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L Yu

Publications and source records attributed to L Yu.

At least 595 records · Page 33Linked to original sources
PubMed1994

Role of subunit IV in the cytochrome b-c1 complex from Rhodobacter sphaeroides.

Rhodobacter sphaeroides mutants lacking subunit IV (M(r) = 14,384) of the cytochrome b-c1 complex (representative mutant strain, RS delta IV-2) have been constructed by site-specific recombination between the wild-type genomic subunit IV structural gene (fbcQ) and a suicide plasmid containing a defective fbcQ sequence. RS delta IV-2 gives rise to a photosynthetically competent phenotype after a period of adaptation. The chemical compositions, spectral properties, and cytochrome b-c1 complex activities in subunit IV-deficient chromatophores from adapted RS delta IV-2 are similar to those in wild-type chromatophores. However, the apparent Km for Q2H2 for the b-c1 complex in subunit IV-deficient chromatophores from adapted RS delta IV-2 cells is about four times higher than that in chromatophores from wild-type cells. The cytochrome b-c1 complex activity in subunit IV-deficient chromatophores of adapted RS delta IV-2 cells is more labile to detergent treatment than that from wild-type cells. The specific activities of dodecylmaltoside-solubilized fractions of RS delta IV-2, based on cytochrome b, are only one-fourth that of the untreated chromatophores. Introducing a wild-type fbcQ operon on a stable low copy number plasmid, pRK415, into RS delta IV-2 restores photosynthetic growth behavior, the apparent Km value for Q2H2, and tolerance to detergent treatment to that of wild-type cells. Cytochrome b-c1 complex purified from adapted RS delta IV-2 contains only three subunits. It has only 25% of the activity of the four-subunit enzyme. This low activity is accompanied by an increase of the apparent Km for Q2H2 from 3 to 13 microM, suggesting that subunit IV may be involved in quinone binding in addition to its structural role.

Base Sequence↗
PubMed1994

Delta opioid receptor gene expression in lymphocytes.

Previous studies have shown that morphine stimulates simian immunodeficiency virus (SIV) replication in SIV-infected human CEM x174 cells as well as in monkey lymphocytes through a mechanism of delaying the lysis of infected cells (Biochem. Biophys. Res. Commun. 195:1165-1173, 1993). The present study describes the identification of brain-like opioid receptor sequences in RNA transcripts of both CEM x174 cells and monkey lymphocytes. Study of the gene sequence of a lymphocyte opioid receptor encompassing the third transmembrane domain and the third cytoplasmic loop indicates a 96% homology in amino acid composition to the delta opioid receptor in brain cells. Expression of such an opioid receptor sequence in lymphocytic cells is constitutive, since it could be detected in both saline-treated and morphine-treated monkeys as well as in morphine-treated monkeys after detoxification.

Amino Acid Sequence↗
PubMed1994

Expression of mu opioid receptor mRNA in rat brain: an in situ hybridization study at the single cell level.

The mu (mu) opioid receptors, which mediate the effects of morphine, are widely distributed in brain. We have examined the distribution of mRNA encoding a mu opioid receptor in rat brain with in situ hybridization histochemistry at the single-cell level to obtain information about the cell types synthesizing this receptor. Only neurons, not glia, were labeled in discrete brain regions. High levels of labeling were detected in the thalamus, striosomes of the caudate-putamen, globus pallidus, and brain regions involved in nociception, arousal, respiratory control, and, possibly, addiction. The general distribution of the receptor mRNA paralleled that of mu opioid binding sites with some notable exceptions. These include the cerebral cortex, which contains binding sites, but very few labeled neurons. No labeling was observed in the cerebellum, a region devoid of mu binding sites. Three main findings emerged from these experiments: 1) the mRNA was present in regions mediating both the therapeutic (analgesia) and the unwanted (respiratory depression, addiction) effects of morphine, 2) the mRNA was very densely expressed by neurons known to receive dense enkephalin-containing inputs, and 3) the dissociation between the presence of binding sites and absence of mRNA in some brain regions supports a presynaptic localization of mu opioid receptors in these areas. Alternatively, other subtypes of mu opioid receptors may be encoded by a different mRNA. These results provide new insights into the receptor types and neuronal circuits involved in the effects of endogenous opioids and morphine.

Animals↗
PubMed1994

Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family.

