Characterization of polyclonal antibody-catalyzed hydrolysis.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
Y Yu
Publications and source records attributed to Y Yu.
Dimer and Néel order-parameter fluctuations in the spin-fluid phase of the s=1/2 spin chain with first- and second-neighbor couplings.
Explore the source record for details and available documents.
Journal Article↗
GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF.
We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.
Amino Acid Sequence↗
Differential mu opiate receptor phosphorylation and desensitization induced by agonists and phorbol esters.
mu opiate receptors, the principal sites for opiate analgesia and reward, can display compensatory responses to opiate agonist drug administration. Agonist-induced K+ channel responses mediated by these receptors desensitize when examined in Xenopus oocyte expression systems. Mechanisms underlying such processes could include phosphorylation events similar to those reported to desensitize other G-protein-linked receptors. We used C-terminally directed anti-mu receptor antibodies to immunoprecipitate a phosphoprotein with size appropriate for the mu receptor from stably expressing Chinese hamster ovary cells. Phosphorylation of this mu opiate receptor protein was enhanced approximately 5-fold by treatment with the mu agonist morphine. The time course and dose-response relationships between mu receptor phosphorylation and agonist-induced desensitization display interesting parallels. Phosphorylation of mu opiate receptor protein is also enhanced approximately 5-fold by treatment with the protein kinase C activator phorbol 12-myristate 13-acetate. The protein kinase inhibitor staurosporine blocked the effect of phorbol 12-myristate 13-acetate on mu receptor phosphorylation. However, staurosporine failed to block morphine-induced phosphorylation. These observations suggest that several biochemical pathways can lead to mu receptor phosphorylation events that may include mechanisms involved in mu receptor desensitization.
Analgesics↗
The exit from G(0) into the cell cycle requires and is controlled by sarco(endo)plasmic reticulum Ca2+ pump.
The intracellular calcium pump sarco(endo)plasmic reticulum Ca2+ (SERCA) is responsible for the formation of the Ca2+ gradient across the endoplasmic reticulum membrane, and this gradient is used to generate the Ca2- signal during agonist-stimulated cell growth. In the present study, the role of SERCA in both cell cycle and growth control was investigated using cultured rat aortic endothelial cells (RAEC). Using a novel DNA transfection approach, cell lines were established that showed varying degree of SERCA activity through the down-regulation of the endogenous SERCA gene (B. F. Liu, X. Xu, R. Fridman, S. Muallem, and T. H. Kuo, J. Biol. Chem. 271, 1--9, 1996). Cell proliferation studies indicated that the lower SERCA expressing cells exhibited a slower growth pattern without altering the doubling time which was similar for both parental and transfected RAEC lines. G1 to S phase transition was prolonged with a smaller proportion of cells entering DNA synthesis as indicated by thymidine incorporation assay. Comparison of transfected cell lines indicated a tight coupling of SERCA activity and the length of the G1 period. Down-regulation of SERCA gene expression was accompanied by increased mRNA levels of p21 (WAF1/CIP1), a universal cell cycle inhibitor. The delay in G1 to S progression also coincided with the up-regulation of p53 mRNA and underphosphorylation of the retinoblastoma protein. This study suggests that Ca2+ metabolism in the agonist mobilizable pool controls the cell cycle through the regulation of genes operating in the critical G1 to S checkpoint.
Animals↗
Investigation of electrostatic interactions in two-stranded coiled-coils through residue shuffling.
The effects of electrostatic interactions on the stability of coiled-coils were investigated using the strategy of shuffling the sequence without changing the overall content of amino acid residues in the peptides. Shuffling the sequence provides peptides with thermodynamically similar unfolded states. Therefore, the unfolded state can be used as a universal reference state in comparing the thermodynamic properties of the folded coiled-coil structure of the peptides, while varying the configuration of ionized groups, that is, changing the types and number of potential electrostatic interactions. The relative stabilities of these states were determined by monitoring the temperature-induced folding/unfolding of the peptides in solutions with different pH and ionic strength by circular dichroism spectroscopy and scanning microcalorimetry. It was found that, in solutions with low ionic strength, ionic pairs contribute significantly to the stability of the coiled-coil conformation. The stability increases with an increase in the number of ionized groups in the peptide upon changing pH from acidic to neutral. In contrast, in the solutions with high ionic strength, the coiled-coil becomes less stable at neutral pH than at acidic pH. Most surprisingly, the increase in Gibbs energy of stabilization of the coiled-coil state with increasing pH at low ionic strength proceeds with a decrease in the enthalpy and entropy of unfolding. This observation can be explained only by hydration of ionized groups upon unfolding of coiled-coils which is associated with significant negative enthalpy and entropy effects.
