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Biomedical subjects

X Zhao

Publications and source records attributed to X Zhao.

At least 595 records · Page 33Linked to original sources
PubMed1995

[Analysis of genetic properties of meiotic products from an interspecific triploid fusion-hybrid itself constructed by protoplast fusion in Saccharomvces].

A genetically stable interspecific triploid fusion-hybrid between a diploid Saccharomyces cerevisiae var. ellopsoideus and a haploid Saccharomyces diastaticus was constracted by protoplast fusion technique. Experimental results indicate that such hybrid can be induced to sporulate as a sexual hybrid. Genetic analysis of the complete and non-complete tetrads demonstrates that the prototrophic interspecific triploid fusion-hybrid HU-KDF-240 selected by nutritional complementation experienced segregation and exchange for the genetic markers, and showed parental ditype and recombination in the meiotic process of sporulation. The results of the tetrad analysis of the hybrid HU-KDF-240 showed a 1:2 segregation ratio for soluble starch fermenting and non-fermenting characters and a 1:1 segregation ratio for genetic markers of the auxotrophic character in 349 complete and non-complete tetrads from 186 asci of the hybrid.

Cloning, Molecular↗
PubMed1994

Molecular characterization of human and mouse photoreceptor guanylate cyclase-activating protein (GCAP) and chromosomal localization of the human gene.

Guanylate cyclase-activating protein (GCAP) is a novel Ca(2+)-binding protein that stimulates synthesis of cGMP in photoreceptors. Molecular cloning of human and mouse GCAP cDNA revealed that the known mammalian GCAPs are more than 90% similar, consist of 201-205 amino acids, and contain three identically conserved EF hand Ca2+ binding sites. The sequence homology with recoverin, a related photoreceptor Ca(2+)-binding protein, is less than 35%. In situ hybridization in primate retinas shows that the GCAP gene is expressed exclusively in photoreceptor inner segments. To investigate the GCAP gene structure, we probed 10 eucaryotic genomic DNAs with a bovine GCAP cDNA under stringent conditions. The results demonstrate that the GCAP gene has been well conserved during evolution of vertebrate species and that each gene is most likely present as a single copy. By genomic cloning, polymerase chain reaction, mapping, and direct sequencing, we show that the human GCAP gene spans approximately 6 kilobases of genomic DNA, and consists of four exons (> 250, 146, 94, and 800 base pairs) separated by three introns (4.5 kilobases, 370 base pairs, and 347 base pairs). Using human/hamster hybrid panels and fluorescent in situ hybridization, the GCAP gene was localized to the short arm of chromosome 6 (p21.1).

Amino Acid Sequence↗
PubMed1994

Optimizing liposome-mediated gene transfer in primary rat septo-hippocampal cell cultures.

Although liposomes have been widely employed to transfect DNA into a variety of cell types, no previous studies have systematically examined conditions producing optimal liposomal-mediated transfection of DNA into central nervous system (CNS) cells. Thus, we used the beta-galactosidase (beta-gal) reporter gene to examine factors influencing the efficiency of liposome-mediated gene transfection in CNS cell cultures. Our results indicate that without increasing the amounts of DNA, increased liposome concentrations within certain limits enhanced transfection efficiency. However, higher liposome levels could produce cell lysis. Without increasing liposome concentrations, increased amounts of DNA did not improve transfection efficiency. Employing the optimal concentration (1 microgram DNA/3 microliters liposomes/well), beta-gal gene expression was sustained for at least two weeks after transfection in primary septo-hippocampal cultures.

Animals↗
PubMed1994

Sustained expression of functional nerve growth factor in primary septo-hippocampal cell cultures by liposome-mediated gene transfer.

We examined liposome-mediated gene transfection of nerve growth factor (NGF) in primary central nervous system cultures. RT-PCR analyses detected increased expression of NGF mRNA one day after liposome-mediated NGF gene transfection. ELISA studies detected large increases in NGF protein in cells and in culture medium after NGF gene transfection. Cells continued to secrete NGF into the medium for at least 2 weeks. NGF bioassays confirmed that the NGF secreted after gene transfection was biologically active.

Animals↗
PubMed1994

1H NMR investigation of distal mutant deoxy myoglobins. Interpretation of proximal His contact shifts in terms of a localized distal water molecule.

