPIV in patients with CML may be a useful tool for the diagnosis and monitoring of the platelet dysfunction. The release of internal TSP pools is hindered by either beta-TG or PF4 in sera.

CD36 Antigens↗

PubMed1997

A peptide derived from neutrophil inhibitory factor (NIF) blocks neutrophil adherence to endothelial cells.

OBJECTIVE AND DESIGN: Peptides derived from neutrophil inhibitory factor (NIF), a known antagonist of Mac-1, were evaluated as inhibitors of neutrophil adherence. MATERIAL: In vitro assays of adherence employed: 1) human polymorphonuclear cells (PMN), 2) human umbilical vein endothelial cells (HUVEC), and 3) CHO cells expressing ICAM-1 (CHO-ICAM cells). TREATMENT: Cells, pretreated with NIF-derived peptides (0.1-100 microM) for 10 minutes, were permitted to adhere for 20 min in the continued presence of peptide. METHODS: Cell-based assays: 1) PMN adherence to HUVEC, 2) PMN adhesion to immobilized human serum proteins, and 3) adherence of CHO-ICAM cells to immobilized Mac-1. RESULTS: A NIF-derived peptide of 29 amino acids blocked PMN adherence to HUVEC, but behaved somewhat differently than the parent NIF protein. NIF specifically antagonized Mac-1 dependent adherence, but the peptide blocked neutrophil adherence that was dependent upon both Mac-1 and LFA-1 integrins. CHO-ICAM adherence to Mac-1 was blocked by NIF, but not by the peptide. Binding studies with NIF and the peptide indicate that the molecules bind to different sites. CONCLUSIONS: A peptide derived from NIF blocks PMN adherence but, unlike NIF, the mechanism of action is not mediated by direct antagonism Mac-1.

Amino Acid Sequence↗

PubMed1997

Evaluation of an extracorporeal membrane oxygenation system using a nonporous membrane oxygenator and a new method for heparin coating.

A new heparin binding method was applied to a miniature extracorporeal membrane oxygenation (ECMO) system with a nonporous membrane oxygenator (the priming volume, 45 mL; the membrane surface area, 0.4 m2; maximal flow rate, 2 L/min) that is resistant to plasma leakage. The authors evaluated the stability of the immobilized heparin in vitro and the feasibility of this system in animals. Samples of hollow fibers and tubing were rinsed at 40 degrees C for 4 days in normal saline, Ringer's lactate, and 1 mol/L NaCl solution. Heparin activities on hollow fibers after rinsing were 99 +/- 2.3% (mean +/- SD), 96 +/- 3.9%, and 93 +/- 2.0% of the control in each solution, while those of the tubing were 87 +/- 4.1%, 86 +/- 3.1%, and 76 +/- 8.6%, respectively. Veno-arterial ECMO using this heparin-coated system was performed on five beagles (8 to 12 kg) for 10 hours. Neither major thrombus formation nor plasma leakage was detected during the procedure in spite of a low flow rate (300 mL/min) and a reduced activated clotting time (mean, 128 seconds). Platelets decreased to 52% of the control (P < .01) at 1 hour, but no progressive decrease was seen thereafter. Antithrombin-III decreased (P < .01) and thrombin/antithrombin III complex increased (P < .05 at 4 hours and P < .01 at 6, 8, and 10 hours) during bypass, but the changes of fibrinogen and fibrinopeptide A were not significant. Fibrinogen/fibrin degeneration products, fibrinopeptide B beta 15-42, and plasma-free hemoglobin levels did not rise significantly. O2 transfer of the oxygenators at a flow rate of 300 mL/min were 12.3 +/- 0.4 mL/min at 30 minutes, 14.3 +/- 1.2 mL/min at 5 hours, and 14.7 +/- 1.7 mL/min at 10 hours (no statistical difference). Histological examination of the brains and the kidneys showed no evidence of thromboembolic sequela in any of the animals. These results suggest that this new system is a promising device for long-term ECMO in which the amount of systemic heparinization can be reduced with the minimal possibility of plasma leakage.

Animals↗

PubMed1997

The apoptotic death of neuroblastoma cells caused by serum from patients with insulin-dependent diabetes and neuropathy may be Fas-mediated.

Immunoglobulins from patients with diabetic neuropathy are toxic to neuroblastoma cells. The cell death has characteristics of apoptosis: condensed chromatin, shrunken cytoplasm, elevation of [Ca2+]i and DNA fragmentation. N1E-115 cell membranes contain Fas, a regulator of apoptosis that recently has been shown to be involved in pancreatic beta-cell destruction leading to diabetes. Fas-specific antibodies bind to the surface of N1E-115 cells and induce apoptosis. Serum from patients with diabetic neuropathy block Fas-antibody binding. We conclude that sera from patients with diabetic neuropathy contain an activator of Fas-regulated apoptosis that may contribute to the pathogenesis of diabetic neuropathy.

Adult↗

PubMed1997

Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by 'inverse' sequencing.

