Biomedical subjects

Y Zhu

Publications and source records attributed to Y Zhu.

At least 19 recordsLinked to original sources

PubMed1995

Thrombin-bound structures of designed analogs of human fibrinopeptide A determined by quantitative transferred NOE spectroscopy: a new structural basis for thrombin specificity.

The mutation of Gly12 to Val12 in the A alpha chains of human fibrinogen Rouen is associated with a delayed proteolytic release of fibrinopeptide A (FpA or A alpha 1 to 16 of fibrinogen) by thrombin, leading to a bleeding disorder. Analogs of FpA and FpA Rouen have been designed that include a Pro15 to replace Val15 in natural FpA and to mimic the frequent occurrences of a proline residue at equivalent positions of other protein substrates of thrombin. The Pro15 analogs of FpA and FpA Rouen bind specifically to the active site of thrombin as shown by thrombin-induced differential line broadening and transferred nuclear Overhauser effects (transferred NOEs). Pro15 is well tolerated by the thrombin-bound structures of both FpA and FpA Rouen in solution, resulting in enhanced conformational stabilities of the thrombin-FpA complexes. The Val12 mutation in FpA Rouen causes backbone conformational changes in residues Val12 and Gly13 accompanied by an expansion of the hydrophobic cluster of FpA to accommodate the bulky side-chain of Val12. The single turn of helical structure between residues Asp7 and Glu11 is stabilized by hydrogen bonds from the side-chain carboxylate of Asp7 to the exposed backbone NH groups of Ala10 and/or Leu9 (N-capping), and by hydrogen bonds between the exposed backbone carbonyl groups of residues Phe8 and Leu9, and the backbone NH groups of Gly12/Val12 and Gly13 (C-capping). The bound structure of FpA Rouen may be further stabilized by a non-polar (i,i + 4) interaction between the aromatic side-chain of Phe8 and the aliphatic side-chain of Val12. Despite these optimized intrapeptide interactions, the thrombin-peptide interactions are highly dynamic as indicated by the fast rate of dissociation (koff > 100 s-1) of the peptide ligands from the thrombin complexes. Sequence comparison between mammalian fibrinopeptides A and B suggests that the specificity of thrombin is dictated by a four-residue consensus motif, Phe(P4)-Xxx(P3)-Pro(P2)-Arg(P1) or FXPR, when Xxx at P3 can be a charged or a neutral polar residue capable of specific interactions with residues near the active site of thrombin.

Amino Acid Sequence↗

PubMed1995

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

Amino Acid Sequence↗

PubMed1995

Structural organization of mouse peroxisome proliferator-activated receptor gamma (mPPAR gamma) gene: alternative promoter use and different splicing yield two mPPAR gamma isoforms.

To gain insight into the regulation of expression of peroxisome proliferator-activated receptor (PPAR) isoforms, we have determined the structural organization of the mouse PPAR gamma (mPPAR gamma) gene. This gene extends > 105 kb and gives rise to two mRNAs (mPPAR gamma 1 and mPPAR gamma 2) that differ at their 5' ends. The mPPAR gamma 2 cDNA encodes an additional 30 amino acids N-terminal to the first ATG codon of mPPAR gamma 1 and reveals a different 5' untranslated sequence. We show that mPPAR gamma 1 mRNA is encoded by eight exons, whereas the mPPAR gamma 2 mRNA is encoded by seven exons. Most of the 5' untranslated sequence of mPPAR gamma 1 mRNA is encoded by two exons, whereas the 5' untranslated sequence and the extra 30 N-terminal amino acids of mPPAR gamma 2 are encoded by one exon, which is located between the second and third exons coding for mPPAR gamma 1. The last six exons of mPPAR gamma gene code for identical sequences in mPPAR gamma 1 and mPPAR gamma 2 isoforms. The mPPAR gamma 1 and mPPAR gamma 2 isoforms are transcribed from different promoters. The mPPAR gamma gene has been mapped to chromosome 6 E3-F1 by in situ hybridization using a biotin-labeled probe. These results establish that at least one of the PPAR genes yields more than one protein product, similar to that encountered with retinoid X receptor and retinoic acid receptor genes. The existence of multiple PPAR isoforms transcribed from different promoters could increase the diversity of ligand and tissue-specific transcriptional responses.

