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Biomedical subjects

L Yu

Publications and source records attributed to L Yu.

At least 649 records · Page 36Linked to original sources
PubMed1993

Targeted inactivation of the gene psaL encoding a subunit of photosystem I of the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I is a multisubunit pigment-protein complex that functions as a light-driven plastocyanin-ferredoxin oxidoreductase in thylakoid membranes of cyanobacteria and higher plants. A 16-kDa protein subunit of photosystem I complex was isolated from the cyanobacterium Synechocystis sp. PCC 6803. The sequence of its NH2-terminal residues was determined and a corresponding oligonucleotide probe was used to isolate the gene encoding this subunit. The gene, designated as psaL, codes for a protein of 16,605 Da. The deduced amino acid sequence is homologous to the subunit PsaL of barley photosystem I. There are two conserved hydrophobic regions in the subunit PsaL that may cross or interact with thylakoid membranes. The gene psaL exists as a single copy in the genome and is expressed as a monocistronic RNA. Stable mutant strains in which the gene psaL was interrupted by a gene conferring resistance to chloramphenicol, were generated by targeted mutagenesis. Growth and photosynthetic characteristics of a selected mutant strain under photoautotrophic conditions were similar to those of the wild type, suggesting that the function of PsaL is dispensable for photosynthesis in Synechocystis sp. PCC 6803. Western analysis and subunit composition of purified photosystem I revealed that the mutant strain contained other subunits of photosystem I in thylakoid membranes and in the assembled complex. When photosystem II activity was inhibited and glucose was supplied in the medium, mutant strains grew faster than the wild type. Under these conditions of growth, re-reduction of P700 in the mutant cells, but not in the wild type cells, showed a component with an uncharacteristically rapid half-time.

Amino Acid Sequence↗
PubMed1993

The mitochondrial targeting presequence of the Rieske iron-sulfur protein is processed in a single step after insertion into the cytochrome bc1 complex in mammals and retained as a subunit in the complex.

The amino acid sequence of subunit 9 of the bovine heart cytochrome bc1 complex is identical to the 78-amino acid presequence that is removed post-translationally from the Rieske iron-sulfur protein as it is imported and targeted to the mitochondrial cytochrome bc1 complex. Iron-sulfur protein precursor, generated by in vitro transcription and translation, is processed to mature size in a single step when incubated with rat liver mitochondria, and generates a peptide that comigrates on SDS-polyacrylamide gel electrophoresis with subunit 9. These results suggest that the Rieske protein is processed in a single proteolytic step after it is inserted into the cytochrome bc1 complex in mammals, and that the processed presequence remains as a subunit of the complex. This is apparently the first instance in which a cleaved targeting presequence has been shown to be retained in the cell, possibly exhibiting a second function in addition to its function in protein trafficking.

Amino Acid Sequence↗
PubMed1993

Three distinct nuclear protein binding sites in the promoter of the murine multidrug resistance mdr1b gene.

Multidrug resistance in mammalian cells is often associated with the overproduction of a membrane glycoprotein, P-glycoprotein, that is encoded by mdr genes. Multidrug resistance cell lines selected with either vinblastine, colchicine, or taxol from the drug-sensitive murine macrophage-like cell line J774.2 overexpress the mdr1a and/or mdr1b genes, and overproduce P-glycoprotein. To elucidate the mechanisms of mdr1b gene expression, the mdr1b 5'-flanking sequences have been isolated from a normal mouse liver genomic library and analyzed by gel shift and DNase I footprinting assays. These analyses have demonstrated three nuclear protein binding sites, from -82 to -59 (site 1), from -123 to -101 (site 2), and from -272 to -249 (site 3), which interact with proteins present in nuclear extracts from both sensitive and resistant cells. Although site 1 contains a partially conserved AP-2 consensus sequence, our results indicate that the nuclear protein binding to site 1 is not AP-2 protein. The sequence of site 2 is conserved in the murine mdr1a, human mdr1, and hamster pgp1 promoters. Such conservation suggests that this sequence may have an important role in mdr gene expression. The use of a transient chloramphenicol acetyltransferase expression vector containing the basal promoter for herpes simplex virus thymidine kinase (tkCAT) and either site 1 or site 2 or both revealed that the sequences of sites 1 and 2 enhanced tkCAT activity. DNase I footprinting analyses demonstrated that site 3 is recognized by human AP-1 protein, indicating that the nuclear protein binding to this site is an AP-1-like protein. These observations suggest that mdr1b gene expression is mediated by preexisting transcription factors present in sensitive and resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Solution structure of the cyclosporin A/cyclophilin complex by NMR.

