Biomedical subjects

F Wang

Publications and source records attributed to F Wang.

At least 163 records · Page 9Linked to original sources

PubMed2000

Hammerhead ribozyme as a therapeutic agent for hyperlipidemia: production of truncated apolipoprotein B and hypolipidemic effects in a dyslipidemia murine model.

In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we used adenovirus-mediated vector to target hammerhead ribozyme at GUA(6679) downward arrow of apoB mRNA (designated AvRB15) in the liver of a dyslipidemic mouse model that is deficient in apoB mRNA editing enzyme and overexpresses human apoB100. In this study, we delivered approximately 4 x 10(11) virus particles of AvRB15 (active ribozyme) or AvRB15-mutant (inactive ribozyme) to the animals. Using Southern blot analysis, we readily detected RB15 DNA in the mouse liver as long as day 35 after injection. This result was correlated with the RNA expression of RB15 by RNase protection assay. Using reverse ligation-mediated polymerase chain reaction, the 3' cleavage product of apoB mRNA was detected, and the exact cleavage site was confirmed by sequencing. Importantly, the levels of human and mouse apoB mRNA decreased approximately 80% after AvRB15 transduction. There was a marked decrease in plasma cholesterol, triglyceride, and human apoB of 42, 51, and 62%, respectively, when compared with the inactive ribozyme-treated group. Moreover, ribozyme cleavage of apoB mRNA generated a truncated protein of the expected size (apoB48.1), which was associated with lipoprotein particles in the very low density, low density, and high density lipoprotein fractions. Taken together, these results indicate that apoB mRNA-specific hammerhead ribozyme can be used as a potential therapeutic agent to modulate apoB gene expression and to treat hyperlipidemia.

Animals↗

PubMed2000

Absolute configurations, predominant conformations and tautomeric structures of enantiomeric tert-butylphenylphosphine oxides

Enantiomeric tert-butylphenylphosphine oxides have been isolated via resolution of the racemate with mandelic acid and investigated by using vibrational circular dichroism (VCD). Vibrational absorption and circular dichroism spectra of dextrorotatory, levorotatory, and racemic mixture of tert-butylphenylphosphine oxide have been measured in CDCl(3) and CHCl(3) solutions in the 2000-900 cm(-)(1) region. Experimental spectra are compared with the ab initio predictions of absorption and VCD spectra obtained with density functional theory using B3LYP/6-31G basis set for different tautomeric structures and conformers of (S)-tert-butylphenylphosphine oxide. This comparison indicates that (-)-tert-butylphenylphosphine oxide is of the (S)-configuration and indicates only one tautomeric structure and one conformation predominant for tert-butylphenylphosphine oxide in CDCl(3) and CHCl(3) solutions.

Journal Article↗

PubMed2000

The nonsense-mediated decay pathway and mutually exclusive expression of alternatively spliced FGFR2IIIb and -IIIc mRNAs.

Exons IIIb and IIIc of the FGFR2 gene are alternatively spliced in a mutually exclusive manner in different cell types. A switch from expression of FGFR2IIIb to FGFR2IIIc accompanies the transition of nonmalignant rat prostate tumor epithelial cells (DTE) to cells comprising malignant AT3 tumors. Here we used transfection of minigenes with and without alterations in reading frame and with and without introns to examine how translation affects observed FGFR2 splice products. We observed that nonsense mutations in other than the last exon led to a dramatic reduction in mRNA that is abrogated by removal of downstream introns in both DTE and AT3 cells. The mRNA, devoid of both IIIb and IIIc exons (C1-C2), is a major splice product from minigenes lacking an intron downstream of the second common exon C2. From these observations, we suggest that repression of exon IIIc and activation of exon IIIb inclusion in DTE cells lead to the generation of both C1-IIIb-C2 and C1-C2 products. However, the C1-C2 product from the native gene is degraded due to a frameshift and a premature termination codon caused by splicing C1 and C2 together. Derepression of exon IIIc and repression of exon IIIb lead to the generation of both C1-IIIc-C2 and C1-C2 products in AT3 cells, but the C1-C2 product is degraded. The C1-IIIb-IIIc-C2 mRNA containing a premature termination codon in exon IIIc was present, but at apparently trace levels in both cell types. The nonsense-mediated mRNA decay pathway and cell type-dependent rates of inclusion of exons IIIb and IIIc result in the mutually exclusive expression of FGFR2IIIb and IIIc.

