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Biomedical subjects

J Zhao

Publications and source records attributed to J Zhao.

At least 487 records · Page 27Linked to original sources
PubMed1997

The effects of angiotensin II and specific angiotensin receptor blockers on embryonic cardiac development and looping patterns.

The role of angiotensin II (Ang II) in the early embryonic development of the heart has not been examined. We have used RT-PCR to identify mRNA for angiotensinogen, angiotensin-converting enzyme, and the Ang II AT1 and AT2 receptors in embryonic day 10.25 Sprague-Dawley rats, and have used confocal microscopy to localize the AT1 receptor to the greater curvature of the developing ventricle in these animals at embryonic days (ED) 9.25 and 10.25. The antibodies used in immunolocalization studies did not distinguish between the AT1a and AT1b receptor subtypes. In whole embryo culture, Ang II added to the culture media resulted in increased ventricular growth and myocyte hypertrophy when treated embryos were compared to cultured littermate controls. Use of Losartan and PD123,319 to block the Ang II AT1 and AT2 receptors resulted in reduced ventricular development and cardiac dilation when compared to control and Ang II-treated embryos. Addition of Ang II and PD123,319 to the culture media also resulted in cardiac loop inversions which may be associated with disruption of normal myofibrillar development. These results clearly indicate an important role for Ang II in the early embryonic development of the heart.

Actins↗
PubMed1997

Progressive cyclic nucleotide-induced conformational changes in the cGMP-dependent protein kinase studied by small angle X-ray scattering in solution.

Small angle scattering data from bovine lung type Ialpha cGMP-dependent protein kinase (PKG) in the absence of cGMP show the protein to have a highly asymmetric structure with a radius of gyration (Rg) of 45 A and a maximum linear dimension (dmax) of 165 A. The addition of cGMP induces a marked conformational change in PKG. The Rg and dmax increase 25-30%, and the protein's mass moves further away from the center of mass; this results in an even more asymmetric structure. Fourier transform infrared spectroscopy data suggest that the conformational change induced by cGMP binding is primarily due to a topographical movement of the structural domains of PKG rather than to secondary structural changes within one or more of the individual domains. Each monomer of the dimeric PKG contains one high and one low affinity cGMP-binding site. A prominent increase in the asymmetry of PKG occurs with binding to high affinity cGMP-binding sites alone, but the full domain movements require the binding to both sets of sites. These conformational changes occurring in PKG with the progressive binding of cGMP to both sets of cGMP-binding sites correlate with past data, which have indicated that cGMP binding to both sets of sites is required for the full activation of the enzyme. These results provide the first quantitative measurement of the overall PKG structure, as well as an assessment of the structural events that accompany the activation of a protein kinase upon binding a small molecular weight ligand.

Animals↗
PubMed1997

Characterization of the scoliosis that develops after pinealectomy in the chicken and comparison with adolescent idiopathic scoliosis in humans.

STUDY DESIGN: The characteristics of the scoliosis that develops after pinealectomy in young chickens were determined from weekly posteroanterior radiographs. These data were compared with similar data collected from human patients with adolescent idiopathic scoliosis. OBJECTIVES: To characterize the scoliosis produced in young chickens after pinealectomy and to compare these characteristics with those seen in human patients with adolescent idiopathic scoliosis. SUMMARY OF BACKGROUND DATA: Although it has been recognized that pinealectomy produces scoliosis in chickens, the characteristics of these curves have never been well described other than by simple visual descriptions. METHODS: The characteristics of the scoliosis produced in chickens after pinealectomy done 3 days after hatching were measured from radiographs taken at weekly intervals. These characteristics were compared with similar data collected from human patients with adolescent idiopathic scoliosis. RESULTS: Similarities included development of single and double curves, degree of curvature, stability of the curve, numbers of vertebrae involved, direction of rotation, and progression characteristics. Differences included wedged vertebrae in the chickens, in conjunction with curve development and increased variability in vertebrae involved. CONCLUSIONS: There are many similarities in the development of scoliosis in young chickens after pinealectomy and in children with adolescent idiopathic scoliosis. The few differences might be related to the different biomechanical properties associated with the spine in the two species.

