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Biomedical subjects

M Luo

Publications and source records attributed to M Luo.

At least 109 records · Page 6Linked to original sources
PubMed1997

Crystallization and preliminary X-ray diffraction studies of the Saccharomyces cerevisiae phospholipid-transfer protein Sec14p.

The Saccharomyces cerevisiae phosphatidylinositol-transfer protein Secl4p catalyzes the exchange of phosphatidylinositol or phosphatidylcholine between membrane bilayers in vitro, and is an essential protein required for the budding of secretory vesicles from the yeast Golgi complex in vivo. At issue is the fundamental question of how the dual phospholipid ligand specificity of Sec 14p translates to in vivo function. In an attempt to determine the structural basis for how Secl4p binds each of its phopholipid ligands, Secl4p occupied with phosphatidylcholine has been purified and the complex crystallized in the presence of the mild detergent n-octyl beta-D-glucopyranoside. The Secl4p crystals diffract to 2.7 A and belong to space group P3(1)21 or P3(2)21 with unit-cell dimensions of a = b = 88.79, c = 111.21 A, alpha = beta = 90, gamma = 120 degrees. As Secl4p exhibits significant primary sequence homology to mammalian retinaldehyde binding proteins and the noncatalytic domain of human MEG2 protein tyrosine phosphatase, is is anticipated that solution of the Secl4p crystal structure will provide new functional insights for a family of interesting proteins.

Journal Article↗
PubMed1997

Crystallization and preliminary X-ray crystallographic studies of type A influenza virus matrix protein M1.

The matrix protein, M1, of influenza virus strain A/PR/8/34 has been purified from virions and crystallized. The crystals consist of a stable fragment (18 kDa) of the M1 protein. X-ray diffraction studies indicated that the crystals are in space group P3(1)21 or P3(2)21, with a = 66.17, c = 135.30 A. V(m) calculations showed that there are two monomers in the asymmetric unit.

Journal Article↗
PubMed1997

GABP cooperates with c-Myb and C/EBP to activate the neutrophil elastase promoter.

Neutrophil elastase (NE) is a serine protease that is transcriptionally regulated during early myeloid differentiation. The murine NE (mNE) promoter contains functionally important c-Myb, C/EBP, and ets binding sites. Deletion of the ets site reduced promoter activity by 90%. Although the ets transcription factor, PU.1, bound to this ets site, it only modestly activated the mNE promoter. Here, we show that a second transcription factor from myeloid cells-GABP-binds to the mNE ets site but strongly activates the mNE promoter. GABP is a heteromeric transcription factor complex that consists of GABP alpha, an ets factor, and GABP beta, a Notch-related protein. GABP alpha bound to the mNE ets site and, in turn, recruited GABP beta to form a transcriptionally active complex. GABP alpha and PU.1 competed with each other for binding to the mNE ets site. GABP increased the activity of the mNE promoter sevenfold in U937 myeloid cells. GABP cooperated with c-Myb and C/EBP alpha to activate the mNE promoter more than 85-fold in otherwise nonpermissive, nonhematopoietic NIH 3T3 cells. Thus, GABP binds to the crucial mNE promoter ets site and powerfully activates its expression alone and in cooperation with the transcription factors c-Myb and C/EBP.

Animals↗
PubMed1997

Guanidinobenzoic acid inhibitors of influenza virus neuraminidase.

The active site of influenza virus neuraminidase (NA) is formed by 11 universally conserved residues. A guanidino group incorporated into two unrelated NA inhibitors was previously reported to occupy different negatively charged sites in the NA active site, A new inhibitor containing two guanidino groups was synthesized in order to utilize both sites in an attempt to acquire a combined increase in affinity. The X-ray crystal structures of the complexes show that the expected increase in affinity could not be achieved even though the added guanidino group binds to the negatively charged site as designed. This suggests that the ligand affinity to the target protein is contributed both from ligand-protein interactions and solvation/conformation energy of the ligand.

Binding Sites↗
PubMed1997

The effects of gliclazide and other sulfonylureas on low-density lipoprotein oxidation in vitro.

