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Biomedical subjects

S Yao

Publications and source records attributed to S Yao.

At least 19 recordsLinked to original sources
PubMed1998

NS398, a selective cyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells.

Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins and other eicosanoids from arachidonic acid, is constitutively expressed in LNCaP human prostate cancer cell line. To evaluate the potential role of COX-2 in prostate cancer, LNCaP cells were treated with NS398, a selective COX-2 inhibitor, and the effects on cell viability and apoptosis were determined. NS398 treatment induced apoptosis in LNCaP cells in a time- and dose-dependent fashion. Treatment with 100 microM NS398 caused a down-regulation in bcl-2 protein expression, followed by chromatin condensation, chromosomal DNA fragmentation, and changes in nuclear morphology detected by 4,6-diamidino-2-phenylindole staining, DNA fragmentation assay, and terminal deoxynucleotidyl transferase-mediated UTP-biotin nick end-labeling assay. In contrast, NS398 treatment had no effect on either cell viability or nuclear function and morphology in human fetal prostate fibroblasts. These results demonstrate that NS398 induces apoptosis in LNCaP cells but not in human fetal prostate fibroblasts, and that this induction is associated with a decreased level of bcl-2 protein.

Apoptosis↗
PubMed1998

Topotecan lactone selectively binds to double- and single-stranded DNA in the absence of topoisomerase I.

We report the first experimental observation that a clinically important camptothecin [CPT; topotecan (TPT), a water-soluble CPT] binds directly and noncovalently to double-stranded DNA and single-stranded DNA structures in the absence of topoisomerase I, but only in the lactone form. We observed clear DNA sequence specificity of the TPT lactone binding to duplex DNA, which was comprised of alternating purine-pyrimidine sequences that contained dT. These structural studies of direct TPT lactone-DNA binding support several important considerations involving possible mechanism(s) of anticancer activity of CPT-type drugs containing a 20(S) lactone moiety.

Antineoplastic Agents↗
PubMed1998

Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa.

Usher syndrome type IIa (OMIM 276901), an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa, maps to the long arm of human chromosome 1q41 between markers AFM268ZD1 and AFM144XF2. Three biologically important mutations in Usher syndrome type IIa patients were identified in a gene (USH2A) isolated from this critical region. The USH2A gene encodes a protein with a predicted size of 171.5 kilodaltons that has laminin epidermal growth factor and fibronectin type III motifs; these motifs are most commonly observed in proteins comprising components of the basal lamina and extracellular matrixes and in cell adhesion molecules.

Amino Acid Sequence↗
PubMed1998

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest.

Vpr, one of the accessory gene products encoded by HIV-1, is a 96-residue protein with a number of functions, including targeting of the viral pre-integration complex to the nucleus and inducing growth arrest of dividing cells. We have characterized by 2D NMR the solution conformations of bioactive synthetic peptide fragments of Vpr encompassing a pair of H(F/S)RIG sequence motifs (residues 71-75 and 78-82 of HIV-1 Vpr) that cause cell membrane permeabilization and death in yeast and mammalian cells. Due to limited solubility of the peptides in water, their structures were studied in aqueous trifluoroethanol. Peptide Vpr59-86 (residues 59-86 of Vpr) formed an alpha-helix encompassing residues 60-77, with a kink in the vicinity of residue 62. The first of the repeated sequence motifs (HFRIG) participated in the well-defined alpha-helical domain whereas the second (HSRIG) lay outside the helical domain and formed a reverse turn followed by a less ordered region. On the other hand, peptides Vpr71-82 and Vpr71-96, in which the sequence motifs were located at the N-terminus, were largely unstructured under similar conditions, as judged by their C(alpha)H chemical shifts. Thus, the HFRIG and HSRIG motifs adopt alpha-helical and turn structures, respectively, when preceded by a helical structure, but are largely unstructured in isolation. The implications of these findings for interpretation of the structure-function relationships of synthetic peptides containing these motifs are discussed.

Amino Acid Sequence↗
PubMed1998

Improved Estimation of Protein Rotational Correlation Times from 15N Relaxation Measurements

In the study of protein backbone dynamics by 15N relaxation measurements, an initial estimation of the isotropic global correlation time, taum, is usually obtained from the average T1/T2 ratio of nuclear spins that do not exhibit slow internal motion and with T2 values not significantly shortened by chemical or conformational exchange processes. Different methods have been used for identification of the rates of internal motion. However, the number of nuclear spins included in the taum estimation is often larger than the number that ultimately can be fitted to a single-order parameter, S2, implying that some nuclear spins involved in the initial taum estimation actually have an effective internal correlation time, taue, not as fast as assumed. As a consequence, taum is underestimated, since internal motion reduces the T1/T2 ratio. This situation becomes more obvious if the molecule has a large taum value because the reduction in T1/T2 ratio arising from internal motion is more significant than for molecules with smaller taum and the same degree of internal motion. This Communication describes a more reliable method for identifying nuclear spins which should be excluded from the taum estimation because of insufficiently rapid internal motion. This results in an improved taum value, giving a much better agreement between the number of nuclear spins fitted successfully to a single-order parameter, S2, and those used in the taum estimation. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

A rapid method for determination of in vitro susceptibility to antibiotics with a bulk acoustic wave bacterial growth biosensor.