1H NMR spectra of a series of distal point mutants of human and sperm whale deoxy myoglobin have been recorded and their spectral parameters compared with those of wild type. The substitutions investigated include His64(E7)-->Gly, Ala, Val, Leu, Ile, and Gln and Val68(E11)-->Ala, Ile. The three resonances from the proximal His F8 imidazole ring, as well as two heme methyl signals, are identified in each of the proteins. Significant perturbations of the NMR spectra of mutant deoxy myoglobins (Mbs) occurred only upon substitution of His64(E7) by any non-polar residue, with only minor variation in parameters throughout the range of side chains. These spectral changes are attributed to the elimination of a non-coordinated ordered water molecule in the distal pocket found hydrogen bonded to His64(E7) in crystals of wild-type deoxy Mb, but abolished in the His64(E7)-->Leu mutant deoxy Mb crystal (Quillin, M. L., Arduini, R. M., Olson, J. S., and Philips, G. N., Jr. (1993) J. Mol. Biol. 234, 140-155). The observed spectral changes, increased His F8 ring spin delocalization, and decreased heme in-plane asymmetry, can be directly attributed to the weakening of the effective axial field and a decrease in the asymmetry in the rhombic ligand field resulting from removal of the water molecule. The hyperfine shift patterns for the mutants His64(E7)-->Gln and Val68(E11)-->Ile deoxy Mbs are minimally perturbed from that of wild type and are interpreted to reflect a conserved distal water-binding site. In the point mutant Val68(E11)-->Ala, the decreased covalency to the axial His F8 is interpreted as reflecting a conserved distal water molecule that can interact more strongly with the iron due to the reduced steric bulk of the E11 side chain. The differential 1H NMR spectral parameters for the His F8 resonances in the two subunits of T state deoxy Hb A are shown to be similarly consistent with the known occupation of the distal water binding site in the alpha-, but not beta-subunit.

Animals↗
PubMed1994

Mechanism of homophilic binding mediated by the neural cell adhesion molecule NCAM. Evidence for isologous interaction.

We have previously shown that a decapeptide sequence between Lys-243 and Glu-252 (KYSFNYDGSE) in the third immunoglobulin (Ig)-like domain of chick neural cell adhesion molecule NCAM is directly involved in NCAM-to-NCAM binding. To identify the domain that interacts with this decapeptide sequence, the Ig-like domain 3 of NCAM was expressed in bacteria and the refolded protein was assayed for its NCAM binding activity. Covaspheres conjugated with domain 3 protein bound to a substratum coated with either NCAM or the domain 3 protein, suggesting that NCAM-NCAM binding is mediated by interactions between domain 3 sequences on apposing molecules. Further studies were carried out using a cell-to-substratum binding assay. Mouse L cells stably transformed with different deletion constructs of NCAM were assayed for their ability to attach to substratum coated with different peptide conjugates. The results indicated that a site in NCAM Ig-like domain 3 bound specifically to the decapeptide sequence. To identify this site, cells expressing mutant NCAMs with alterations in the amino acid sequence of the homophilic binding site were subjected to the same cell-to-substratum assay. Mutant NCAMs that had lost their homophilic binding activity also failed to attach to the peptide substrate. Taken together, these results suggest that the NCAM homophilic binding site interacts isologously with the same sequence on apposing molecules.

Amino Acid Sequence↗
PubMed1994

Induction of apoptosis by quercetin: involvement of heat shock protein.

Quercetin, a widely distributed bioflavonoid, inhibits the growth of tumor cells. The present study was designed to investigate the possible involvement of apoptosis and heat shock protein in the antitumor activity of quercetin. Treatment with quercetin of K562, Molt-4, Raji, and MCAS tumor cell lines resulted in morphological changes, including propidium iodide-stained condensed nuclei (intact or fragmented), condensation of nuclear chromatin, and nuclear fragmentation. Agarose gel electrophoresis of quercetin-treated tumor cells demonstrated a typical ladder-like pattern of DNA fragments. In addition, the hypodiploid DNA peak of propidium iodide-stained nuclei was revealed by flow cytometry. Quercetin induced apoptosis in cells at G1 and S in a dose- and time-dependent manner. The apoptosis-inducing activity of quercetin was enhanced by cycloheximide and actinomycin D. A nuclease inhibitor, aurintricarboxylic acid, inhibited quercetin-induced apoptosis, whereas deprivation of intracellular calcium by EGTA had no effect. 12-O-Tetradecanoylphorbol-13-acetate and H-7 did not affect the induction of apoptosis by quercetin. The synthesis of HSP70 was inhibited by quercetin when determined by immunocytochemistry, Western blot analysis, and Northern blot analysis. Quercetin-treated tumor cells were not induced to show aggregation of HSP70 in the nuclei and nucleolus in response to heat shock, resulting in apoptosis. By contrast, when tumor cells were first exposed to heat shock, no apoptosis was induced by quercetin. In addition, pretreatment of tumor cells with HSP70 antisense oligomer that specifically inhibited the synthesis of HSP70 enhanced the subsequent induction of apoptosis by quercetin. These results suggest that quercetin displays antitumor activity by triggering apoptosis and that HSP70 may affect quercetin-induced apoptosis.