Diabetes is associated with increased oxidant stress. This may contribute to the development of diabetic macrovascular complications through increased oxidation of low-density lipoprotein (LDL), which is thought to be a crucial step in the development of atherosclerosis. The sulfonylurea gliclazide has been shown to have free radical-scavenging activity in vitro, but its effects on LDL oxidation, and these effects of other sulfonylureas, are unknown. To investigate this we studied the effects of in vitro supplementation with gliclazide 1 mumol/L on copper-induced oxidation of LDL isolated from 20 control subjects and 22 type II diabetic patients. The effects of 1 mumol/L vitamin C, a known water-soluble antioxidant, were studied simultaneously. The resistance to oxidation, expressed as the lag time between the addition of copper and commencement of oxidation, was significantly increased by both gliclazide and vitamin C, and the effect was similar for LDL from diabetic and control subjects. The baseline oxidation lag time was 63.4 +/- 2.1 minutes, and increased to 108 +/- 4.4 minutes with gliclazide and 88.7 +/- 5.6 minutes with vitamin C (P = .0001, baseline v either treatment). The increase in lag time with gliclazide of 70% +/- 3% was greater than the 30% +/- 5% increase with vitamin C (P < .0005). In a separate experiment, LDL isolated from eight control and 10 diabetic subjects was supplemented with 1 mumol/L gliclazide, glibenclamide, glipizide, and tolbutamide. For each LDL sample, all drugs were studied simultaneously and the oxidation lag time was compared against that of untreated LDL. Gliclazide increased the lag time from 53.7 +/- 2.4 minutes to 108.4 +/- 4.5 minutes (P = .0001). None of the other sulfonylureas had any effect on lag time. These findings demonstrate that gliclazide is an effective inhibitor of in vitro LDL oxidation, and in this respect, it is more potent on a molar basis than vitamin C. This antioxidant property of gliclazide was not shared by the other sulfonylureas studied.

Administration, Oral↗
PubMed1997

Multiple transcription start sites of the carrot dihydrofolate reductase-thymidylate synthase gene, and sub-cellular localization of the bifunctional protein.

The analysis of clones obtained by rapid amplification of the 5' end and by primer extension of the mRNA for carrot bifunctional dihydrofolate reductase-thymidylate synthase showed transcripts of differing lengths that belonged to two sub-populations. The longer transcripts were found to contain a translation start site 147 nt upstream of, and in frame with, the one which is present in the shorter transcripts. The ORF that begins at this ATG codes for a protein of 64714 Da, which is much larger than mature DHFR-TS subunit. The N-terminus region of this polypeptide shows features typical of plant transit peptides. Immunogold labelling studies and immunorecognition of the plastid-containing sub-cellular fraction suggested a plastidial localisation of the bifunctional protein. Although plant cells were shown to contain folate pools in plastids, in mitochondria and in the cytosol, few enzymes of the folate pathway have been associated with any sub-cellular compartment. Thus, this is the first indication for the presence of an enzyme of the folate biosynthetic pathway in plastids. The longer transcripts revealed the presence of a TC microsatellite at the 5'-untranslated end.

Amino Acid Sequence↗
PubMed1997

Cloning and characterisation of a carrot cDNA coding for a WD repeat protein homologous to Drosophila fizzy, human p55CDC and yeast CDC20 proteins.

The present study describes the isolation of a cDNA coding for a carrot protein of 450 amino acids that contains WD repeats (DcWD1) and is homologous to Drosophila melanogaster fizzy protein, mammalian p55CDC and yeast Cdc20p. As for the known related proteins, sequence conservation concerned the majority of the polypeptide except the far N-terminus. Results of Southern blot analysis with genomic DNA under high stringency conditions showed the occurrence of a single gene. Northern blot analyses revealed the accumulation of DcWD1 mRNA in all tested tissues (leaves, petioles and hypocotyls, apical meristems, roots and suspension cultured cells), though at a different extent. Lack of induction of relevant transcripts in proliferating auxin-stimulated hypocotyls suggests a mode of expression not strictly related to the cell proliferation.

Amino Acid Sequence↗
PubMed1997

Structure of a bifunctional membrane-RNA binding protein, influenza virus matrix protein M1.

Matrix protein (M1) of influenza virus is a bifunctional protein that mediates the encapsidation of RNA-nucleoprotein cores into the membrane envelope. It is therefore required that M1 binds both membrane and RNA simultaneously. The X-ray crystal structure of the N-terminal portion of type A influenza virus M1-amino acid residues 2-158-has been determined at 2.08 A resolution at pH 4.0. The protein forms a dimer. A highly positively charged region on the dimer surface is suitably positioned to bind RNA while the hydrophobic surface opposite the RNA binding region may be involved in interactions with the membrane. The membrane-binding hydrophobic surface could be buried or exposed after a conformational change.

