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Biomedical subjects

L Yu

Publications and source records attributed to L Yu.

At least 469 records · Page 26Linked to original sources
PubMed1996

Backbone dynamics of the C-terminal domain of Escherichia coli topoisomerase I in the absence and presence of single-stranded DNA.

The backbone dynamics of the C-terminal DNA-binding domain of Escherichia coli topoisomerase I has been characterized in the absence and presence of single-stranded DNA by NMR spectroscopy. 15N spin-lattice relaxation times (T1), spin-spin relaxation times (T2), and heteronuclear NOEs were determined for the uniformly 15N-labeled protein. These data were analyzed by using the model-free formalism to derive the model-free parameters (S2, tau e, and R(ex)) for each backbone N-H bond vector and the overall molecular rotational correlation time (tau m)., The molecular rotational correlation time tau m was determined to be 7.49 +/- 0.36 ns for the free and 12.7 +/- 1.07 ns for the complexed protein. Several residues were found to be much more mobile than the average, including 11 residues at the N-terminus, 2 residues at the C-terminus, and residues 25 and 31-35 which are located in a region of the protein that binds to DNA. The binding of ssDNA to the free protein causes a slight increase in the order parameters (S2) for a small number of residues and a slight decrease in the order parameters (S2) for the majority of the residues. In particular, upon binding to ssDNA, the mobility of the first alpha-helix and the two beta-sheets was slightly increased, and the mobility of a few specific residues in the loops/turns was restricted. These results differ from the previous studies on the backbone dynamics of molecular complexes in which reduced mobilities were typically observed upon ligand binding.

Base Sequence↗
PubMed1996

Crystallization and preliminary structure of beef heart mitochondrial cytochrome-bc1 complex.

The method reported for isolation of ubiquinol-cytochrome-c reductase complex from submitochondrial particles was modified to yield a preparation for crystallization. The cytochrome bc1 complex was first crystallized in large thin plate form and diffracts X-rays to 7 A resolution in the presence of mother liquor. This crystalline complex was enzymatically active and contains ten protein subunits. It had 33 mol phospholipid and 0.6 mol ubiquinone per mol protein. With slightly modified crystallization conditions, different crystal forms were obtained. Crystals grown in the presence of 20% glycerol diffracted X-rays up to 2.9 A resolution using a synchrotron source. Four heavy atom derivatives have been obtained. The 3-D structure of the cytochrome bc1 complex was solved to 3.4 A resolution. Crystalline cytochrome bc1 complex is a dimer: most of the masses of core proteins I and II protrudes from the matrix side of the membrane, whereas the cytochrome b protein is located mainly within the membrane. There are 13 transmembrane helices in each monomer. Most of the mass of cytochrome c1 and iron-sulfur protein including their redox centers are located on the cytoplasmic side of the membrane. The distances between these redox centers have been determined, and several electron transfer inhibitor binding sites in the complex have been located.

Animals↗
PubMed1996

Cloning and characterization of cDNAs encoding the epsilon-subunit of eukaryotic initiation factor-2B from rabbit and human.

A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the epsilon-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two V8 fragments of purified nonrecombinant eIF-2B epsilon in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2B epsilon. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2B epsilon mRNA were obtained by 5' RACE. A human eIF-2B epsilon cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in lambda gt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2B epsilon polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single approximately 2.8 kilobase mRNA species which is ubiquitously expressed.

Amino Acid Sequence↗
PubMed1996

The weaver mutation changes the ion selectivity of the affected inwardly rectifying potassium channel GIRK2.

The weaver mutation in mice has recently been identified as a single base-pair mutation in the Girk2 gene, which encodes a G-protein-activated inwardly rectifying potassium channel, GIRK2. The mutation results in a Gly to Ser substitution at residue 156, in the putative pore-forming region of the potassium channel. In the present study, we used Xenopus oocytes to express mutant GIRK2, and to characterize the effects of the mutation on the channel. The mutation results in a loss of the normal high selectivity for K+ over Na+, with little effect on other channel properties such as activation by the mu opioid receptor. The resulting increase in basal Na+ permeability causes a marked depolarization of oocytes expressing the mutant GIRK2 protein. This result was observed even when the mutant GIRK2 was coexpressed with GIRK1, a situation more analogous to that seen in vivo. Thus, the increased Na+ permeability and resulting depolarization may contribute to the pathology of cerebellar granule cells and substantia nigra dopaminergic neurons observed in the weaver mice.