Alternative Splicing↗

PubMed1995

Long-term (5-year) changes in HDL cholesterol in cardiac rehabilitation patients. Do sex differences exist?

BACKGROUND: It is unknown whether the benefits of a cardiac rehabilitation program on HDL cholesterol (HDL-C) are equally achieved in men and women. To study this, we compared changes in HDL-C and other lipids in a large group of men and women participating in a cardiac rehabilitation program for up to 5 years. METHODS AND RESULTS: We compared changes in HDL-C and other fasting lipids in 553 men and 166 women participating in a cardiac rehabilitation program at baseline and then annually for up to 5 years. Patients exercised 3 days a week at 70% to 85% of their maximum heart rate predetermined by a symptom-limited treadmill test. Aerobic capacity was estimated in metabolic equivalents (METs), and percent body fat was determined by skin-fold measurements. Baseline HDL-C, LDL cholesterol (LDL-C), and total cholesterol were significantly higher in women, whereas the ratio of total cholesterol to HDL-C was lower. Although both men and women showed an increase in HDL-C after 1 year (10% and 7%, respectively), only the women's level continued to increase over 5 years (20% versus 5% for men, P = .03). The sex difference in change in HDL-C remained after adjustment for age and smoking. A nonsignificant trend toward a greater change in HDL-C in women existed after adjustment for baseline percent body fat and estimated METs. The change in the ratio of total cholesterol to HDL-C was also more favorable in women, with a 38% decrease over 5 years compared with a 14% decrease in men (P = .01). Total cholesterol decreased by 20% in women and 8% in men (P = .001), whereas LDL-C dropped by 34% in women and 15% in men (P = .0001). There was no sex difference in change in triglycerides. CONCLUSIONS: Women with heart disease who participate in a cardiac rehabilitation program may achieve greater lipid benefits over longer periods of time than previously demonstrated in men.

Aged↗

PubMed1995

Study of dosimetric penumbra due to multileaf collimation on a medical linear accelerator.

PURPOSE: Investigations of dosimetric penumbra produced by multileaf collimation on a medical linear accelerator are presented. METHODS AND MATERIALS: Multileaf collimators (MLCs) can be designed with at least three different shaped leaf-end profiles: straight, divergent, and curved. Assessment of the dosimetric effects of the collimator edge profiles was implemented using a fast Fourier transform (FFT) convolution algorithm. Accelerator source intensity was considered to have a Gaussian distribution. The calculated dose profile, for a source-to-surface distance of 100 cm and at depth of 10 cm in a water phantom, was fitted to a penumbral-forming function from which the penumbral width between 80% and 20% of the central axis dose was obtained. RESULTS: Calculation performed at various field sizes showed that curved collimator leaf-end produces a wider penumbra than the diverging collimator leaf-side. Film measurements agreed with the calculations within an uncertainty of less than 2 mm. The effect of backup jaws for the MLC and of the lower pair of diverging diaphragms on dosimetric penumbra was also investigated. CONCLUSIONS: This study is useful for characterizing collimator edge effects and for optimizing new collimator designs.

Algorithms↗

PubMed1995

An effector site that stimulates G-protein GTPase in photoreceptors.

Heterotrimeric G-proteins mediate between receptors and effectors, acting as molecular clocks. G-protein interactions with activated receptors catalyze the replacement of GDP bound to the alpha-subunit with GTP. alpha-Subunits then modulate the activity of downstream effectors until the bound GTP is hydrolyzed. In several signal transduction pathways, including the cGMP cascade of photoreceptor cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with corresponding effectors. In the phototransduction cascade the GTPase activity of photoreceptor G-protein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. Here we characterize the stimulation of transducin GTPase by a set of 23 mutant phosphodiesterase gamma-subunits (PDE gamma) containing single alanine substitutions within a stretch of the 25 C-terminal amino acid residues known to be primarily responsible for the GTPase regulation. The substitution of tryptophan at position 70 completely abolished the acceleration of GTP hydrolysis by transducin in a complex with this mutant. This mutation also resulted in a reduction of PDE gamma affinity for transducin, but did not affect PDE gamma interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDE gamma to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDE gamma in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1995

Construction of a panel of transgenic mice containing a contiguous 2-Mb set of YAC/P1 clones from human chromosome 21q22.2.