Apoptosis↗
PubMed1994

Specificity of DnaK-peptide binding.

The sequence specificity of DnaK substrate binding has been studied using a peptide display library. Based on the amino acid patterns that appeared in this selection, short peptides were synthesized for direct measurements of DnaK affinity. The results show that peptides enriched in internal hydrophobic residues are preferential DnaK substrates, and negatively charged peptides have poor affinity. The isolated C-terminal domain of DnaK binds peptides. Peptide dissociation studies indicate that bound peptides are released from the C-terminal fragment and from DnaK at identical rates. ATP stimulates peptide dissociation from DnaK but not from the C-terminal fragment.

Amino Acid Sequence↗
PubMed1994

Myosin filament ATPase is enhanced by intramolecularly cross-linked actin.

Reaction of rabbit skeletal muscle F-actin with the lysine-directed photolabile cross-linker, N-5-azido-2-nitrobenzoyloxy succinimide was limited to Lysine-328 and Lysine-326, with Lysine-328 being labelled to a greater extent. Photolysis of the modified actin enhanced the actin-activated MgATPase activity of filamentous scallop myosin 3-4-fold more than unmodified actin, without affecting calcium sensitivity. Unphotolysed modified actin behaved as untreated actin, indicating that photolysis was essential for the effect. The actin-activated ATPase of filamentous rabbit myosin was similarly increased by photolysed N-5-azido-2-nitrobenzoyloxy succinimide-modified actin. After photolysis in either the monomeric (G-) or filamentous (F-) form, N-5-azido-2-nitrobenzoyloxy succinimide-modified actin moved as a monomeric (42 kDa) species on SDS gels, and depolymerized and polymerized readily, demonstrating that any cross-linking event produced by photolysis must be intramolecular. In contrast to the substantial increase in actin-activated ATPase activity observed when photolysed ANB-NOS-modified actin was added to filamentous myosin, the enhancement was not observed with the soluble HMM and S-1 fragments of myosin. Photolysed modified actin showed only poor movement on a rabbit HMM-coated surface in vitro motility assays. These results can be explained if the internally cross-linked G-actin subunits which comprise only a fraction of the actin population, either weaken the actin-actin contacts or have an increased affinity for myosin.

Actins↗
PubMed1994

Suitability of autoclaved tap water for preparation of ELISA reagents and washing buffer.

The suitability of autoclaved tap water for the preparation of ELISA reagents and washing buffer was compared with that of ultrapure water, in a standard indirect ELISA for the detection of antibodies to pseudorabies virus (PRV). The performance of the assay, using autoclaved tap water (AT-ELISA) compared favourably to that of the standard assay, using ultrapure water (UP-ELISA) in detecting anti-PRV antibodies in sequential serum samples from a pig experimentally infected with PRV. While both the UP-ELISA and AT-ELISA proved reliable in detecting anti-PRV antibodies in a coded proficiency serum panel (n = 60), the AT-ELISA detected fewer positive sera than the UP-ELISA in evaluating a limited number (n = 80) of field samples. The results suggest that autoclaved tap water may be substituted for ultrapure water for the preparation of ELISA reagents when or where ultrapure water may not be available.

Animals↗
PubMed1994

Somatotropin and insulin-like growth factor-I concentrations in plasma and milk after daily or sustained-release exogenous somatotropin administrations.

Effects of daily injectable or sustained-release bovine somatotropin (bST) administrations on plasma and milk bST and insulin-like growth factor-I (IGF-I) concentrations were monitored in 74 lactating cows through early, mid- and late lactation. Treatments beginning at wk 4 of lactation were excipient (CO, 24 cows) at 2 wk intervals, daily injections of 10.3 mg bST (DI, 25 cows) and 350 mg sustained-release bST at 2 wk intervals (SR, 25 cows). The duration of treatments was 40 wk. Data were first analyzed for the overall mean concentrations covering the 40 wk treatment period. Overall mean plasma bST, milk bST and plasma IGF-I concentrations were significantly increased by both bST treatments (p < 0.05). On the other hand, milk IGF-I concentrations were significantly increased (p < 0.05) only in the DI group. Next, data were analyzed according to stage of lactation. The bST treatments resulted in significant increases (p < 0.05) in plasma and milk bST concentrations for all early, mid- and late lactation periods. Even though plasma IGF-I concentrations were higher (p < 0.05) in all lactation periods for bST treatment groups, higher milk IGF-I concentrations (p < 0.05) occurred only in mid- and late lactation periods for the DI group. The patterns of bST and IGF-I concentrations in milk follows those of the plasma after bST treatments.