Adolescent↗
PubMed1997

Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel.

For a cystic fibrosis transmembrane conductance regulator (CFTR) channel to enter its open state, serine residues in the R domain must be phosphorylated by cAMP-dependent protein kinase, and intracellular ATP must bind to the nucleotide-binding folds and subsequently be hydrolyzed. CFTR with its R domain partially removed, DeltaR(708-835)-CFTR, forms a chloride channel that opens independently of protein kinase A phosphorylation, with open probability approximately one-third that of the wild type CFTR channel. Deletion of this portion of the R domain from CFTR alters the response of the channel to 5'-adenylylimidodiphosphate, pyrophosphate, and vanadate, compounds that prolong burst duration of the wild type CFTR channel but fail to do so in the DeltaR-CFTR. In addition, the addition of exogenous unphosphorylated R domain protein, which blocks the wild type CFTR channel, has no effect on the DeltaR-CFTR channel. However, when the exogenous R domain is phosphorylated, significant stimulation of the DeltaR-CFTR channel results; Po increases from 0.10 to 0.22. These data are consistent with a model for CFTR function in which the R domain in the unphosphorylated state interacts with the first nucleotide binding fold to inhibit either binding or hydrolysis of ATP or transduction of the effect to open the pore, but when the R domain is phosphorylated, it undergoes conformational change and interacts at a separate site in the first nucleotide binding fold to stimulate either binding or hydrolysis of ATP or transduction of the effect to open the pore.

Adenosine Triphosphate↗
PubMed1997

Determination of the genome composition of influenza virus reassortants using multiplex reverse transcription-polymerase chain reaction followed by fluorescent single-strand conformation polymorphism analysis.

The influenza virus genome is composed of eight negative-strand RNA segments. In cells coinfected with two or more influenza strains, the genomic RNAs reassort at random, resulting in progeny viruses (reassortants) that contain genes derived from each parent. Genetic reassortment among influenza viruses occurs naturally and plays an important role in viral epidemiology and pathogenicity. The reassortment process is also utilized for the annual production of influenza vaccines. Each year, the two gene segments that encode the major surface antigens of the current virulent, wild-type viruses are reassorted with the remaining six gene segments of a laboratory-derived vaccine or "master donor" strain. As the gene reassortment appears to be random, identifying a progeny virus with the desired gene constellation can be labor-intensive. We developed a streamlined, cost-effective method to genotype influenza viruses that combines multiplex reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent single-strand conformation polymorphism (SSCP) analysis. This method utilizes oligonucleotide primers labeled with one of three fluorescent dyes to generate RT-PCR products for each gene segment in a multiplex configuration. The RT-PCR products of the reassortants, wild-type, and master donor viruses are then electrophoresed under SSCP conditions. The viral origin of each gene segment can be identified by fluorescence and mobility shift patterns of the corresponding RT-PCR products. We demonstrate the utility of this method in differentiating the genes of a master donor strain, several wild-type viruses, and vaccine reassortants.

Animals↗
PubMed1997

Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3'-end formation in yeast.

In yeast, four factors (CF I, CF II, PF I, and PAP) are required for accurate pre-mRNA cleavage and polyadenylation in vitro. CF I can be separated further into CF IA and CF IB. Here we show that CF IB is the 73-kD Hrp1 protein. Recombinant Hrp1p made in Escherichia coli provides full CF IB function in both cleavage and poly(A) addition assays. Consistent with the presence of two RRM-type motifs, Hrp1p can be UV cross-linked to RNA, and this specific interaction requires the (UA)6 polyadenylation efficiency element. Furthermore, the CF II factor enhances the binding of Hrp1p to the RNA precursor. A temperature-sensitive mutant in HRP1 yields mRNAs with shorter poly(A) tails when grown at the nonpermissive temperature. Genetic analyses indicate that Hrp1p interacts with Rna15p and Rna14p, two components of CF 1A. The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export. Like Npl3p, Hrp1p shuttles between the nucleus and cytoplasm, providing a potential link between 3'-end processing and mRNA export from the nucleus.