A cDNA was isolated from rat brain by low stringency hybridization with the rat mu opioid receptor cDNA. Sequence analysis of this clone indicated that it contains an open reading frame capable of encoding a 367 amino acid protein. The deduced amino acid sequence of this protein shows high degrees of homology to all three opioid receptors, mu, kappa, and delta, suggesting that it is a member of the opioid receptor gene family. RNA blot analysis detected high level expression of the receptor mRNA in the brain. Southern blot analysis suggests that it is a single-copy gene, and mapping studies localized the gene on mouse chromosome 2. Despite the high sequence homologies between this protein and the other opioid receptors, expression studies of this clone in COS-7 cells did not show binding to [3H]diprenorphine, a ligand that binds to the other three opioid receptors. Furthermore, co-expression of this receptor with a G protein-activated potassium channel in Xenopus oocytes did not show functional coupling upon stimulation with mu, kappa and delta agonists. Given the similar degrees of high homology to the mu, kappa and delta opioid receptors and the lack of apparent affinity for their ligands, this receptor does not appear to belong to any of the three known classes of opioid receptors. Rather, it represents a novel member of the opioid receptor gene family, not identified from previous pharmacological studies.

Amino Acid Sequence↗
PubMed1994

Differential regulation by cAMP-dependent protein kinase and protein kinase C of the mu opioid receptor coupling to a G protein-activated K+ channel.

A mu opioid receptor and a G protein-activated K+ channel were coexpressed in Xenopus oocytes. Stimulation of the mu opioid receptor induced an inwardly rectifying current that was blocked by opioid receptor antagonist naloxone, indicating that the mu opioid receptor is functionally coupled to the K+ channel. The coupling is mediated by G proteins, since pertussis toxin treatment reduced the K+ current and injection of GTP gamma S (guanosine 5'-O-(thiotriphosphate)) enhanced it. Repeated stimulation of the mu receptor leads to desensitization, as the K+ current from the second stimulation was reduced to 70% of that from the first one. Both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) regulate this process, but in opposite direction. Activation of PKC by treatment of the oocyte with phorbol ester potentiated the desensitization of the mu receptor-induced current. However, incubation of the cell with a membrane-permeable cAMP analog, 8-chlorophenylthio-cAMP, completely abolished the desensitization. The cAMP effect appears to be mediated by PKA, since injection of a PKA catalytic subunit showed the same effect as cAMP incubation. These results suggest that PKA and PKC differentially regulate the mu opioid receptor coupling to the G protein-activated K+ channel.

Animals↗
PubMed1994

Nitric oxide: a mediator in rat tubular hypoxia/reoxygenation injury.

Nitric oxide (NO), among several other functions, may play a role in hypoxia and reoxygenation injury due to its free radical nature and high reactivity with the superoxide radical to yield peroxynitrite, an oxidant molecule. The present study was undertaken to evaluate a potential role for NO, either endogenous or exogenous, in a model of hypoxia/reoxygenation (H/R) in freshly isolated rat proximal tubules. NO synthase activity, as assessed by conversion of L-[3H]arginine to L-[3H]citrulline, was detected in normoxic tubules. This activity could be inhibited by N-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor, and was stimulated by 15 min of hypoxia. The injury in proximal tubules caused by 15 min of hypoxia followed by 35 min of reoxygenation was completely prevented by L-NAME as assessed by release of lactate dehydrogenase, whereas D-NAME, which does not inhibit NO synthase, had no effect. In contrast, L-arginine (NO substrate) enhanced the H/R injury. These effects were paralleled by nitrite/nitrate production. In separate experiments, the addition of sodium nitroprusside, a NO donor, to proximal tubules enhanced the H/R injury; this effect could be blocked by hemoglobin, a NO scavenger. Also, addition of nitroprusside reversed L-NAME protection against H/R injury. These results demonstrate that NO is synthesized in rat proximal tubules and participates as one of the mediators in rat tubular H/R injury.

Amino Acid Oxidoreductases↗
PubMed1994

1-Hexadecyl-2-arachidonoylthio-2-deoxy-sn-glycero-3-phosphorylcholine as a substrate for the microtiterplate assay of human cytosolic phospholipase A2.

Human cytosolic phospholipase A2 (cPLA2) is an 85-kDa protein which displays a preference for arachidonoyl phospholipids as substrates. This substrate preference and the assay characteristics of the enzyme are quite different from those of the smaller, more well-studied extracellular PLA2s. We now report the development of a nonradioactive, spectrophotometric, microtiterplate assay for human cPLA2 using a novel synthetic thio-phospholipid analog as a substrate. This substrate is a phosphatidylcholine derivative with an arachidonoylthioester in the sn-2 position and an alkyl-ether in the sn-1 position. The use of an sn-1 alkyl-ether in the substrate ensures that the assay will only measure PLA2 activity and will not be complicated by the metabolism of the lysophospholipid product by the enzyme's lysophospholipase activity. cPLA2 is assayed at pH 7.4 and 37 degrees C with a mixed micellar substrate consisting of 2 mM thio-phospholipid and 4 mM Triton X-100 in 30% glycerol. Under these conditions, the assay is fairly linear for over 1 h.

Cytosol↗
PubMed1994

Enediyne-mediated DNA damage in nuclei is modulated at the level of the nucleosome.