A novel bulk acoustic wave (BAW) bacterial growth biosensor was developed to study in vitro susceptibility by continuous monitoring of disturbances of bacterial growth at low antibiotic concentrations, followed by the accurate and rapid estimation of growth kinetic parameters and minimum inhibitory concentrations (MICs). The susceptibilities of bacteria, e.g. Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Pr. morganii and Pr. mirabilis, to various antibiotics, e.g. penicillin, streptomycin, gentamicin and cefotaxime, were investigated, respectively, and the MICs were rapidly determined with a higher reproducibility than the conventional broth micro-dilution technique (BMDT). The effects of cell constant of conductivity electrode, pH and temperature on bacterial growth and biosensor signals were discussed in detail. The proposed method offers an effective alternative to the conventional methods.

Acoustics↗
PubMed1998

Conformational and topological requirements of cell-permeable peptide function.

Cell-permeable peptide import recently was developed to deliver synthetic peptides into living cells for studying intracellular protein functions. This import process is mediated by an N-terminal carrier sequence which is the hydrophobic region of a signal peptide. In this study, the conformational consequence of the interaction of cell-permeable peptides with different mimetic membrane environments was investigated by circular dichroism analysis. We showed that cell-permeable peptides adopted alpha-helical structures in sodium dodecyl sulfate (SDS) micelles or aqueous trifluoroethanol (TFE). The potency of these peptides in forming helical structures is higher in an amphiphilic environment (SDS) than in a hydrophobic environment (TFE), suggesting that some hydrophilic molecules associated with the cell membrane may be involved in peptide import. We also studied topological requirements of cell-permeable peptide function. We demonstrated that peptides containing the carrier sequence in their C-termini can also be imported into cells efficiently. This important discovery can avoid repetitious synthesis of the membrane-translocating sequence for peptides with different functional cargoes and is potentially useful for developing a cell-permeable peptide library. Finally, we showed that, when a retro version of the carrier sequence was used, the peptide lost its translocating ability despite retaining a high content of alpha-helical structure in mimetic membrane environments. This suggests that the propensity of peptides to adopt a helical conformation is required but not sufficient for cellular import and that other structural factors such as the side-chain topology of the carrier sequence are also important. Our studies together contribute to the more rational design of useful cell-permeable peptides.

3T3 Cells↗
PubMed1998

Modulation of platinum-induced toxicities and therapeutic index: mechanistic insights and first- and second-generation protecting agents.

Platinum-type drugs have proven to be valuable in the treatment of a variety of solid tumors, beginning with the commercial approval of cisplatin 18 years ago. There are several clinically important toxicities commonly associated with the administration of these drugs. Despite the extensive use of cisplatin and carboplatin, the fundamental chemical transformations and mechanisms that underlie their antitumor and toxic effects have not been fully characterized. Several first-generation protective thiols have been clinically studied in an attempt to reduce the toxicity of platinum-type drugs; while some of these agents appear to protect against certain toxicities, nearly all platinum-protecting drugs have their own intrinsic toxicities, which can be additive to the toxicity of platinum-type drugs. Tumor protection by platinum-protecting drugs is an additional untoward effect that is associated with certain types of agents and must be addressed with care. Recent advances in theoretical and laboratory methods and the use of supercomputers have extended our understanding of the possible major mechanisms underlying platinum drug antitumor activity and toxicity; we present strong evidence that there are two classes of chemical species of platinum drug. One class appears to predominantly account for the antitumor activity, and the other class of chemical species produces many of the toxic effects of platinum drugs. We have discovered a new nontoxic, second-generation platinum-protecting agent, known as BNP7787, which appears to selectively inactivate and eliminate toxic platinum species. BNP7787 has recently entered phase I clinical testing in cancer patients.

Amifostine↗
PubMed1997

An alternative to phosphotyrosine-containing motifs for binding to an SH2 domain.