Amino Acid Sequence↗
Purification and molecular characterization of a novel b5-type cytochrome of the parasitic nematode, Ascaris suum.
A b5-type cytochrome was extracted from Ascaris suum muscle at pH 4.5 with 0.3% aluminum sulfate and purified by ammonium sulfate fractionation, ion-exchange chromatography on CM-500 Cellulofine, and gel filtration on Sephadex G-75. The hemoprotein displayed a typical absorption spectrum of cytochrome b with a midpoint redox potential of 78 mV. The N-terminal amino acid sequence was determined and revealed the N-terminus to be highly homologous to the heme-binding domain of vertebrate cytochrome b5. Using an oligonucleotide probe synthesized based on the amino acid sequence of the purified protein, the cDNA clone encoding A. suum cytochrome b5 was isolated from the lambda ZAP II cDNA library. The entire nucleotide sequence of 563 bases comprised an open reading frame of 339 bases encoding a precursor protein of 112 amino acid residues. The purified cytochrome B5 was predicted to contain 82 amino acids with a molecular mass of 9141 Da, matching the 9140 Da obtained from electrospray ionization mass spectometry, and to lack of membrane-anchor domain at the C-terminus. In contrast, an N-terminal extension of 30 amino acids, characteristic of signal peptides, was apparent. Immunoblots revealed the presence of an A. suum cytochrome b5 of 82 amino acids, but no protein with an N-terminal extension. These results demonstrate a novel cytochrome b5 possessing a presequence.
Amino Acid Sequence↗
Differential regulation of IRP1 and IRP2 by nitric oxide in rat hepatoma cells.
Iron-regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that bind to stem-loop structures known as iron-responsive elements (IREs). IREs are located in the 5'- or 3'-untranslated regions (UTRs) of specific mRNAs that encode proteins involved in iron homeostasis. The binding of IRPs to 5' IREs represses translation of the mRNA, whereas the binding of IRPs to 3' IREs stabilizes the mRNA. IRP1 and IRP2 binding activities are regulated by intracellular iron levels. In addition, nitric oxide (NO.) increases the affinity of IRP1 for IREs. The role of NO. in the regulation of IRP1 and IRP2 in rat hepatoma cells was investigated by using the NO.-generating compound S-nitroso-N-acetylpenicillamine (SNAP), or by stimulating cells with multiple cytokines and lipopolysaccharide (LPS) to induce NO. production. Mitochondrial and IRP1 aconitase activities were decreased in cells producing NO(.). NO. increased IRE binding activity of IRP1, but had no effect on IRE binding activity of IRP2. The increase in IRE binding activity of IRP1 was coincident with the translational repression of ferritin synthesis. Transferrin receptor (TfR) mRNA levels were increased in cells treated with NO.-generating compounds, but not in cytokine- and LPS-treated cells. Our data indicate that IRP1 and IRP2 are differentially regulated by NO. in rat hepatoma cells, suggesting a role for IRP1 in the regulation of iron homeostasis in vivo during hepatic inflammation.
Animals↗
The cAMP receptor subtype cAR2 is restricted to a subset of prestalk cells during Dictyostelium development and displays unexpected DIF-1 responsiveness.