Alternative Splicing↗

PubMed2000

Predicting the cumulative risk of false-positive mammograms.

BACKGROUND: The cumulative risk of a false-positive mammogram can be substantial. We studied which variables affect the chance of a false-positive mammogram and estimated cumulative risks over nine sequential mammograms. METHODS: We used medical records of 2227 randomly selected women who were 40-69 years of age on July 1, 1983, and had at least one screening mammogram. We used a Bayesian discrete hazard regression model developed for this study to test the effect of patient and radiologic variables on a first false-positive screening and to calculate cumulative risks of a false-positive mammogram. RESULTS: Of 9747 screening mammograms, 6. 5% were false-positive; 23.8% of women experienced at least one false-positive result. After nine mammograms, the risk of a false-positive mammogram was 43.1% (95% confidence interval [CI] = 36.6%-53.6%). Risk ratios decreased with increasing age and increased with number of breast biopsies, family history of breast cancer, estrogen use, time between screenings, no comparison with previous mammograms, and the radiologist's tendency to call mammograms abnormal. For a woman with highest-risk variables, the estimated risk for a false-positive mammogram at the first and by the ninth mammogram was 98.1% (95% CI = 69.3%-100%) and 100% (95% CI = 99.9%-100%), respectively. A woman with lowest-risk variables had estimated risks of 0.7% (95% CI = 0.2%-1.9%) and 4.6% (95% CI = 1. 1%-12.5%), respectively. CONCLUSIONS: The cumulative risk of a false-positive mammogram over time varies substantially, depending on a woman's own risk profile and on several factors related to radiologic screening. By the ninth mammogram, the risk can be as low as 5% for women with low-risk variables and as high as 100% for women with multiple high-risk factors.

Adult↗

PubMed2000

The preparation and characterization of an immobilized l-glutamic decarboxylase and its application for determination of l-glutamic acid.

This paper is to study the preparation and characterization of an immobilized L-glutamic decarboxylase (GDC) and develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N'-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO(2) electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The dynamic response of Na(2)HPO(4)-citric acid buffer system selected is much better than that of the others, 0.10 M HAc-0.10 M NaAc and 0.10 M sodium citrate-0.10 M citric acid. The initial rate of the enzyme reaction v(0) in this buffer system is 1.76 mol. l(-1) min(-1), moreover, the rate of the enzyme reaction appears linear in the first 4 min. The optimum adsorption equilibrium time is around 6 h. The amount of enzyme adsorbed on CM-CADB resin affects the response to substrate L-glutamic acid, the widest range of linearity is obtained with over 30 mg (GDC)/g(resin). The GDC activity immobilized on CM-CADB reaches a maximum when the immobilization temperature was kept around 40 degrees C. pH was kept at 4.4 when measuring the activity of the immobilized GDC. No variation of the activity of immobilized GDC is observed when the capacity is over 2.5 meq/g.(CM-CADB resin). The properties of the immobilized enzyme on CM-CADB were characterized. No significant improvement can be achieved when the substrate concentration exceeds 12.00 mmol/l, where the activity of immobilized GDC is equal to 1.58 mmol/l.min.g. The optimum pH is found to be 5.2, which changes 0.2 unit, comparing with that of the free GDC (5.0). The optimum temperature is found to be around 48 degrees C, which is lower than that of free GDC (55 degrees C). The critical temperature of the free GDC and the immobilized GDC is approximately 50 degrees C and 45 degrees C, respectively. The half-life of the activity is 127 days when the immobilized enzyme was stored in the cold (4 degrees C). An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO(2) electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamic acid. The determination conditions are that the buffer solution is 0.10 M Na(2)HPO(4)-0.05 M citric acid at pH 4.4 and t = 37 degrees C. The limit of detection is 1.0 x 10(-)(5) M. The linearity response is in the range of 5 x 10 (-2) - 5 x 10 (-5) M. The equation of linear regression of the calibration curve is y = 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamic acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

Journal Article↗

PubMed2000

Nuclear localization and intramolecular cleavage of N-terminally deleted NS5A protein of hepatitis C virus.