Amino Acid Sequence↗
PubMed1997

Carboxyl terminus of delta opioid receptor is required for agonist-dependent receptor phosphorylation.

The wild-type delta opioid receptor (DOR) and a carboxyl terminus-truncated mutant DOR lacking the last 31 amino acids (DOR-T) were expressed in neuroblastoma x glioma hybrid NG108-15 cells to investigate the role of the carboxyl terminus of DOR in agonist-dependent receptor phosphorylation. Stimulation of the cells with delta specific agonists significantly induced DOR phosphorylation whereas no phosphorylation of DOR-T was detected under the same conditions. Neither overexpression of G protein-coupled receptor kinases (GRK2 or GRK5) nor activation of protein kinase C promoted agonist-induced phosphorylation of DOR-T, in contrast to their strong stimulatory effect on the agonist-dependent phosphorylation of DOR. Furthermore, DOR-T failed to be internalized after agonist stimulation, probably due to its inability to be phosphorylated. Our results indicate that the carboxyl terminus of DOR is required for agonist-dependent receptor phosphorylation and the phosphorylation site(s) of DOR is likely located at its carboxyl terminus.

Animals↗
PubMed1997

Molecular cloning of human plasma membrane phospholipid scramblase. A protein mediating transbilayer movement of plasma membrane phospholipids.

The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca2+ is thought to play a key role in expression of platelet procoagulant activity and in clearance of injured or apoptotic cells. We recently reported isolation of a approximately 37-kDa protein in erythrocyte membrane that mediates Ca2+-dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca2+ in the cytosol (Bassé, F., Stout, J. G., Sims, P. J., and Wiedmer, T. (1996) J. Biol. Chem. 271, 17205-17210). Based on internal peptide sequence obtained from this protein, a 1,445-base pair cDNA was cloned from a K-562 cDNA library. The deduced "PL scramblase" protein is a proline-rich, type II plasma membrane protein with a single transmembrane segment near the C terminus. Antibody against the deduced C-terminal peptide was found to precipitate the approximately 37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca2+-dependent PL scramblase activity was also demonstrated in recombinant protein expressed from plasmid containing the cDNA. Quantitative immunoblotting revealed an approximately 10-fold higher abundance of PL scramblase in platelet ( approximately 10(4) molecules/cell) than in erythrocyte ( approximately 10(3) molecules/cell), consistent with apparent increased PL scramblase activity of the platelet plasma membrane. PL scramblase mRNA was found in a variety of hematologic and nonhematologic cells and tissues, suggesting that this protein functions in all cells.

Amino Acid Sequence↗
PubMed1997

Production and characterization of a mutant cell line defective in aminophospholipid translocase.

Phospholipids are normally asymmetrically distributed between leaflets of the plasma membrane, due to the activity of aminophospholipid translocase (APT), a putative plasma membrane Mg2(+)-ATPase which is thought to selectively transport phosphatidylserine (PS) and other aminophospholipids from outer to inner membrane leaflet. Although several candidate proteins have been proposed to serve this function, positive identification awaits demonstration of their capacity to restore APT activity to a cell line that is deficient in this process. This study describes a simple and rapid protocol for the production and selection of mutant cell lines that are defective in APT activity and suitable for expression cloning of cDNAs coding for candidate APT enzymes. By flow cytometry, we demonstrate the time-dependent uptake of NBD-labeled PS, but not phosphatidylcholine (PC), by the mouse fibroblast cell line SV-T2. This uptake was inhibited by known inhibitors of APT, including o-vanadate and N-ethylmaleimide, and by ATP-depletion. SV-T2 cells were mutagenized with ethyl methanesulfonate, and APT-deficient cells were isolated by fluorescence activated cell sorting using NBD-PS as substrate. From a total of 7.2 x 10(6) cells passed through the flow cytometer, 98 clones exhibited APT activity that was less than 50% of that observed for wild-type SV-T2 cells. One clone which exhibited < or = 25% of that observed for wild-type cells, mutant M2711, was further characterized. The defect in mutant M2711 was specific for NBD-PS, and cellular ATP was unchanged, suggesting that the defect in APT activity was not due to a decrease in cellular ATP levels. Mutant M2711 exhibited a growth pattern indistinguishable from that of wild-type SV-T2 cells, and SV-40 large T antigen, which is needed for efficient episomal replication of plasmids containing the SV40 origin of replication, was unchanged. Finally, transfection of M2711 with cDNAs for marker membrane proteins consistently resulted in the same high level of protein expression as that observed for identically-transfected wild-type SV-T2. Thus, flow cytometry can be used for rapid identification of mutants with defects in phospholipid transport that are suitable for functional reconstitution by transfection with candidate APT cDNAs.