Libraries of the entire human genome, or regions of the genome, have been made in bacteria, yeast, and somatic cells. We have expanded this strategy using overlapping YACs and P1s from human 21q22.2 (the Down syndrome region) to create a panel of transgenic mice containing DNA that encompasses this region of the human genome. Together the members of the in vivo library, each with a unique transgene (four YACs and four P1s), contain approximately 2 Mb of contiguous DNA. The integrity, stable inheritance, and expression of a coding sequence for each member of the YAC panel are demonstrated, and the uses of the panel are described.

Animals↗

PubMed1995

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase.

Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated Mr of 143,808. The deduced amino acid sequence of the E. coli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.

Amino Acid Sequence↗

PubMed1995

Erythropoietin-induced cellular differentiation requires prolongation of the G1 phase of the cell cycle.

Erythropoietin (EPO), like many other hematopoietic growth factors, can induce either growth or differentiation of hematopoietic cells. Little is known about the molecular basis of this cellular decision, in part because of a paucity of cell lines in which these two phenomena can be dissociated. Ectopic expression of the EPO receptor (EPO-R) in Ba/F3, a murine interleukin 3 (IL-3)-dependent progenitor cell line, confers EPO-dependent cell growth. In these cells (Ba/F3-EPO-R), EPO also induces beta-globin mRNA, a specific marker of erythroid differentiation. Here we show that the induction of erythroid differentiation by EPO requires a delay in cell growth and a prolongation of the (G1) phase of the cell cycle. Interestingly, this effect on G1 prolongation was concentration dependent. At low EPO concentrations (0.05-0.1 unit of EPO per ml; 1 pM EPO = 0.01 unit of EPO per ml), EPO prolonged G1 and induced differentiation; at high concentrations (0.5-10.0 units per ml), EPO shortened G1 and preferentially stimulated growth. IL-3 stimulated Ba/F3 growth but not differentiation at all growth factor concentrations ranging from 0.1 to 500 pM. Moreover, IL-3 suppressed EPO-induced beta-globin induction in a dose-dependent manner. This suppression correlated with the shortening of G1 by IL-3. Taken together, these data demonstrate distinct effects of EPO and IL-3 and a balance between erythroid growth and differentiation that is cell cycle dependent.

Animals↗

PubMed1995

A set of inter-Alu PCR markers for chromosome 21 generated from pulsed-field gel-fractionated NotI restriction fragments.

Genomic probes can be efficiently obtained for specific chromosomal regions by PCR amplification of gel slices containing fractionated restriction enzyme-cleaved DNA. Here, single-copy, human-specific DNA sequences were amplified using inter-Alu PCR on gel slices containing a NotI digest of DNA from hybrid cell line WAV17. Rodent cell line WAV17 contains human chromosome 21. About 75% of the 0.15- to 3-kb inter-Alu PCR products could be regionally assigned, en masse, by hybridization experiments using inter-Alu PCR probes generated from cell lines containing portions of chromosome 21. This work produced 10 new chromosome 21 markers that came from regions of 21q containing few useful markers. These markers were needed to finish a NotI restriction map for 21q (Wang and Smith (1994) Genomics 20: 441). This approach provides markers needed to close map gaps and for top-down mapping approaches.

Animals↗

PubMed1995

Aflatoxin B1, 2-aminoanthracene, and 7,12-dimethylbenz[a]anthracene-induced frameshift mutations in human APRT.

Aflatoxin B1, 2-aminoanthracene, and 7,12-dimethylbenz[a]anthracene have been implicated in the etiology of human cancers. In this study, we demonstrate that these three chemicals can be activated by rat liver homogenate S9 coupled with NADPH coenzymes to produce a dose-dependent increase in the frequency of APRT reversion in the APRT-deficient human cell line HTD114. HTD114 contains single nucleotide insertions at different positions in each APRT allele and the spontaneous reversion frequency is < 10(-8). However, the highest reversion frequency induced by these chemicals is 1.2-2.0 x 10(-5), at least a 10(3)-fold increase over the frequency of spontaneous reversion. Reversion of either mutant allele was observed to be a consequence of a frame-restoring loss of a single nucleotide, which indicates that these three chemicals can function as frameshift mutagens in human cells.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1995

Rapid screening of genetic polymorphisms using buccal cell DNA with detection by matrix-assisted laser desorption/ionization mass spectrometry.