The full-size NS5A (NS5A-F) of hepatitis C virus is localized in the cytoplasm despite the presence of a functional nuclear localization signal (NLS) in its C-terminal region (amino acids (aa) 354-362). In the present study, we demonstrated that a short stretch of sequence near the N-terminus of NS5A (aa 27-38) masked the functional NLS, preventing NS5A from being transported to the nucleus. This sequence, referred to as an NLS-masking sequence, was distinct from a nuclear export signal, as it did not actively target a protein to the cytoplasm. We also found that other sequences located at either an N- (aa 1-21) or a C-terminal region (aa 353-447) were responsible for targeting NS5A to the cytoplasm. Western blot analysis of the transfected cells revealed that NS5A mutants that had been N-terminally deleted by 66 aa or more were cleaved at a certain cleavage site, generating a common fragment of ca. 40 kDa. This result implies the possible presence of a cleavage site in the NS5A sequence around aa 150, which is exposed through conformational alteration upon the N-terminal deletions.

Animals↗

PubMed2000

Inhibition of nuclear factor-kappab activation in mouse macrophages and the RAW 264.7 cell line by a synthetic adenyl carbocyclic nucleoside.

Adenyl carbocyclic nucleosides have potent anti-inflammatory effects on a number of cell types. Notable in this regard is their ability to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) by mouse macrophages that have been activated with bacterial lipopolysaccharide (LPS). Because the transcriptional activation of the mouse TNF-alpha gene is highly dependent on kappaB enhancers, the present study determined whether the synthetic carbocyclic nucleoside 9-[(1S,3R)-cis-cyclopentan-3-ol]adenine (cPA) inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation in these cells. Stimulation of either mouse peritoneal macrophages or RAW 264. 7 macrophage-like cells with LPS led to the appearance of four distinct kappaB-binding nucleoprotein complexes detected by gel mobility shift assays. Cells treated with 100 microM cPA showed significantly reduced levels of NF-kappaB activation as evidenced by measurements of nucleoprotein kappaB-binding activity and diminished kappaB-dependent transcriptional activation. However, both the LPS-induced degradation of the cytoplasmic NF-kappaB inhibitor IkappaBalpha and the nuclear translocation of the NF-kappaB p50, p65, and c-Rel peptides were unaffected by treatment of the cells with the nucleoside. These findings suggest that certain adenyl carbocyclic nucleosides inhibit the activation of NF-kappaB/Rel complexes by a novel mechanism that results in an inhibition of their DNA-binding activities, without blocking their dissociation from IkappaBalpha or their nuclear translocation.

Adenine↗

PubMed2000

Cyclin-dependent kinases as a therapeutic target for stroke.

Cyclin-dependent kinases (CDKs) are commonly known to regulate cell proliferation. However, previous reports suggest that in cultured postmitotic neurons, activation of CDKs is a signal for death rather than cell division. We determined whether CDK activation occurs in mature adult neurons during focal stroke in vivo and whether this signal was required for neuronal death after reperfusion injury. Cdk4/cyclin D1 levels and phosphorylation of its substrate retinoblastoma protein (pRb) increase after stroke. Deregulated levels of E2F1, a transcription factor regulated by pRb, are also observed. Administration of a CDK inhibitor blocks pRb phosphorylation and the increase in E2F1 levels and dramatically reduces neuronal death by 80%. These results indicate that CDKs are an important therapeutic target for the treatment of reperfusion injury after ischemia.

Animals↗

PubMed2000

Differential modulation of proliferation in the neocortical ventricular and subventricular zones.

Recent studies have implicated the classical neurotransmitters GABA and glutamate in the regulation of neural progenitor proliferation. We now show that GABA and glutamate have opposite effects on the two neural progenitor populations in the ventricular zones (VZs) and subventricular zones (SVZs) of the embryonic cerebrum. Application of either molecule to organotypic slice cultures dramatically increases proliferation in the VZ by shortening the cell cycle, whereas proliferation in the SVZ is decreased. These disparate effects, measured both by bromodeoxyuridine uptake and the expansion of retrovirally labeled progenitor clones, are mimicked by the application of specific GABA and glutamate agonists and are blocked by antagonists. Thus, the relative contributions of the VZ and SVZ to neocortical growth may be regulated by differential responsiveness to GABA and glutamate.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2000

Modelling the cumulative risk for a false-positive under repeated screening events.