Animals↗
PubMed1997

Initial genomic scan of the NIMH genetics initiative bipolar pedigrees: chromosomes 3, 5, 15, 16, 17, and 22.

As part of the four-center NIMH Genetics Initiative on Bipolar Disorder we carried out a genomic scan of chromosomes 3, 5, 15, 16,17, and 22. Genotyping was performed on a set of 540 DNAs from 97 families, enriched for affected relative pairs and parents where available. We report here the results of the initial 74 markers that have been typed on this set of DNAs. The average distance between markers (theta) was 12.3 cM. Nonparametric analysis of excess allele sharing among affected sibling pairs used the SIBPAL program of the S.A.G.E. package to test three hierarchical models of affected status. D16S2619 gave some evidence of linkage to bipolar disorder, with P = 0.006 for Model II (in which bipolar 1, bipolar 2 and schizoaffective-bipolar type individuals are considered affected). Nearby markers also showed increased allele sharing. A second interesting region was toward the telomere of chromosome 5q, where D5S1456 and nearby markers showed increased allele sharing; for D5S1456, P = 0.05, 0.015 and 0.008 as the models of affected status become more broad. MOD score analysis also supported the possible presence of a susceptibility locus in this region of chromosome 5. A pair of adjacent markers on chromosome 3, D3S2405 and D3S3038, showed a modest increased allele sharing in the broad model. Several isolated markers had excess allele sharing at the P < 0.05 level under a single model. D15S217 showed a MOD score of 2.37 (P < 0.025). Multipoint analysis flagged the region of chromosome 22 around D22S533 as the most interesting. Thus, several regions showed modest evidence for linkage to bipolar disorder in this initial genomic scan of these chromosomes, including broad regions near previous reports of possible linkage.

Alleles↗
PubMed1997

Endogenous opioid receptor-like receptor in human neuroblastoma SK-N-SH cells: activation of inhibitory G protein and homologous desensitization.

Endogenous expression of opioid receptor-like receptor (ORL1), in human neuroblastoma SK-N-SH cells was demonstrated by binding with nociceptin/ orphanin FQ (N/OFQ). Scatchard analysis of [3H]N/ OFQ saturation binding data gave Kd = 1.3 +/- 0.1 nM and Bmax = 1.58 +/- 2.5 fmol/mg protein. N/OFQ stimulation increased [35S]GTP gamma S binding to cell membranes and attenuated forskolin-induced cAMP accumulation in a concentration-dependent manner. The effects of N/OFQ were eliminated by the pretreatment of pertussis toxin (PTX) but not by the antagonists of opioid receptors, revealing mediation of N/OFQ signal transduction by ORL1 receptor and PTX sensitive G protein(s). The ability of N/OFQ to inhibit cAMP production was greatly reduced after prechallenging with N/OFQ, indicating that ORL1 undergoes homologous desensitization in neuronal cells.

Brain Neoplasms↗
PubMed1997

The Ca2+/calmodulin binding domain of the Ca2+-ATPase linked to the Na+,K+-ATPase alters transport stoichiometry.