Animals↗
PubMed1994

Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein.

Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.

Amino Acid Oxidoreductases↗
PubMed1994

The first solid-state 171Yb nuclear magnetic resonance spectra.

The first powder and magic-angle spinning (MAS) experiments on 171Yb are reported. The divalent ytterbium compound RbYbI3 was studied by solid-state 171Yb nuclear magnetic resonance (NMR) and a powder spectrum of axial symmetry was obtained. The principal components of the chemical shielding tensor were determined as sigma 11 = sigma 22 = -62 ppm and sigma 33 = 124 ppm. An inversion-recovery experiment showed an anisotropic relaxation behavior with the parallel component relaxing faster than the perpendicular component (T1 perpendicular = 426 ms, T1 parallel = 298 ma). The natural line width of 800 Hz, which corresponds to an effective T2 of 0.4 ms, was obtained from the MAS spectrum.

Anisotropy↗
PubMed1994

Dopachrome conversion activity in Aedes aegypti: significance during melanotic encapsulation of parasites and cuticular tanning.

Phenol oxidase (PO) and dopachrome conversion enzyme (DCE) were partially purified from Aedes aegypti larvae by ammonium sulfate fractionation. PO from A. aegypti functions in the hydroxylation of monophenols (e.g., tyrosine and tyramine) to their related o-diphenols, and the oxidation of o-diphenols (e.g., L-dopa, dopamine, N-acetyldopamine) to their respective o-quinones. Partially purified DCE showed high specificity toward dopachrome generated from dopa with the L-configuration. The combined effects of PO and DCE significantly accelerated melanization pathways when L-dopa was used as substrate. Significant DCE activity also was detected in hemolymph samples from adult, female A. aegypti, and undoubtedly plays a role in melanotic encapsulation reactions.

Aedes↗
PubMed1994

Quantitative single-photon emission computed tomography: basics and clinical considerations.

Although quantitative single-photon emission computed tomography (SPECT) has been the goal of much research effort for a number of years, only recently has it received wide interest, especially for clinical applications. It has been increasingly recognized that the achievement of quantitative SPECT will increase the accuracy of measurements, such as dimensions of specific regions of interest, absolute amount of radioactivity, and dosimetry calculations, and substantially reduce reconstruction image artifacts and distortions, thus, greatly improving clinical diagnosis. This article provides a review of the definition of terms, major factors affecting SPECT quantitation and their degrading effects on SPECT image quality, and methods to compensate for these effects. Compensation methods include those that make certain approximations for ease of implementation and those that provide more accurate compensation by modeling the imaging process more exactly, usually at the cost of increased complexity and computational requirements. Different reconstruction and compensation methods may be compared through the use of phantom cardiac and brain SPECT studies. The clinical efficacy of the methods may be demonstrated by applying them to a clinical thallium-201 myocardial perfusion SPECT study. The results clearly demonstrate that, by modeling the imaging process and/or image degrading factors three-dimensionally, quantitative reconstruction and compensation methods provide the best image quality and quantitative accuracy. Important research efforts and developmental work being conducted currently to bring quantitative SPECT into routine clinical use are also discussed.

Humans↗
PubMed1994

Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ.

The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine kinases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme-specific antibodies revealed that PKC alpha, beta II, delta, epsilon, and zeta are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitted analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC alpha, beta II, delta, and zeta occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue. Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC alpha from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.

Animals↗
PubMed1994

The importance and implementation of accurate 3D compensation methods for quantitative SPECT.

The purpose of this study was to investigate the importance of 2D versus 3D compensation methods in SPECT. The compensation methods included in the study addressed two important degrading factors, namely attenuating and collimator-detector response in SPET. They can be divided into two general categories. The conventional methods are based on the filtered backprojection algorithm, the Chang algorithm for attenuation compensation and the Metz filter for detector response compensation. These methods, which were computationally efficient, could only achieve approximate compensation due to the assumptions made. The quantitative compensation methods provide accurate compensation by modelling the degrading effects at the expense of large computational requirements. Both types of compensation methods were implemented in 2D and 3D reconstructions. The 2D and 3D reconstruction/compensation methods were evaluated using data from simulation of brain and heart, and patient thallium SPECT studies. Our results demonstrate the importance of compensation methods in improving the quality and quantitative accuracy of SPECT images and the relative effectiveness of the different 2D and 3D reconstruction/compensation methods. We concluded that 3D implementation of the quantitative compensation methods provides the best SPECT image in terms of quantitative accuracy, spatial resolution, and noise at a cost of high computational requirements.

Algorithms↗