Dictyostelium discoidium cells express a family of cell surface cAMP receptors, and these G-protein-coupled receptors are each expressed with unique spatial and temporal patterns. One of these receptors, cAR2, is present during the postaggregative stages of development and our previous work suggests that it is preferentially expressed in prestalk cells. We report here the isolation of the promoter for carB, the gene which encodes cAR2. Using this fragment to generate a carB::lacZ, gene fusion construct, we investigated carB expression in detail. Expression is first detected at the tight aggregate stage and subsequently in a pattern reminiscent of the prestalk-specific gene ecmA. There are subtle differences, however, with, ecmA being expressed significantly in the anterior-like cells of the migrating pseudoplasmodium and in the basal disc and lower cup supporting the sorus during terminal development. carB is not expressed in any of these places. The presence of these different prestalk cell subtypes was confirmed by double indirect immunofluorescence using anti-cAR2 and anti-beta-galactosidase antibodies. While virtually all cAR2-expressing cells also express ecmA::lacZ, a substantial fraction of ecmA::lacZ-positive cells do not express cAR2. We also found the regulation of carB gene expression to differ from that of ecmA. carB expression is induced in vitro by extracellular cAMP, but surprisingly, not by DIF-1, a soluble molecule thought to be essential for the initiation of prestalk differentiation. Thus, cAR2 appears to be a cAMP receptor present on a restricted subset of prestalk cells and whose expression does not respond typically to the prestalk inducer DIF-1. DIF-1 sensitivity may, therefore, not be characteristic of all early prestalk differentiation.
Amino Acid Sequence↗
Coinduction of nitric oxide synthase, argininosuccinate synthetase, and argininosuccinate lyase in lipopolysaccharide-treated rats. RNA blot, immunoblot, and immunohistochemical analyses.
Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase (NOS), and citrulline which is generated can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of NOS (iNOS), AS, and AL was analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the LPS treatment but was induced by LPS in the lung, heart, liver, and spleen, and less strongly in the skeletal muscle and testis. AS mRNA was induced in the lung and spleen, and AL mRNA was weakly induced in these tissues. AS and AL mRNAs were abundant in the control liver and remained unchanged after the treatment. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6-12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunoblot analysis, increase of iNOS protein was evident in the lung, liver, and spleen, and there was an increase of AS protein in the lung and spleen. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS, and AL were coinduced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation.
Animals↗
Limitations of the minimum peripheral dose as a parameter for dose specification in permanent 125I prostate implants.
PURPOSE: The objective of this work is to investigate whether the minimum peripheral dose is a practical parameter for dose specification in permanent 125I implants of the prostate. METHODS AND MATERIALS: The investigation was carried out by use of a computer model of ellipsoidal 125I implants in which the average dimension and elongation factor were varied to provide a wide range of geometries. Both ideal and nonideal implants were investigated. The 125I seeds were confined to the target volume except for a portion of the study in which the effect of placing seeds outside the target volume was investigated. RESULTS: The minimum peripheral dose was found to be very sensitive to the seed placement. The irregularities in the seed spacing that inevitably occur in actual implants tend to lower the minimum peripheral dose. As a result, the minimum peripheral dose is generally significantly less than planned by an amount that is unpredictable, and often exceeds 25%. However, the percentage of the target volume that receives a dose less that the prescribed minimum peripheral dose is generally less than 10%. Implanting seeds outside the target volume improves the dose uniformity, but does not appear to offer any advantage in dose coverage, and increases the volume of normal tissue irradiated. CONCLUSION: If a minimum peripheral dose is prescribed for a permanent 125I prostate implant, and the implant is planned using an idealized implant having precisely spaced seeds, the prescribed dose will rarely, if ever, be achieved. Reasonable agreement with the prescribed dose can be achieved only if the requirement for coverage is relaxed from 100 to 90%, or if the total source strength is increased by 20% to compensate for the anticipated imperfections in seed placement.
Brachytherapy↗
Ion pairs significantly stabilize coiled-coils in the absence of electrolyte.
We have used a synthetic coiled-coil peptide model system to address the long perplexing issue as to why coiled-coils are in general more stable at acidic pH than at neutral pH. Contrary to the above expectation, our results show that at low ionic strength (10 mM) the coiled-coil was much more stable at neutral pH than at acidic pH against both thermal and urea unfolding, indicating that the Lys(+)-Glu- ions pairs present around the coiled-coil interface at neutral pH contribute significantly to the stability of the coiled-coil. However, while the addition of NaCl had no significant effect on the coiled-coil stability at neutral pH, its stability at acidic pH increased dramatically. The cross-over point between the stability at acidic pH and neutral pH occurred at around 100 mM salt, above which the coiled-coil became more stable at acidic pH, in agreement with published results. Therefore, salt effect, rather than intrinsic property, such as carboxyl-carboxyl hydrogen bonding, causes this coiled-coil to become more stable at acidic pH. The preferential stabilizing effect of salt on the coiled-coil at acidic pH can be best explained in terms of the condensation of anions to the positively charged groups on the coiled-coil, the net density of which increases as glutamic acid residues become protonated in acidic pH.