Screening examinations are widely utilized in detecting the presence of medical disorders, for instance, screening mammograms and clinical breast examinations for detection of breast cancer. Such procedures are invaluable in enabling early treatment but produce the possibilities of false-positive and false-negative diagnoses. Focusing on false-positive results, with increasing number of screening events, it is clear that the risk of a false-positive increases. The objective of this paper is to quantify the cumulative risk associated with repeated screening. We provide a very general framework within which to investigate this risk, both at the population and the individual level. The latter allows incorporation of evolving patient medical history to permit individualized assessment of risk. We model cumulative risk in terms of the number of screening events until first false-positive. We develop models which are essentially familiar actuarial models for life table data adding a Cox regression to enable individual level modelling. Because it offers several advantages, we employ a Bayesian inference framework and apply our modelling to the analysis of 9773 screening mammograms collected from 2227 women at an HMO serving nearly 300000 adults in and around Boston, MA.

Adult↗

PubMed2000

Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor alpha and Sp3 proteins.

Treatment of HEC1A endometrial cancer cells with 10 nm 17beta-estradiol (E2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA expression, and a similar response was observed using a construct, pVEGF1, containing a VEGF gene promoter insert from -2018 to +50. In HEC1A cells transiently transfected with pVEGF1 and a series of deletion plasmids, it was shown that E2-dependent down-regulation was dependent on wild-type estrogen receptor alpha (ERalpha) and reversed by the anti-estrogen ICI 182, 780, and this response was not affected by progestins. Deletion analysis of the VEGF gene promoter identified an overlapping G/GC-rich site between -66 to -47 that was required for decreased transactivation by E2. Protein-DNA binding studies using electrophoretic mobility shift and DNA footprinting assays showed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter. Coimmunoprecipitation and pull-down assays demonstrated that Sp3 and ERalpha proteins physically interact, and the interacting domains of both proteins are different from those previously observed for interactions between Sp1 and ERalpha proteins. Using a dominant negative form of Sp3 and transcriptional activation assays in Schneider SL-2 insect cells, it was confirmed that ERalpha-Sp3 interactions define a pathway for E2-mediated inhibition of gene expression, and this represents a new mechanism for decreased gene expression by E2.

Base Sequence↗

PubMed2000

Complex formation between hepatitis C virus core protein and p21Waf1/Cip1/Sdi1.

The core protein (Core) of hepatitis C virus (HCV) has been known to play an important role in hepatocarcinogenesis. By using glutathione S-transferase (GST) pull-down assay, we show here that Core formed a complex with p21Waf1/Cip1/Sdi1 (p21) cell cycle regulator. The deletion-mapping analysis revealed that a portion near the N-terminus of Core (amino acids 24-52) and a C-terminal portion of p21 (amino acids 139-164) were involved in the complex formation. The complex formation was not impaired by point mutations of p21 at residues 147, 149, and 150, which have been reported to abrogate interaction of p21 with proliferating cell nuclear antigen (PCNA), discriminating the Core-binding sequence from the PCNA-binding sequence. Due to the close vicinity of the binding sites, however, Core and PCNA competed with each other when interacting with p21. The distinct interaction between Core and p21 may provide a new aspect to the studies of HCV pathogenesis.

Base Sequence↗

PubMed2000

Determination of neurotoxin 3-N-oxalyl-2,3-diaminopropionic acid and non-protein amino acids in Lathyrus sativus by precolumn derivatization with 1-fluoro-2,4-dinitrobenzene.

A rapid and simple method is presented for determining neuro-excitatory nonprotein amino acid 3-N-oxalyl-2,3-diaminopropionic acid (beta-ODAP) and non-protein amino acids in Lathyrus sativus. Seed and foliage extracts of Lathyrus sativus were treated with 1-fluoro-2,4-dinitrobenzene (FDNB) and a reversed-phase high-performance liquid chromatography method (RP HPLC) for the separation of the derivatives in the pmol range is reported. The RP HPLC method and a colorimetric method were compared for measuring ODAP.

Amino Acids↗

PubMed2000

Population genetic implications from sequence variation in four Y chromosome genes.