Shc is an important signalling protein whose overexpression leads to cell transformation in NIH 3T3 fibroblasts. Although the formation of Shc/Grb2 complexes involving Shc tyrosine residue 317 is necessary to induce this transformation, the Shc proteins in these Shc-overexpressing cells are not substantially tyrosine-phosphorylated. This observation led to our hypothesis that the non-phosphorylated Tyr317-containing region of Shc might have specific affinity for the Grb2 protein. We show here that cell-permeable peptides encompassing the Shc Tyr317 region, 312FDD-PSYVNVQNL323, can bind to the SH2 domain of Grb2 regardless of the state of tyrosine phosphorylation. When delivered into cells, both phosphorylated and non-phosphorylated Shc peptides inhibit growth factor-induced Shc/Grb2 protein-protein interaction. The non-phosphorylated Shc peptides with single point mutations at Asp313, Asp314, or Tyr317 are inactive, suggesting that these residues play an important role in Grb2 protein recognition. Our findings represent the first paradigm of the specific interaction between an unphosphorylated tyrosine-containing region and an SH2 domain and have important implications for understanding the mechanism of cell transformation by Shc overexpression.

3T3 Cells↗
PubMed1997

An investigation of the fluidity of concentration polarisation layers in crossflow membrane filtration of an oil-water emulsion using chemical shift selective flow imaging.

A chemical shift selective NMR flow imaging sequence using stimulated echoes for data acquisition is presented. The sequence was tested using a 20% (vol/vol) oil-water emulsion formed from a soluble cutting oil, which was passed through a simple flow phantom to yield two-dimensional velocity distribution maps of the oil droplets and of the water separately. It was then used to investigate the fluidity of concentration polarisation layers formed from the oil droplets during crossflow membrane filtration of a 5% (vol/vol) emulsion of the same cutting oil. A simple membrane filtration module was used for this purpose, with the feedstock emulsion fed into the lumen of a single tubular membrane at a trans-membrane pressure difference P approximately 70 kPa and crossflow Reynolds number, Re, in the range 100-1000. The results confirm a net axial flow rate < 7.5 microns/s (half digital resolution in the velocity dimension) for the oil polarisation layer. Under these conditions, the upper limit for oil flow tangential to the membrane in the polarised layer is less than 15% of the convective flow of oil towards the membrane.

Emulsions↗
PubMed1997

Detection and analysis of the growth characteristics of Proteus vulgaris with a bulk acoustic wave ammonia sensor.

A bulk acoustic wave ammonia sensor to monitor the growth of Proteus vulgaris is described. The corresponding bacterial growth response equation was derived based on the basic growth characteristics of P. vulgaris, and was verified experimentally. The application of the equation to the estimation of kinetic parameters (such as the asymtote, the maximum specific growth rate and the lag time) was accomplished successfully. The generation time is 33.3 min at 37 degrees C. The influence of temperature on bacterial growth was also investigated and the activation energy of P. vulgaris growth was estimated. Furthermore, the initial bacterial concentrations were determined based on the linear relationship between the inflection time, ti, and the logarithm of the initial bacterial concentration in the range 10(2)-10(7) (cells ml-1). The results were in good agreement with those from the pour plate count method and the precision was better than that of the frequency detection time method.

Acoustics↗
PubMed1996

Controlling epidermal growth factor (EGF)-stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor.

Epidermal growth factor (EGF)-stimulated Ras activation involves specific interactions between the EGF receptor (EGFR), the adaptor proteins Grb2 and Shc, and the nucleotide exchange factor Sos-1. Study and control of these protein-protein interactions in vivo can be greatly promoted by introducing intracellular reagents that mimic EGFR functions. Here, we showed that a synthetic phosphopeptide encompassing the autophosphorylation site 1068 of EGFR formed a complex with endogenous Grb2 after this peptide was delivered into intact cells by a cell-permeable peptide import technique. Consequently, this intracellular peptide inhibited EGF-induced EGFR/Grb2 associations but not EGFR/Shc or Shc/Grb2 associations. Peptide-mediated disruption of the EGF/Grb2/Sos-1 cascade led to reduced Ras activation and mitogen-activated protein kinase activation. These results indicate that the binding of Grb2 to the phosphorylated Tyr-1068 of EGFR is crucial to the EGF-induced Ras/mitogen-activated protein kinase signaling pathway. The application of cell-permeable peptides to this study demonstrates a useful biochemical tool to probe and control various intracellular processes involved in signal transduction and gene transcription.

3T3 Cells↗
PubMed1996

Similar conformations of hairpins with TTT and TTTT sequences: NMR and molecular modeling evidence for T.T base pairs in the TTTT hairpin.