Amino Acid Sequence↗
Differential distribution of cAMP receptors cAR2 and cAR3 during Dictyostelium development.
Signal transduction via a family of cAMP receptor subtypes (cARs) is critical for proper development in the cellular slime mold Dictyostelium. Genes encoding four related subtypes have been cloned and their expression, based on RNA accumulation, has been previously reported. Here we report the differential spatial and temporal distribution of cAR2 and cAR3 proteins, based on indirect double immunofluorescence. Cells were transformed with a carB::lacZ construct, and an antibody against beta-galactosidase was used to visualize cAR2 expression. Simultaneously, a cAR3-specific antibody was used to identify cAR3-expressing cells. Results indicate that by the time of tip formation (12-14 hr) both receptors are expressed and distribute in a virtually nonoverlapping pattern, with cAR2 being expressed on anterior, prestalk cells and cAR3 present in the rest of the organism. Differential distribution of these two receptor subtypes may result in distinct cAMP signaling mechanisms in the two major regions of the organism.
Animals↗
Transfection of human ovarian cancer cells with the HER-2/neu receptor tyrosine kinase induces a selective increase in PTP-H1, PTP-1B, PTP-alpha expression.
Protein tyrosine phosphorylation is a key regulator of cell growth regulation and, when aberrant, is linked with the process of oncogenic transformation. Since tyrosine phosphatases (PTP) reverse phosphorylation mediated by tyrosine kinases (PTK), it has been hypothesized, but insufficiently studied to date, that PTPs function as tumor suppressors. Alternatively, PTPs may augment signal transduction by repriming substrates. To begin to assess the role of PTPs in ovarian cancer cell growth regulation, PTP partial cDNAs were cloned from the OVCA 433 ovarian cancer cell line using RT/PCR and degenerate oligonucleotide primers for the PTP consensus domain. Thirteen partial cDNAs were isolated, the sequences of which, when compared to GenBank, suggested that they were derived from PTP family members. These included PTP-alpha, PTP-gamma, LAR, PTP-delta, T-cell PTP, PTP-2A(1D), PTP-1B, PTP-1C, PTP-H1, PTP-PEST, PTP-Gallus, and two isolates which potentially represent novel PTPs. Steady-state expression analyses revealed that PTP-1B expression was undetectable in normal epithelium but was expressed in four of five ovarian cancer cell lines. In contrast, the expression of two SH2-containing PTPs, PTP-1C and PTP-2A, was detected in the normal ovarian epithelium but lost from one or more ovarian cancer cell lines. Finally, PTP-1B, PTP-alpha, and PTP-H1 expression was increased following HER-2/neu transfection of ovarian cancer cell lines. These results suggest that both normal ovarian epithelial cells and ovarian cancer cells express multiple PTPs. Further, some PTPs are differentially expressed between normal ovarian epithelium and ovarian cancer cells. Intriguingly, the transfection and increased expression of the prognostically significant HER-2/neu PTK induced a selective increase in the expression of the PTP-alpha, PTP-1B, and PTP-H1 tyrosine phosphatases.
Base Sequence↗
Production of congenic mouse strains carrying genomic intervals containing SLE-susceptibility genes derived from the SLE-prone NZM2410 strain.
Systemic lupus erythematosus is inherited as a complex polygenic trait. Four genomic intervals containing major SLE-susceptibility loci were previously identified by interval mapping in the NZM2410 mouse model. In this paper, we utilized a marker-assisted selection protocol to produce four congenic mouse strains, each carrying an NZM2410-derived SLE-susceptibility interval on a C57BL/6-resistant background. Each strain carries only one susceptibility allele derived from this polygenic model and consequently can be used to characterize the specific component phenotypes contributed by individual SLE-susceptibility genes. We illustrate the efficacy of this approach with phenotypic data for one of our congenic strains, B6.NZMH2(z). Our results indicate that this single genomic interval from Chromosome (Chr) 17 of NZM2410 can mediate increased levels of IgG autoantibodies specific for chromatin and that, similar to results obtained in our original genetic cross, B6.NZMH2(z/b) heterozygotes are more prone than B6.NZMH2(z) homozygotes to the development of humoral autoimmunity to nuclear antigens. These results illustrate the feasibility of using congenic strains to dissect the complex pathogenic mechanisms that mediate polygenic SLE. These congenic strains will be valuable tools in the genetic analysis of SLE susceptibility. In future studies, these congenic strains will be interbred to produce bi- and tri-congenic strains in order to assess the role of genetic interactions in the expression of specific components of SLE pathogenesis. They will also be instrumental to the positional cloning and identification of the genes responsible for SLE susceptibility, via the production of congenic recombinants.