Analgesics↗
PubMed1996

The effects of nitric oxide on electron transport complexes.

The effect of nitric oxide on mitochondrial electron transfer complexes was studied by comparing the activities of nitric oxide-treated and untreated, deoxygenated samples of purified beef heart succinate-cytochrome c reductase, succinate-ubiquinone reductase, and ubiquinol-cytochrome c reductase. More than 90% of succinate-cytochrome c reductase activity is lost during nitric oxide treatment. The activity of the succinate-ubiquinone reductase component of succinate-cytochrome c reductase decreases 95%, while the ubiquinol-cytochrome c reductase component is unaffected by nitric oxide. This inactivation is due primarily to the destruction of iron-sulfur clusters from succinate-ubiquinone reductase. When purified beef heart succinate-ubiquinone reductase was treated with nitric oxide, virtually all activity was irreversibly lost. The electron paramagnetic resonance (EPR) spectra of the treated complex showed typical iron-nitric oxide complex signals, confirming that inactivation is due to destruction of the iron-sulfur clusters. Similar results were obtained with purified Escherichia coli succinate-ubiquinone reductase. Pure beef heart ubiquinol-cytochrome c reductase treated with nitric oxide loses 40% of its initial activity, but regains most of it (90-100 % after 24 h of incubation at 0 degrees C in the absence of nitric oxide. This suggests that ubiquinol-cytochrome c reductase is protected from nitric oxide when complexed with succinate-ubiquinone reductase or that when split from succinate-ubiquinone reductase, ubiquinol-cytochrome c reductase undergoes a conformational change which allows access of nitric oxide to the Rieske iron-sulfur center. Such access is not possible when ubiquinol-cytochrome c reductase is complexed with succinate-ubiquinone reductase. The loss of ubiquinol-cytochrome c reductase activity correlates with a decrease in the Rieske protein EPR signal intensity without formation of any new EPR signal. The Rieske iron-sulfur cluster signal is recovered after 24 h incubation in the absence of nitric oxide.

Animals↗
PubMed1996

RIG-E, a human homolog of the murine Ly-6 family, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell.

In vivo all-trans-retinoic acid (ATRA), a differentiation inducer, is capable of causing clinical remission in about 90% of patients with acute promyelocytic leukemia (APL). The molecular basis for the differentiation of APL cells after treatment with ATRA remains obscure and may involve genes other than the known retinoid nuclear transcription factors. We report here the ATRA-induced gene expression in a cell line (NB4) derived from a patient with APL. By differential display-PCR, we isolated and characterized a novel gene (RIG-E) whose expression is up-regulated by ATRA. The gene is 4.0 kb long, consisting of four exons and three introns, and is localized on human chromosome region 8q24. The deduced amino acid sequence predicts a cell surface protein containing 20 amino acids at the N-terminal end corresponding to a signal peptide and an extracellular sequence containing 111 amino acids. The RIG-E coded protein shares some homology with CD59 and with a number of growth factor receptors. It shares high sequence homology with the murine LY-6 multigene family, whose members are small cysteine-rich proteins differentially expressed in several hematopoietic cell lines and appear to function in signal transduction. It seems that so far RIG-E is the closest human homolog of the LY-6 family. Expression of RIG-E is not restricted to myeloid differentiation, because it is also present in thymocytes and in a number of other tissues at different levels.

Amino Acid Sequence↗
PubMed1996

Potential induced redox reactions in mitochondrial and bacterial cytochrome b-c1 complexes.