Cyclosporin A, a cyclic undecapeptide, is a potent immunosuppressant that binds to a peptidyl-prolyl cis-trans isomerase of 165 amino acids, cyclophilin. The cyclosporin A/cyclophilin complex inhibits the calcium- and calmodulin-dependent phosphatase, calcineurin, resulting in a failure to activate genes encoding interleukin-2 and other lymphokines. The three-dimensional structures of uncomplexed cyclophilin, a tetrapeptide/cyclophilin complex, and cyclosporin A when bound to cyclophilin have been reported. However, the structure of the cyclosporin A/cyclophilin complex has not been determined. Here we present the solution structure of the cyclosporin A/cyclophilin complex obtained by heteronuclear three-dimensional NMR spectroscopy. The structure, one of the largest determined by NMR, differs from proposed models of the complex and is analysed in terms of the binding interactions and structure/activity relationships for CsA analogues.

Amino Acid Isomerases↗
PubMed1993

Evidence for telluroamino acid in biological materials and some rules of assimilation of inorganic tellurium by yeast.

A natural substance, telluroamino acid, was discovered in tellurium yeast by GC and GC/MS. This substance was obtained for the first time by means of the assimilation of inorganic tellurium by yeast, having 600 ppm of total Te and 150 ppm of telluroamino acid. Some rules for such an assimilation were found: the content of total Te and telluroamino acid in tellurium-yeast is closely related to the kind of culture media used and is proportional to the concentration of Te in culture media. This discovery is of theoretical and practical significance, especially for the production by a simpler microbiological synthesis of rare and expensive organic tellurium compounds which hopefully will prove to be anticarcinogens like the selenium-yeast, and for explaining the biochemical effect of tellurium on the human body.

Amino Acids↗
PubMed1993

Identification, chromosomal location, and genome organization of mammalian G-protein-coupled receptors.

Vertebrate G-protein-coupled receptors are encoded by a diverse multigene family. Thirteen distinct G-protein-coupled receptors (Gpcr) were cloned from mouse germline cDNA following amplification in the polymerase chain reaction (PCR) with degenerate oligonucleotide primers complementary to the third and sixth transmembrane domains. Eleven Gpcr clones were mapped to single sites in the mouse genome following interspecific backcross analysis. One clone was mapped to two sites and another was not polymorphic in the cross and could not be mapped. Gpcr loci were well dispersed throughout the mouse genome and mapped to chromosomes 1, 2, 3, 4, 5, 8, 9, 10, 13, 17, and 18. Six Gpcr clones likely represent mouse homologs of already identified receptors, one Gpcr clone may identify a third type of IL-8 receptor, and three Gpcr clones appear to encode novel G-protein-coupled receptors. Further, three factor crosses and Southern blot analyses demonstrated that Gpcr16 maps proximally within the Spr deletion on mouse chromosome 1, near the Vil and Bcg loci. Human chromosomal locations for most Gpcr loci could be predicted based on linkage homologies that have been identified between human and mouse. Mapping additional G-protein-coupled receptors against the panel of murine interspecific backcrosses should expand our understanding of mammalian Gpcr gene evolution and genome organization.

Amino Acid Sequence↗
PubMed1993

Heteronuclear NMR studies of 13C-labeled yeast cell wall beta-glucan oligosaccharides.

The structures of uniformly 13C-labeled beta-glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear beta(1-->3) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a beta(1-->6) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain beta(1-->6) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain beta(1-->6) linkages. Since isolated fungal membranes only synthesize linear beta(1-->3) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.

Candida albicans↗
PubMed1993

Mitochondrial ubiquinol-cytochrome c reductase complex: crystallization and protein: ubiquinone interaction.