Some insight into human evolution has been gained from the sequencing of four Y chromosome genes. Primary genomic sequencing determined gene SMCY to be composed of 27 exons that comprise 4,620 bp of coding sequence. The unfinished sequencing of the 5' portion of gene UTY1 was completed by primer walking, and a total of 20 exons were found. By using denaturing HPLC, these two genes, as well as DBY and DFFRY, were screened for polymorphic sites in 53-72 representatives of the five continents. A total of 98 variants were found, yielding nucleotide diversity estimates of 2.45 x 10(-5), 5. 07 x 10(-5), and 8.54 x 10(-5) for the coding regions of SMCY, DFFRY, and UTY1, respectively, with no variant having been observed in DBY. In agreement with most autosomal genes, diversity estimates for the noncoding regions were about 2- to 3-fold higher and ranged from 9. 16 x 10(-5) to 14.2 x 10(-5) for the four genes. Analysis of the frequencies of derived alleles for all four genes showed that they more closely fit the expectation of a Luria-Delbrück distribution than a distribution expected under a constant population size model, providing evidence for exponential population growth. Pairwise nucleotide mismatch distributions date the occurrence of population expansion to approximately 28,000 years ago. This estimate is in accord with the spread of Aurignacian technology and the disappearance of the Neanderthals.

Aged↗

PubMed2000

Ligand binding properties of binary complexes of heparin and immunoglobulin-like modules of FGF receptor 2.

Epithelial cells, which express FGFR2IIIb, bind and respond to FGF-1, FGF-7 and FGF-10, but not FGF-2. Stromal cells, which bind and respond to FGF-1 and FGF-2, but not FGF-7 and FGF-10, express FGFR2IIIc or FGFR1IIIc. Here we show that when both isolated FGFR2betaIIIb and FGFR2betaIIIc or their common Ig module II are allowed to affinity select heparin from a mixture, the resultant binary complexes bound FGF-1, FGF-2, and FGF-7 with nearly equal affinity. In addition, FGF-2 and FGF-7 bound to both heparin-Ig module IIIb and IIIc complexes, but FGF-1 bound to neither Ig module III. The results show that in isolation both Ig modules II and III of FGFR2 can interact with heparin and that each exhibits a binding site for FGF. We suggest that the specificity of FGFR2IIIb and FGFR2IIIc is dependent on the cell membrane environment and heparin/heparan sulfate. Ig modules II and III cooperate both within monomers and across dimers with cellular heparan sulfates to confer cell type-dependent specificity of the FGFR complex for FGF.

Amino Acid Sequence↗

PubMed2000

Two-headed binding of a processive myosin to F-actin.

Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation.

Actins↗

PubMed2000

Solid phase syntheses of polyamine toxins HO-416b and PhTX-433. Use of an efficient polyamide reduction strategy that facilitates access to branched analogues.

[structure--se text] Polyamine toxins HO-416b (1) and PhTX-433 (2) isolated from the venom of insects are important lead compounds in neuropharmacology. Their total synthesis has been achieved on a trityl derivatized resin in good yield and purity using a mild borane reduction protocol to access the polyamine chains from polyamide precursors. The synthesis of PhTX isomer 3 demonstrates the potential of this strategy for the generation of branched analogues.

Indoles↗

PubMed2000

[Mechanism of impairment to microtubule polymerization resulting from zinc deficiency during pregnancy and lactation in mice].

To probe into the mechanism of zinc deficiency on microtubule polymerization impairment, the learning ability and the levels of alpha-tubulin, beta-tubulin and microtubule-associated protein 2 expression in the brain of zinc deficient offsprings, maternal ICR mice were fed with experiment diets containing different levels of zinc(1, 5, 30 and 100 mg/kg) during pregnancy and lactation respectively. On the postnatal day 70 of offsprings, the learning ability and the expression of alpha-tubulin, beta-tubulin and microtubule-associated protein 2 in the brain were examined by shuttle box and Western blot assays respectively. The results showed that the number of trials needed to reach the learning criterion for zinc deficient groups (1 and 5 mg/kg) was much higher than that for non zinc deficient groups (30 and 100 mg/kg). The levels of alpha-tubulin, beta-tubulin and microtubule-associated protein 2 expression in the brain of zinc deficient offsprings (1 and 5 mg/kg) were lower than those in the brain of offsprings whose dams fed with zinc adequately supplied diet (30 mg/kg) and zinc supplemented supplied diet (100 mg/kg) respectively. These results suggested that the inhibition of alpha-tubulin, beta-tubulin and microtubule-associated protein 2 expression might be the most important mechanism of microtubule polymerization decline resulting from zinc deficiency, which had close relationship with brain function impairment.

Animals↗