Purified cytochrome b-c1 complexes from beef heart mitochondria and Rhodobacter sphaeroides were reconstituted into potassium-loaded asolectin liposomes for studies of the energy-dependent electron transfer reactions within the complexes. Both complexes in a ubiquinone-sufficient state exhibit antimycin-sensitive reduction of cytochromes b (both low and high potential ones) upon induction of a diffusion potential by valinomycin in the presence of ascorbate. Addition of N,N,N',N'-tet-ramethyl-p-phenylenediamine (TMPD) to the ascorbate-reduced potassium-loaded asolectin proteoliposomes resulted in reduction of cytochrome b262. Upon addition of valinomycin, the induced diffusion potential caused a partial reoxidation of cytochrome b562 and partial reduction of cytochrome b566 in beef heart cytochrome b-c1 complex in the presence of antimycin and/or myxothiazol. Surprisingly, when ubiquinone-depleted beef heart cytochrome b-c1 complex liposomes were treated under the same conditions, no cytochrome b566 reduction was observed but only the oxidation of cytochrome b562, and the oxidation was not oxygen-dependent. We explain this effect by b566, iron-sulfur protein short-circuiting under these conditions, assuming that both antimycin and myxothiazol markedly affect subunit b conformation. The electrochemical midpoint potential of heme b566 appears to be significantly higher than that of heme b562 in the presence of myxothiazol, which cannot be accounted for only by the potential-driven electron transfer between these two hemes plus the shift in chemical midpoint potentials caused by myxothiazol. A model for energy coupling consistent with structural findings by Ohnishi et al. (Ohnishi, T., Schagger, H., Meinhardt, S. W., LoBrutto, R., Link, T. A., and von Jagow, G. (1989) J. Biol. Chem. 264, 735-744) is presented. This model is a compromise between pure "redox-loop" and pure "proton-pump" mechanisms. Reoxidation of high potential heme b is observed in an antimycin- or antimycin plus myxothiazol-inhibited, ascorbate plus TMPD-prereduced R. sphaerodies b-c1 complex, upon membrane potential development, suggesting that a similar electron transfer mechanism is also operating in the bacterial complex.

Animals↗
PubMed1996

Axial heme ligation in the cytochrome bc1 complexes of mitochondrial and photosynthetic membranes. A near-infrared magnetic circular dichroism and electron paramagnetic resonance study.

The combination of EPR and low-temperature near-IR magnetic circular dichroism spectroscopies have been used to investigate the axial ligation of the cytochromes in the cytochrome bc1 complexes from bovine heart mitochondria, Rhodobacter capsulatus, Rhodobacter sphaeroides, and Rhodospirillum rubrum, and the purified cytochromes c1 from bovine heart mitochondria, Rb. capsulatus and Rb. sphaeroides. The possibility of axial ligation of cytochrome c1 by the amino terminus of the polypeptide was also assessed by acetylating the N-terminus of Rb. capsulatus cytochrome c1 and comparing the properties of the acetylated and unmodified samples. The results are consistent with bis-histidine axial ligation for the high- and low-potential b-type cytochromes and histidine/methionine axial ligation for the c1-type cytochrome in the intact cytochrome bc1 complexes. Purified samples of cytochrome c1 are mixtures of two forms, one with histidine/methionine and the other with bis-histidine axial ligation. The form with bis-histidine axial ligation is also assembled in the M183L mutant of the Rb. capsulatus cyt bc1 complex in which the methionine residue coordinating cyt c1 is replaced by a leucine. The bis-histidine form appears to be an artifact of dissociation of cytochrome c1 from the cytochrome bc1 complex and is greatly enhanced particularly in the bacterial cytochromes c1 by sample handling and the addition of 50% (v/v) ethylene glycol or glycerol.

Amino Acid Sequence↗
PubMed1996

Functional expression and signaling properties of cloned human parathyroid hormone receptor in Xenopus oocytes. Evidence for a novel signaling pathway.