The conformations of the d[G(1)C(2)G(3)C(4)-T(a)T(b)T(c)T(d)-G(5)C(6)G(7)C(8)] (T4) and d[G(1)C(2)G(3)C(4)-T(a)T(b)T(c)-G(5)C(6)G(7)C(8)] (T3) DNA hairpins have been studied. The 1H and 31P signals of the two hairpins have been nearly completely assigned by means of two-dimensional NMR spectroscopy in D2O (NOESY (two-dimensional nuclear Overhauser effect and exchange spectroscopy) at mixing times of 5, 50, 100, 300 and 500 ms, double-quantum-filtered correlation spectroscopy (DQF-COSY) and 1H-31P reverse chemical shift correlation (RCSC), and one-dimensional NOE spectra in 90% H2O. Conformational analysis using distance geometry (DG), molecular mechanics (MM) and molecular dynamics (MD) gave model conformations, which were evaluated by comparison of experimental and simulated 2D NOESY spectra. For the T4 sequence in T4, both NMR data and modeling indicated a T(a).T(d) wobble base pair. Although two types of T(a).T(d) base pairs are possible, the one with T(a)NH-T(d)O4 and T(a)O2-T(d)NH H-bonds was calculated to be more stable. Because the T(a).T(d) base pair of T4 extends the stem, there are only two residues (T(b) and T(c) in the loop. Although there are three residues in the T3 loop, the T(c) base projects into the solvent. The resulting conformational models have very similar loop folding patterns (FP): the bases of the two adjacent residues that begin the loop [T(b)T(c) of T4 and T(a)T(b) or T3] have a minor groove/major groove orientation with the first residue each having a trans alpha torsion angle; and the phosphodiester group that links the residues at the 3' end of the loop and the 5' top of the stem [T(c)pT(d) of T4 and T(c)pG(5) of T3] has a gauche+, gauche+ zeta,alpha conformation with a trans gamma angle for the second residue in both. These or similar features appear to be present in most of the few other hairpins studied previously by conformational methods. Thus, we believe that the conformations of the loops in T3 and T4 hairpins have greater similarities than previously recognized.

Base Composition↗
PubMed1996

An oligodeoxyribonucleotide N3'--> P5' phosphoramidate duplex forms an A-type helix in solution.

The solution conformations of the dinucleotide d(TT) and the modified duplex d(CGCGAATTCGCG)2 with N3'--> P5' phosphoramidate internucleoside linkages have been studied using circular dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex conformation is similar to that of isosequential phosphodiester RNA, a A-type helix, and is different from that of DNA, a B-type helix, NMR studies of model dimers d(TpT) and N3'--> P5' phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2'-endo to C3'-endo when the 3'-phosphoester is replaced by a phosphoramidate group. Two-dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide phosphoramidate and confirm that all furanose rings of 3'-aminonucleotides adopt predominantly N-type sugar puckering.

Base Sequence↗
PubMed1996

A new urea sensor based on combining the surface acoustic wave device with urease extracted from green soya bean and its application--determination of urea in human urine.

The urea sensor was prepared by combining a surface acoustic wave (SAW) device, in which a SAW resonator operating at 61 MHz and a pair of parallel electrodes were used in series, with urease extracted from green soya bean. The Michaelis constant and maximum reaction rate of the urease were estimated as 2.14 mM and 27.18 kHz min-1, respectively, at pH 7.0 and 25.0 degrees C. Influences of pH, temperature and effectors on the response properties of the SAW urea sensor were investigated. Recovery of the sensor ranged from 95 to 105% and the detection limit of urea was 1.0 micrograms ml-1 (1.7 x 10(-5) M). The proposed sensor has been successfully applied to the rapid determination of urea in human urine samples. The results are consistent with the reported values and also support the clinical diagnosis.

Acoustics↗
PubMed1996

Behaviour of surface acoustic wave interdigitated array electrode sensor in non-aqueous solution and determination of blood plasma recalcification time.

A novel SAW-IDA sensor system was constructed for the first time by connecting the IDA electrodes in series with a SAW resonator. The frequency characteristics of the SAW-IDA sensor in non-aqueous solution were investigated. The effects of the parallel capacitance and cell constant were studied, and were calculated with the circuit network theory. These calculations provide guiding rules for design of the SAW-IDA sensor system. The SAW-IDA sensor was applied to determination of recalcification time and activated partial thromboplastin time of blood plasma. The sensor offers a new and effective way of studying clinical and laboratory haemostasis.

Acoustic Stimulation↗
PubMed1996

Repair of dGMP hydroxyl radical adducts by verbascoside via electron transfer: a pulse radiolysis study.

The repair activity of verbascoside (VER), isolated from Pedicularis spicata, towards the oxidizing hydroxyl radical adduct of dGMP and its reaction mechanism were studied using pulse radiolysis. Upon pulse radiolysis of nitrous oxide saturated aqueous solution of 2'-deoxyguanosine-5'-monophosphate (dGMP) and VER, it was found that the transient absorption spectrum of the hydroxyl adduct of dGMP decays with the formation of that of the phenoxyl radical of VER, several tens of microseconds after the electron pulse. From the formation kinetics of the phenoxyl radical of VER, the rate constant of the repair reaction was determined to be 1.12 x 10(9) dm(3) mol(-1) s(-1).

Antioxidants↗