A new approach is developed for the rapid and cost-effective detection of human genetic polymorphisms based on matrix-assisted laser description/ionization mass spectrometric (MALDI MS) detection using a nitrocellulose film substrate. This method employs polymerase chain reaction (PCR) amplification using DNA extracted from buccal cells as templates, followed by direct digestion with restriction enzymes and subsequent analysis by MALDI MS. The extraction of DNA from buccal cells provides a rapid and convenient means for sampling PCR-based diagnostic analysis. The amount of DNA was sufficient as the template for both normal PCR amplifications, and amplifications involving the use of mismatched primers and multiple primers. The MALDI MS methodology has been successfully used for the analysis of such PCR products where restriction fragments generated directly in PCR reactions have been used for detection of carbonic anhydrase and cystic fibrosis transmembrane conductance regulator as model genes. The detection of genetic polymorphisms following routine biological and clinical procedures with the MALDI MS method is demonstrated. The results from MALDI MS analysis are shown to be comparable to those obtained from gel electrophoresis but the MALDI MS method is several orders of magnitude faster than gel electrophoretic techniques. The method described herein should also be readily extended to other areas involving DNA screening and testing.

Base Sequence↗

PubMed1995

Interactions of TCR tyrosine based activation motifs with tyrosine kinases.

The tyrosine activating motif (TAM) is a conserved signaling motif present in many hematopoietic receptors. Although the exact definition and the function of these motifs is not known, it is likely that these motifs bind and activate protein tyrosine kinases. Here we summarize the data regarding tyrosine kinase interactions with the T cell receptor TAMs and integrate much of the information into a functional and testable model. We propose that phosphorylated TAMs are important for the activation of tyrosine kinases as well as for the recruitment of critical signaling molecules.

Amino Acid Sequence↗

PubMed1995

Mechanisms of transport of quinapril in Caco-2 cell monolayers: comparison with cephalexin.

PURPOSE: To determine the transport mechanisms of quinapril and cephalexin in Caco-2 cell monolayers, a cell culture model of the human small intestinal epithelium. METHODS: Uptake, transepithelial transport and intracellular accumulations of these two drugs were measured using Caco-2 cell monolayers grown onto Millicells and magnetically stirred diffusion chambers. RESULTS: Transepithelial transport, apical (AP)4 uptake and intracellular accumulation of both drugs depended on the maintenance of a transepithelial proton gradient and temperature of the medium. However, quinapril transport and accumulation, which did not display a maximum at approximately pH 6, was more sensitive to proton gradient change, whereas cephalexin transport was more sensitive to concentration change (range 0.5-5 mM). In addition, quinapril (1 mM) transport was decreased significantly (p < 0.05) by 10 mM cephalexin, loracarbef, Gly-Pro and Phe-Pro, but not by enalapril; whereas cephalexin (0.1 mM) transport was decreased significantly (p < 0.05) by all four compounds. Similarly, AP quinapril (1 mM) uptake was also decreased by 10 mM loracarbef, Gly-Pro, cephalexin, and enalapril, but these inhibitory effects (20-50%) were quantitatively less than their inhibitory effects on cephalexin uptake (50-90%). Finally, the AP uptake of quinapril was also significantly (p < 0.05) inhibited by FCCP (10 micrograms/ml), amiloride (0.5 mM), DEP (0.5 mM), and staurosporine (5 nM). CONCLUSIONS: The transport of quinapril in the Caco-2 cells is via a combination of the carrier-mediated proton gradient-dependent peptide transporter and passive diffusion.

Amino Acid Sequence↗

PubMed1995

Conformational analysis of molecular chains using nano-kinematics.