Animals↗
Molecular and functional properties of cytochrome c from adult Ascaris suum muscle.
Mitochondrial cytochrome c was isolated at high purity from adult Ascaris suum muscle and its molecular properties were investigated. The molecular weight of A. suum cytochrome c was determined to be 13,119 by electrospray ionization mass spectrometry. The oxidation-reduction potential of nematode cytochrome c was measured to be +248 mV; this value is comparable to those for cytochrome c from mammalian sources. The A. suum cytochrome c, like bovine heart cytochrome c, showed biphasic kinetics against bovine heart cytochrome c oxidase. Comparative kinetic studies revealed species-specificity in the reaction between cytochrome c and cytochrome c oxidase from A. suum and bovine sources. The cytochrome c content in mitochondria was highest at the second larval stage, in which the respiratory chain is the most aerobic among various developmental stages of A. suum. These data clearly show that adult A. suum cytochrome c, as isolated, is a bona fide substrate for cytochrome c oxidase in the aerobic respiratory chain of second-stage larvae.
Age Factors↗
Ginseng extract scavenges hydroxyl radical and protects unsaturated fatty acids from decomposition caused by iron-mediated lipid peroxidation.
This study was conducted to investigate whether or not the antioxidation effect of ginseng extract directly inhibits decomposition of unsaturated fatty acid caused by iron and hydrogen peroxide-induced lipid peroxidation, and whether this effect involves a hydroxyl radical-scavenging mechanism. Thiobarbituric acid-reactive substances (TBARS), gas chromatography, and electron spin resonance (ESR) spectrometer were used to measure lipid peroxidation, unsaturated fatty acid, and hydroxyl radical. The results showed TBARS formed and the loss of arachidonic acid during lipid peroxidation, and that hydroxyl radical formed by the Fenton reaction were completely inhibited by ginseng extract. This antioxidant effect of ginseng may be responsible for its wide pharmacological actions in clinical practice by a free radical reaction-inhibition mechanism.
Arachidonic Acid↗
Elevated serum neopterin level: its relation to endotoxaemia and sepsis in patients with major burns.
The present study was conducted to determine the relationship between levels of neopterin and endotoxin in the circulation, and whether the neopterin level was related to the development of severe sepsis after extensive burns. This prospective study included 35 patients with burn size greater than 30% (30-98%), and 22 healthy volunteers who served as a comparison group. Neopterin levels increased in most patients on day 3 post-burn, but they were not significantly correlated with the extent of the burn surface (P > 0 center dot 05). A high serum neopterin level was found in patients with sepsis (n = 15), and a marked elevation persisted throughout the observation period. The difference between septic and non-septic patients (n = 20) became significant on 14 and 28 days post-burn. Although the presence of early endotoxaemia did not influence the alterations in serum neopterin, patients with endotoxaemia had much higher neopterin values than those who showed no endotoxaemia from the second week onward (P < 0 center dot 05-0 center dot 01). In addition, circulating endotoxin and neopterin levels were positively correlated in patients who developed endotoxaemia on day 14 (r = 0 center dot 368, P < 0 center dot 05) and day 21 (r = 0 center dot 439, P < 0 center dot 01) after major burns. These results suggest that thermal injury can lead to an elevation of serum neopterin independent of the burn surface area. The initial increase in the neopterin level may be a part of the acute-phase response to tissue injury itself, whereas the endotoxin release in the circulation may be responsible for the continuous induction of neopterin during the late stage. In addition, the presence of a constant high neopterin level is associated with a critical event in the development of severe burn sepsis.
Adolescent↗