Using Xenopus oocytes as an expression system, we have investigated ion-transport and ouabain-binding properties of a chimeric ATPase (alpha1-CBD; Ishii and Takeyasu (1995) EMBO J. 14, 58-67) formed by the alpha1-subunit of chicken Na+,K(+)-ATPase (alpha1) and the calmodulin binding domain (CBD) of the rat plasma membrane Ca2(+)-ATPase. alpha1-CBD can be expressed and transported to the oocyte plasma membrane without the beta-subunit, and shows ouabain binding. In contrast to ouabain binding, this chimera requires the beta-subunit for its cation (Na+ and K+) transport activity. alpha1-CBD exhibits an altered stoichiometry of Na(+)-K+ exchange. A detailed analysis of 22Na+ efflux, 86Rb+ uptake, pump current and ouabain binding suggests that the chimeric molecule can operate in an electrically silent 2Na(+)-2K+ exchange mode and, with much lower probability, in its normal 3Na(+)-2K+ exchange mode.

Animals↗
PubMed1997

Cleavage factor II of Saccharomyces cerevisiae contains homologues to subunits of the mammalian Cleavage/ polyadenylation specificity factor and exhibits sequence-specific, ATP-dependent interaction with precursor RNA.

Cleavage of pre-mRNA during 3'-end formation in yeast requires two protein factors, cleavage factor I (CF I) and cleavage factor (CF II). A 5300-fold purification of CF II indicates that four polypeptides of 150, 105, 100, and 90 kDa copurify with CF II activity. The 150-kDa protein is recognized by antibodies against Cft1, the yeast homologue of the 160-kDa subunit of the mammalian cleavage/polyadenylation specificity factor (CPSF). The 100-kDa subunit is identical to Brr5/Ysh1, a yeast protein with striking similarity to the 73-kDa subunit of CPSF. The 105-kDa protein, designated Cft2 (cleavage factor two) exhibits significant homology to the CPSF 100-kDa subunit. Cft2 is cross-linked to pre-mRNA substrate containing the poly(A) site and wild type upstream and downstream flanking sequences, but not to precleaved RNA lacking downstream sequences or to substrate in which the (UA)6 processing signal has been deleted. The specific binding of Cft2 to the RNA substrate is ATP-dependent, in agreement with the requirement of ATP for cleavage. The sequence-specific binding of Cft2 and the similarities of CF II subunits to those of CPSF supports the hypothesis that CF II functions in the cleavage of yeast mRNA 3'-ends in a manner analagous to that of CPSF in the mammalian system. These results provide additional evidence that certain features of the molecular mechanism of mRNA 3'-end formation are conserved between yeast and mammals, but also highlight unexpected differences.

Adenosine Triphosphate↗
PubMed1997

Comparative studies on parallel and antiparallel duplex and triplex DNA.

Parallel strand models for base sequences d(A)10.d(T)10,d(AT)5.d(TA)5, d(G5C5).d(C5G5), d(GC)5.d(CG)5 and d(CTATAGGGAT).d(GATATCCCTA), where reverse Watson-Crick A-T pairing with two H-bonds and reverse Watson-Crick G-C pairing with one H-bond or with two H-bonds were adopted, and three models of d(T)14.d(A)14.d(T)14 triple helix with different strand orientations were built up by molecular architecture and energy minimization. Comparisons of parallel duplex models with their corresponding B-DNA models and comparisons among the three triple helices showed: (i) conformational energies of parallel AT duplex models were a little lower, while for GC duplex models they were about 8% higher than that of their corresponding B-DNA models; (ii) the energy differences between parallel and B-type duplex models and among the three triple helices arose mainly from base stacking energies, especially for GC base pairing; (iii) the parallel duplexes with one H-bond G-C pairs were less stable than those with two H-bonds G-C pairs. The present paper includes a brief discussion about the effect of base stacking and base sequences on DNA conformations.