We present algorithms for 3-D manipulation and conformational analysis of molecular chains, when bond lengths, bond angles and related dihedral angles remain fixed. These algorithms are useful for local deformations of linear molecules, exact ring closure in cyclic molecules and molecular embedding for short chains. Other possible applications include structure prediction, protein folding, conformation energy analysis and 3D molecular matching and docking. The algorithms are applicable to all serial molecular chains and make no assumptions about their geometry. We make use of results on direct and inverse kinematics from robotics and mechanics literature and show the correspondence between kinematics and conformational analysis of molecules. In particular, we pose these problems algebraically and compute all the solutions making use of the structure of these equations and matrix computations. The algorithms have been implemented and perform well in practice. In particular, they take tens of milliseconds on current workstations for local deformations and chain closures on molecular chains consisting of six or fewer rotatable dihedral angles.

Algorithms↗

PubMed1995

A simple data transformation for estimating benchmark doses in developmental toxicity experiments.

Developmental anomalies induced by toxic chemicals may be identified using laboratory experiments with rats, mice or rabbits. Multinomial responses of fetuses from the same mother are often positively correlated, resulting in overdispersion relative to multinomial variation. In this article, a simple data transformation based on the concept of generalized design effects due to Rao-Scott is proposed for dose-response modeling of developmental toxicity. After scaling the original multinomial data using the average design effect, standard methods for analysis of uncorrelated multinomial data can be applied. Benchmark doses derived using this approach are comparable to those obtained using generalized estimating equations with an extended Dirichlet-trinomial covariance function to describe the dispersion of the original data. This empirical agreement, coupled with a large sample theoretical justification of the Rao-Scott transformation, confirms the applicability of the statistical methods proposed in this article for developmental toxicity risk assessment.

Abnormalities, Drug-Induced↗

PubMed1995

Accuracy requirements of the primary x-ray spectrum in dose calculations using FFT convolution techniques.

The photon beam dose calculation methods using fast Fourier transform (FFT) convolution require that the primary beam energy spectrum is accurately known; however, a quantitative assessment of the required accuracy is not yet provided. In this study, the sensitivity of various parameters such as central axis depth dose and dose under attenuators to the incident photon spectrum was simulated and evaluated for 6- and 18-MV photon beams. The central axis depth-dose data using the modified spectrum were compared with those calculated using the original spectrum. Maximum errors on the central axis between these two sets of depth-dose data were found by changing the weighting of each energy bin of the primary spectrum. The maximum change in percent depth dose is proportional to the change in average incident energy relating to the quality of the x ray. To keep the maximum depth-dose error to less than 1%, the relative change in average energy brought about by changes in single spectral bins should be less than 2%. The accuracy required for each energy bin varies dramatically, dependent on its deviation from the original average energy and its relative weighting. For example, for the 6-MV x-ray spectrum, the energy bins centered around 0.76 MeV (first bin) and 6.8 MeV (fifth bin) need to have accuracies of 5% and 40%, respectively, to obtain a 1% accuracy in percent depth dose.

Fourier Analysis↗

PubMed1995

Portal dosimetry using a liquid ion chamber matrix: dose response studies.

Current intensive investigations of electronic portal imaging devices (EPIDs) have prompted their potential application to portal dosimetry. In this paper, the progress made in using a commercial liquid ion chamber matrix EPID for portal dosimetry is discussed. The pixel value of the liquid ion chamber element was calibrated against dose by exposing the imager to 6-MV x-ray beams of various intensities obtained with various thicknesses of lead attenuators and a range of source to detector distances. Absolute dose values were determined using an ion chamber on the central axis at the depth of maximum dose in a solid water phantom. The pixel values of the matrix were determined for various field sizes in order to evaluate the dependence of pixel value on dose at those field sizes. It was confirmed that the pixel value was proportional to the square root of the dose rate and was nearly independent of the field size. The 2D pixel values were converted to 2D dose maps in the water phantom after applying a correction for the effect of horns in the flood calibration field. The flood calibration field was used to obtain the relative sensitivity of each pixel. Good agreement was observed (normally better than 1% in relative standard deviation) between the converted dose distribution obtained from the pixel matrix and the direct dose measurement using an ion chamber scanned in a water phantom in regions of shallow dose gradient. For application to on-line portal dosimetry, both the short- and long-term stability of this EPID system were found to be within 1% relative standard deviation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biophysical Phenomena↗