The ubiquinol-cytochrome c reductase complex was crystallized in a thin plate form, which diffracts X-rays to 7 A resolution in the presence of mother liquor. This crystalline complex contains ten protein subunits and 140 nmol phospholipid per milligram protein. Over 90% of the phospholipid and ubiquinone in the reductase can be removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The delipidated complex has no enzymatic activity and shows significant changes in the circular dichroism spectrum in the near UV region and in the EPR characteristics of both cytochromes b. Enzyme activity and spectral characteristics can be restored by replenishing the phospholipid and ubiquinone. The structural requirements of ubiquinone for electron transport were studied by measuring the ability of a variety of synthetic ubiquinone derivatives to restore the enzymatic activity and native spectroscopic signatures to the delipidated complex. Q-binding proteins and binding domains were identified using photoaffinity labeled Q-derivatives and HPLC separation of photolabeled peptides. Interaction between ubiquinol-cytochrome c reductase and succinate-Q reductase was established by differential scanning calorimetry and saturation transfer EPR using spin-labeled ubiquinol-cytochrome c reductase. Involvement of iron-sulfur protein in proton translocation by ubiquinol-cytochrome c reductase was investigated by hematorporphyrin-promoted photoinactivation of the complex. The cDNAs encoding the Rieske iron-sulfur protein and a small molecular mass Q-binding protein (QPc-9.5 kDa) were isolated and their nucleotide sequences determined. These will be useful in future structural and mechanistic studies of ubiquinol-cytochrome c reductase via in vitro reconstitution between an over-expressed, mutated subunit and a specific subunit-depleted reductase.

Amino Acid Sequence↗
PubMed1993

[The change of T lymphocyte subpopulations, interleukin-2 receptor and HLA-DR antigen in lupus nephritis patients].

In this study peripheral blood mononuclear cells were tested for T lymphocyte subpopulations, interleukin-2 receptor (IL-2R) and HLA-DR antigen by monoclonal antibodies in 14 LN patients. In comparison with normal controls, 14 LN patients showed a decrease of T4 lymphocyte (0.01 < P < 0.05) and increase of T8 cells (P < 0.01) with reduction of T4/T8 ratio (P < 0.01). The expression of HLA-DR cells increased (P < 0.01), the expression of IL-2R activated by PHA significantly decreased (P < 0.01), while nonactivated IL-2R expression significantly increased (P < 0.01). T4/T8 ratio was significantly correlated with activated IL-2R expression (P < 0.01, r = 0.6599). The results indicated that there was a immunological defect in LN patients. The pathogenesis of LN might be heterogeneous.

Adult↗
PubMed1993

Atrial natriuretic factor enhances the hydroosmotic response of toad bladder to submaximal doses of vasopressin.

Using the toad urinary bladder, we examined the effects of submaximal levels of atrial natriuretic factor (ANF) upon the hydroosmotic water flux caused by physiologic concentrations of vasopressin (VP). Pretreatment with ANF prior to the addition of VP (10(-9)M) significantly enhanced water transport (123 +/- 23%) compared to tissues exposed to VP alone. Pre-treatment with ANF also significantly enhanced the hydroosmotic response (143 +/- 43%) to cyclic-3',5'-adenosine monophosphate (cyclic-AMP). When the concentration of VP was progressively increased during time course experiments, an inhibitory effect of ANF on water transport followed the early stimulatory response to this peptide. The data support a novel, dose-dependent modulatory role for ANF early in the response of transporting epithelia to VP. Moreover, the stimulatory effect of submaximal doses of ANF to cyclic-AMP mediated water transport suggest the possibility that modulation by ANF may occur at a site following the VP receptor-linked adenylate cyclase system.

Animals↗
PubMed1993

Analysis of the promoter sequence and the transcription initiation site of the mouse 5-HT1C serotonin receptor gene.

The serotonin 1c (5-HT1C) receptor is found in many brain regions, but is particularly enriched on the epithelial cells of the choroid plexus. A major challenge in neurobiology is to delineate the molecular processes that regulate the specific pattern of neuronal gene expression in the brain. As an initial step towards identifying cis-acting DNA sequences that control the expression of the 5-HT1C receptor, we have isolated the promoter sequence of its gene. Sequence analysis of a 1.8 kb fragment indicated that the 3' end of this fragment overlaps with the 5' untranslated region of the 5-HT1C receptor mRNA, and primer extension using mouse brain poly(A)+ RNA mapped the transcription initiation site within this fragment. There are a number of sequence elements upstream from the transcription initiation site that are homologous to regulatory elements found in other eucaryotic genes. To determine the promoter activity, a plasmid was constructed that contains this fragment as promoter region and the cDNA for the 5-HT1C receptor as the reporter. When injected into the nucleus of Xenopus oocytes, this construct resulted in functional expression of the reporter gene. Primer extension using the RNA extracted from the injected oocytes indicated a single transcription initiation site of the reporter mRNA. These results suggest that the 5-HT1C receptor was functionally expressed under the promoter activity of the 1.8 kb 5' sequence of its gene. This system will be useful for further analysis of the cis-acting elements in the promoter region of the 5-HT1C receptor gene and the trans-acting factors that regulate tissue-specific expression of the receptor.

Animals↗