DNA damage in HeLa nuclei and isolated nucleosome core particles has been examined for several members of the enediyne family of antitumor antibiotics: calicheamicin gamma 1I (CAL), esperamicin A1 (ESP A1), esperamicin C (ESP C), and neocarzinostatin (NCS). In nuclei, both NCS and ESP A1 produced DNA damage limited to the linker region of the nucleosome, while CAL and ESP C, an analog of ESP A1 missing the deoxyfucose-anthranilate moiety, damaged both the core and linker DNA. DNA fragments produced by CAL and ESP C in the nucleosome core occurred with a 10-11-nucleotide periodicity similar to that produced by DNase I, while damage produced by NCS and ESP A1 appeared to be limited to the terminal portions of the core DNA. The damage in nuclei is shown to be caused directly by the drugs with little contribution from endogenous factors, such as nucleases and topoisomerases. Features of drug structure that may limit damage to the nucleosome core include the presence of substituents on both sides of the CAL/ESP-type core, and the presence of an intercalating moiety, such as the naphthoate of NCS and possibly the anthranilate of ESP A1.

Aminoglycosides↗
PubMed1994

Function and organization of photosystem I in a cyanobacterial mutant strain that lacks PsaF and PsaJ subunits.

Photosystem I functions as a light-driven plastocyanin-ferredoxin oxidoreductase in the photosynthetic membranes of cyanobacteria and chloroplasts. A mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 that contains a deletion of the psaF gene and a transcriptionally inactive psaJ gene has assembled photosystem I complexes that lack PsaF, a lumenal protein and PsaJ, a 4-kDa hydrophobic protein. The cells of the mutant and wild type strains have similar rates of photosynthetic electron transfer and P700+ rereduction under linear and cyclic electron transfer conditions. Analysis of flash-induced absorption transients at 700 nm demonstrate that the absence of PsaF in purified mutant photosystem I did not affect the rate of P700 rereduction by cytochrome c553. Therefore, PsaF is not essential for docking of cytochrome c553. We also studied the organization of the proteins of mutant and wild type photosystem I by comparing their accessibility to digestion by thermolysin or to removal by 1 M NaI. The PsaA-PsaB subunits were more easily degraded by thermolysin in the mutant photosystem I. Thermolysin cleavage of PsaB yielded two major fragments that were immunoreactive with an antibody raised against the C terminus of PsaB. The N termini of these PsaB peptides mapped at Ile482 and Ile498 residues, thus identifying a surface-exposed domain of the core of photosystem I. The PsaE subunit could be removed by 1 M NaI and was rapidly digested by thermolysin in the mutant but not in the wild type photosystem I. Therefore, PsaF and PsaJ subunits of photosystem I have dispensable accessory roles in the function and organization of the complex.

Amino Acid Sequence↗
PubMed1994

Protein-ubiquinone interaction: synthesis and biological properties of ethoxy ubiquinone derivatives.

For investigation of the protein-ubiquinone interaction in the succinate-cytochrome c reductase region of the bovine heart mitochondrial electron-transport chain, ethoxy-substituted ubiquinone derivatives, 2-ethoxy-3-methoxy- or 3-ethoxy-2-methoxy-5-methyl-6-decyl-1,4-benzoquinone (EtOQ0C10) and 2,3-diethoxy-5-methyl-6-decyl-1,4-benzoquinone [(EtO)2Q0C10], were synthesized and characterized. These compounds were synthesized from 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (Q0C10) by reaction with sodium ethoxide/ethanol in hexane under anaerobic conditions. The products, EtOQ0C10 and (ETO)2Q0C10, were separated by thin-layer chromatography using hexane/ether (3.5:1) as the developing solvent. The Rf values for diethoxy and monoethoxy derivatives are 0.7 and 0.6, respectively. The spectral and redox properties of EtOQ0C10 and (ETO)2Q0C10 are very similar to those of Q0C10. The reducibility of these derivatives by succinate was measured with succinate-Q reductase (SQR), and their oxidizability was measured by ubiquinol-cytochrome c reductase (QCR). Ethoxy ubiquinone derivatives exhibit concentration-dependent inhibition of SQR activity, with (ETO)2Q0C10 being the more potent inhibitor. These derivatives do not inhibit QCR and are reduced by succinate-cytochrome c reductase in an antimycin-insensitive manner. When used as substrate for QCR, EtOQ0C10H2 has about 55%, and (ETO)2Q0C10H2 about 15%, of the activity of Q0C10H2, but with lower apparent Km values. The low efficiency of these compounds as electron donors is apparently not due to their weak binding to QCR. These results indicate that the binding environment of the benzoquinone ring in succinate-Q reductase is very specific and differs from that in ubiquinol-cytochrome c reductase.

Animals↗