Expression of human parathyroid hormone receptor (hPTHR) was obtained in Xenopus oocytes. Receptor function was detected by hormone stimulation of endogenous Ca2+-activated Cl- current. This current was blocked by injected, but not by extracellular, EGTA, confirming that the hPTHR activates cytosolic Ca2+ signaling pathways. PTH responses were acutely desensitized but were regained in 6 12 h. Injection of cAMP or analogues had no effect on either responsiveness or desensitization to hPTH. The hPTH response was more sluggish than seen with serotonin 5-hydroxytryptamine (5-HT2C) receptor. In oocytes co-expressing both hPTHR and 5-HT2C receptors, homologous desensitization was seen, but cross-desensitization was not observed. Injection of inositol 1,4,5-trisphosphate (InsP3) elicited a fast inward current similar to that induced by serotonin, and complete cross-desensitization occurred between the InsP3 and 5-HT2C responses. Desensitization by hPTH did not affect responses to either InsP3 or serotonin, but cells desensitized to injected InsP3 still responded strongly to PTH. Oocytes did not respond to either cADPR or NAADP+, but NADP+ and analogues were found to be potent inhibitors of PTH signaling. We suggest that PTH cytosolic Ca2+ signaling in oocytes either involves a novel signaling system or proceeds through a Ca2+ compartment whose responsiveness is regulated in a novel way.

Animals↗
PubMed1996

Radiation therapy of breast carcinoma: confirmation of prescription dose using diodes.

PURPOSE: To quantitate the dose delivered during tangential breast radiation therapy and measure the scatter dose to the contralateral breast for three different breast setup techniques. METHODS AND MATERIALS: A commercial semiconductor diode system is used for dose measurements. The diode characteristics were studied by comparing the diode response against a standard ionization chamber response in a reference configuration. In vivo dose measurements on 11 patients undergoing tangential breast radiation therapy with 6 MV photons were performed. Medial and lateral field entrance and exit doses were measured and compared with the expected values from the treatment planning system. Scatter doses to the contralateral breast for three breast setup techniques were measured and documented as a function of distance from the field edge and various beam modifiers commonly used in breast radiation therapy. RESULTS: The diodes used in this study exhibited excellent linearity, dose reproducibility, and minimal anisotropy. The in-phantom measurements resulted in dose accuracy within +/- 1.5%. Dose measurements on patients resulted in standard deviations of 1.2 and 2.3% for the medial entrance and exit doses and 1.7 and 2.2% for the lateral entrance and exit doses, respectively. In patients, the scatter doses to the opposite breast at a 5 cm perpendicular distance from the medial field edge resulted in cumulative scatter doses of 2.47 to 5.30 Gy from the tangential fields and an additional 0.50 Gy from the supraclavicular or axillary field, if included. CONCLUSION: Quantitative verification of the prescribed daily dose is important in breast radiation therapy to ensure precision in patient setup and accuracy in dose delivery. Diodes provide a convenient way of real-time patient dose verification and are easy to use by the therapists.

Breast Neoplasms↗
PubMed1996

Functional analysis of dsRNAs (L1, L3, L5, and M2) associated with isometric 34-nm virions of Agaricus bisporus (white button mushroom).

cDNA clones of dsRNAs associated with La France disease of Agaricus bisporus were isolated. Clones corresponding to L1 and L5 dsRNAs were sequenced. The deduced amino acid sequence of L1 dsRNA (1078 amino acids, Mr 121K) showed significant homology with RNA-dependent RNA polymerases of other dsRNA viruses. The deduced amino acid sequence of L5 dsRNA (724 amino acids, Mr 82K) showed no homology with known proteins. Amino acid sequences of tryptic digests of three virion-associated proteins were determined. The 34-nm virion-associated protein of Mr 115K was encoded by the L1 dsRNA, thus identifying this protein as the RNA-dependent RNA polymerase. The virion-associated protein of Mr 90K was encoded by the previously sequenced L3 dsRNA. A cDNA clone of the previously sequenced M2 dsRNA was expressed in Escherichia coli and antibodies raised against this protein reacted only with a protein present in the cytoplasm of diseased A. bisporus fruit bodies but not in the 34-nm virions.

Agaricus↗
PubMed1996

Functional expression of subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex and reconstitution of recombinant protein with three-subunit core complex.

Subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex was over-expressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-RSIV) using the expression vector, pGEX/RSIV. Maximum yield of soluble active recombinant fusion protein was obtained from cells harvested 3 h after induction of growth at 37 degrees C in LB medium. Subunit IV was released from the fusion protein by proteolytic cleavage with thrombin. When subjected to SDS-polyacrylamide gel electrophoresis, isolated recombinant subunit IV of R. sphaeroides cytochrome b-c1 complex. Although the isolated recombinant subunit IV is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1000 kDa. The addition of detergent deaggregates the isolated protein, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. When the three-subunit core cytochrome b-c1 complex, purified from RS delta IV-adapted chromatophores containing a fraction of the wild type cytochrome b-c1 complex activity, was reacted with varying amounts of recombinant subunit IV, the activity increased as the subunit IV concentration increased. Maximum activity restoration was reached when 1 mol of subunit IV/mol of three-subunit core complex was used. The reconstituted cytochrome b-c1 complex is similar to the wild-type complex in molecular size, apparent Km for Q2H2, and inhibitor sensitivity, indicating that recombinant subunit IV is properly assembled into the active cytochrome b-c1 complex. A tryptophan residue in subunit IV was found to be involved in the interaction with the three-subunit core complex.

Base Sequence↗
PubMed1996

Irreversible binding of cis-(+)-3-methylfentanyl isothiocyanate to the delta opioid receptor and determination of its binding domain.

Binding of cis-(+)-3-methylfentanyl isothiocyanate (SUPERFIT) to cloned opioid receptors stably expressed in Chinese hamster ovary cells was characterized. SUPERFIT inhibited [3H]diprenorphine binding with much higher affinity for the delta than the mu or kappa receptor. Pretreatment with SUPERFIT followed by extensive washing reduced delta binding with an IC50 value of 7.1 nM, yet it did not affect mu and kappa binding up to 0.1 microns. The reduction in delta binding by SUPERFIT pretreatment was due to a decrease in Bmax with no change in Kd. These results indicate that SUPERFIT is a highly selective delta irreversible ligand. We then determined the region in the delta receptor that confered binding selectivity for SUPERFIT by examining its binding to six mu/delta chimeric receptors. SUPERFIT bound to delta, mu/delta 1 (amino acids mu 1-94/delta 76-372), delta/mu 3 (delta 1-134/mu 154-398), and delta/mu 4 (delta 1-187/mu 207-398) receptors with high affinity but to mu, delta/mu 1 (delta 1-75/mu 95-398), mu/delta 3 (mu 1-153/delta 135-372), and mu/delta 4 (mu 1-206/delta 188-372) receptors with low affinity. Pretreatment with SUPERFIT potently inhibited [3H]diprenorphine binding to delta, mu/delta 1, delta/mu 3, and delta/mu 4 but affected binding to mu, delta/mu 1, mu/delta 3, and mu/delta 4 only at much higher concentrations. Thus, the segment from the beginning of the first intracellular loop to the middle of the third transmembrane helix of the delta receptor is important for selective binding of SUPERFIT.

Amino Acid Sequence↗
PubMed1996

Preparation of an intra-laboratory reference material--determination of the aluminum content of a pooled 5% albumin (human) solution by ETAAS, MFS and ICP-AES.

Human albumin solutions as well as other biological products intended for human use may contain aluminum as a result of intentional addition due to manufacturing protocol or through inadvertent contamination occurring during the manufacturing process. Because of the implication of aluminum in human disease, it is desirable that aluminum levels in these products be monitored. To satisfy the need for a reference material that is representative of albumin products that are intended for therapeutic use, a pooled 5% human albumin solution was prepared in bulk to serve as a reference for aluminum during analytical procedures. Aliquots of the albumin pool were analyzed for their aluminum content by three independent analytical methods, namely, electrothermal atomic absorption, molecular fluorescence and inductively coupled plasma-atomic emission spectrometry. Measured values for aluminum in the albumin solution showed good agreement between the different analytical methods. The concentration of aluminum was determined to be 0.512 microgram Al/ml by the atomic absorption method, 0.502 microgram Al/ml by molecular fluorescence, and 0.498 microgram Al/ml by the inductively coupled plasma-atomic emission method. The overall average concentration was calculated to be 0.507 microgram/ml +/- 0.009 micrograms Al/ml. The preparation of the albumin reference and its subsequent aluminum analysis is described.

Aluminum↗