BACKGROUND: The nonpolar nucleoside of difluorotoluene (F) was previously found to behave similarly to thymidine in single-site deoxynucleoside triphosphate (dNTP) insertion experiments with the Klenow fragment (KF) of DNA polymerase I. Further study was needed, first to see whether F-A base pairs could be replicated in more than one sequence context; second to investigate whether specific base pair replication occurs in the presence of four dNTPs; and third to confirm the presence of F in a replicated DNA strand. RESULTS: A primer bound to a template strand containing eight F residues was extended by KF using the four natural dNTPs at 20 microM. Similarly, the complement (containing eight adenines) was extended using dATP, dGTP, dCTP and dFTP. Comparison of the new strands to authentic strands using standard and 'inverse' chemical sequencing showed identical composition within +/- 5%. CONCLUSIONS: The results confirm that F in a template strand encodes the insertion of dATP and that adenine in a template encodes the insertion of dFTP with good specificity in at least six different nearest neighbor contexts. The results confirm that analog F behaves similarly to thymidine despite its poor hydrogen-bonding ability.

Adenine↗

PubMed1997

Improved laboratory diagnosis of ovine footrot: an update.

Ovine footrot is a complex clinical disease syndrome primarily resulting from infection by the anaerobic bacterium Dichelobacter nodosus. In order to aid clinical diagnosis, various laboratory tests based on the detection and measurements of phenotypic properties of D. nodosus have been developed for genus-specific detection as well as virulence determination. However, these tests are generally time-consuming, and tend to be variable with external factors that affect the growth and metabolism of the bacterium. A new generation of diagnostic reagents, such as monoclonal antibodies, gene probes and polymerase chain reaction, has been developed recently. Preliminary assessment of these reagents has shown potential to vastly improve the laboratory identification and determination of the virulence of D. nodosus. It is important that these new reagents are vigorously assessed against existing laboratory tests, such as the elastase test and gelatin gel test, prior to their adoption for the routine diagnosis of footrot.

Animals↗

PubMed1997

The button test: a small scale method using microdialysis cells for assessing protein solubility at concentrations suitable for NMR.

A simple method has been developed for screening solution conditions to determine conditions under which a protein is soluble at the high concentrations typically used for NMR spectroscopy. The method employs microdialysis cells or 'buttons'. The low sample volume (5 microliters) required for each microdialysis button permits testing of a wide range of solution conditions and temperatures with high protein concentrations, using a small amount of protein. Following precipitation of several NMR samples of the C-terminal core domain of human TFIIB, the microdialysis button screen facilitated identification of conditions in which precipitation of the TFIIB core domain was eliminated. The microdialysis button method for screening solution conditions is generally applicable and has been used to permit rapid identification of suitable NMR sample solution conditions for proteins involved in transcription and cell adhesion.

Magnetic Resonance Spectroscopy↗

PubMed1997

The time course of malondialdehyde production following impact injury to rat spinal cord as measured by microdialysis and high pressure liquid chromatography.

This paper reports a highly sensitive, specific, and reproducible method for the analysis of malondialdehyde (MDA) from microdialysates. The microdialysates were reacted with 2-thiobarbituric acid, and the TBA adducts were separated by HPLC and detected using a fluorescence detector. Butylated hydroxytoluene was used as an antioxidant to minimize formation of artifacts. The time course of MDA production following impact injury to the rat spinal cord was obtained using this improved method. MDA concentrations in the extracellular space gradually increased from a basal level of 20 +/- 3.6 nM to 44 +/- 18.1 nM during the first 2 hr, reached a maximum of 95 +/- 19.8 nM at 5 hr, and then decreased to 36 +/- 9.5 nM at 9 hr. The findings support the hypothesis that spinal cord injury leads to increased membrane lipid peroxidation.

Animals↗

PubMed1997

Differential level of DSB repair fidelity effected by nuclear protein extracts derived from radiosensitive and radioresistant human tumour cells.

A cell-free plasmid reactivation assay was used to determine the fidelity of DNA double-strand break (DSB) repair in a panel of eight DSB repair-proficient human tumour cell lines. Nuclear protein extracts derived from radiosensitive tumour cells were less capable of correctly rejoining EcoRI-induced DSBs than were similar extracts from radioresistant tumour cells. Linear regression analysis suggests that there was a significant (r2 = 0.84, P = 0.001, d.f. = 6) correlation between the fidelity of DSB rejoining and the SF2 values of the cell lines studied. This cell-free assay is clearly sensitive to differences in the nuclear protein composition that reflect the clinically relevant radiosensitivity of these cell lines. The fact that our cell-free assay yielded similar results to previous studies that used intracellular plasmid reactivation assays suggests that those differences in DSB mis-rejoining frequencies in radiosensitive and radioresistant cell lines may be due to inherent differences in nuclear protein composition and are not directly attributable to differences in proliferation rates between cell lines. The underlying cause for this association between DSB mis-rejoining frequencies and radiosensitivity is presently unknown, however restriction endonuclease mapping and polymerase chain reaction (PCR) amplification analysis revealed that approximately 40% of the mis-rejoined DSBs arose as a result of the deletion of between 40 and 440 base pairs. These data raise the possibility that the radiosensitivity of DSB repair-proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways.

Animals↗