Animals↗
PubMed1997

Determination of delta-opioid in NG108-15 cells.

The delta-opioid receptors in mouse neuroblastoma x rat glioma NG108-15 cells were characterized by receptor binding and cAMP assays. Saturation binding assays using [3H][D-Pen5]enkephalin (DPDPE) or [3H][D-Ser2, Leu5, Thr6]enkephalin (DSLET) gave similar binding capacities (Bmax). Competition binding assays showed that DPDPE and DSLET have similar affinity for the [3H]DPDPE or 3[H]DSLET binding sites. The rank order of potency of competition with [3H]DPDPE and [3H]DSLET was similar: naltriben approximately DSLET > or = DPDPE > 7-benzylidenenaltrexone (BNTX). Both DPDPE and DSLET were found to decrease cAMP formation. The action of DSLET was antagonized by naltriben but not BNTX, while the action of DPDPE was reversed by both antagonists. Therefore, the delta-opioid receptor in NG108-15 cells has similar affinity for the agonists DPDPE and DSLET, and a higher affinity for the antagonist naltriben than BNTX.

Animals↗
PubMed1997

Microsample determination of lovastatin and its hydroxy acid metabolite in mouse and rat plasma by liquid chromatography/ionspray tandem mass spectrometry.

A sensitive and specific method was developed and validated to quantitate lovastatin and its hydroxy acid in mouse and rat plasma. This method employs a solid-phase extraction procedure to isolate lovastatin and its hydroxy acid metabolite from the biological matrices (0.1 ml of mouse or rat plasma). The reconstituted extracts were analyzed by liquid chromatography/ionspray tandem mass spectrometry (LC/MS/MS). Simvastatin and simvastatin hydroxy acid were used as internal standards for lovastatin and lovastatin hydroxy acid, respectively. The assay has a lower limit of quantitation (LLQ) of 0.50 ng ml-1 in mouse and rat plasma for both lovastatin and its hydroxy acid based on 0.1 ml aliquots of plasma. The intra- and inter-assay precision (RSD), calculated from quality control (QC) samples, was < 7% for lovastatin and < 6% for lovastatin hydroxy acid in both matrices. The inter-assay accuracy as determined from QC samples was less than 6% for lovastatin and less than 8% for lovastatin hydroxy acid in both matrices. The overall recovery of lovastatin was 54% in mouse plasma and 55% in rat plasma, and the overall recovery of lovastatin hydroxy acid was 100% in mouse plasma and 67% in rat plasma.

Animals↗
PubMed1997

A comparative laboratory study of liquid nitrogen and argon gas cryosurgery systems.

Cryotherapy can now be applied using a variety of delivery systems and cryogens. We compared the Cryotech LCS 3000 liquid nitrogen system (Spembly, Andover, UK) with the CRYOcare argon gas-based system (Irvine, CA, U.S.A.) using three different 3-mm cryoprobes: an old liquid nitrogen probe (N-probe), a new N-probe featuring gas bypass and an argon gas probe. Each probe was tested in two models: (i) fresh sheep liver at 20 degrees C--the probe was inserted to a depth of 1.5 cm; the rate of ice ball formation was monitored by recording radial temperatures every 15 s at 5, 10, 15, and 20 mm from the cryoprobe, and the ice-ball diameter was measured every 2.5 min. After 10 min, the probe was warmed and the time taken until it could be extracted from the liver was recorded. (ii) Warm water bath--the probe was immersed in warm water (42 degrees C) for 15 min and the ice-ball diameter was measured at 5-min intervals. Radial temperatures in liver declined more rapidly (P < 0.001) and time to probe extraction was less (P < 0.01) when the argon gas system was used. The new N-probe performed better than its older counterpart, but was still slower than the argon gas system. In liver (20 degrees C), ice-ball diameters were similar after 10 min, but in warm water, they were larger when the new N-probe was used (P < 0.02). It would appear that the argon gas system is initially faster, but it does not achieve as large an ice ball in a warm environment as the liquid nitrogen system.

Animals↗