Amino Acid Sequence↗
PubMed1997

Design of aromatic inhibitors of influenza virus neuraminidase.

Structure-based drug design, a terminology used to describe rational drug design by complementing the structure, spatially and chemically, of the target macromolecule, is rapidly developing as one of the innovative approaches to drug discovery. A growing volume of protein structure data and new techniques of protein structure determination make this all possible. The method of structure-based drug design and a specific example of the design of influenza virus neuraminidase is briefly presented. A whole new class of influenza virus neuraminidase inhibitors has been designed that can potentially be developed as antiinfluenza drugs.

Antiviral Agents↗
PubMed1997

Identification and characterization of human genes encoding Hprp3p and Hprp4p, interacting components of the spliceosome.

Nuclear RNA splicing occurs in an RNA-protein complex, termed the spliceosome. U4/U6 snRNP is one of four essential small nuclear ribonucleoprotein (snRNP) particles (U1, U2, U5 and U4/U6) present in the spliceosome. U4/U6 snRNP contains two snRNAs (U4 and U6) and a number of proteins. We report here the identification and characterization of two human genes encoding U4/U6-associated splicing factors, Hprp3p and Hprp4p, respectively. Hprp3p is a 77 kDa protein, which is homologous to the Saccharomyces cerevisiae splicing factor Prp3p. Amino acid sequence analysis revealed two putative homologues in Caenorhabditis elegans and Schizosaccharomyces pombe. Polyclonal antibodies against Hprp3p were generated with His-tagged Hprp3p over-produced in Escherichia coli . This splicing factor can co-immunoprecipitate with U4, U6 and U5 snRNAs, suggesting that it is present in the U4/U6.U5 tri-snRNP. Hprp4p is a 58 kDa protein homologous to yeast splicing factor Prp4p. Like yeast Prp4p, the human homologue contains repeats homologous to the beta-subunit of G-proteins. These repeats are called WD repeats because there is a highly conserved dipeptide of tryptophan and aspartic acid present at the end of each repeat. The primary amino acid sequence homology between human Hprp4p and yeast Prp4p led to the discovery of two additional WD repeats in yeast Prp4p. Structural homology between these human and yeast splicing factors and the beta-subunit of G-proteins has been identified by sequence-similarity comparison and analysis of the protein folding by threading. Structural models of Hprp4p and Prp4p with a seven-blade beta-propeller topology have been generated based on the structure of beta-transducin. Hprp3p and Hprp4p have been shown to interact with each other and the first 100 amino acids of Hprp3p are not essential for this interaction. These experiments suggest that both Hprp3p and Hprp4p are components of human spliceosomes.

Amino Acid Sequence↗
PubMed1997

Role of sialyloligosaccharide binding in Theiler's virus persistence.

Theiler's murine encephalomyelitis viruses (TMEVs) belong to the Picornaviridae family and are divided into two groups, typified by strain GDVII virus and members of the TO (Theiler's original) group. The highly virulent GDVII group causes acute encephalitis in mice, while the TO group is less virulent and causes a chronic demyelinating disease which is associated with viral persistence in mice. This persistent central nervous system infection with demyelination resembles multiple sclerosis (MS) in humans and has thus become an important model for studying MS. It has been shown that some of the determinants associated with viral persistence are located on the capsid proteins of the TO group. Structural comparisons of two persistent strains (BeAn and DA) and a highly virulent strain (GDVII) showed that the most significant structural variations between these two groups of viruses are located on the sites that may influence virus binding to cellular receptors. Most animal viruses attach to specific cellular receptors that, in part, determine host range and tissue tropism. In this study, atomic models of TMEV chimeras were built with the known structures of GDVII, BeAn, and DA viruses. Comparisons among the known GDVII, BeAn, and DA structures as well as the predicted models for the TMEV chimeras suggested that a gap on the capsid surface next to the putative receptor binding site, composed of residues from VP1 and VP2, may be important in determining viral persistence by influencing virus attachment to cellular receptors, such as sialyloligosaccharides. Our results showed that sialyllactose, the first three sugar molecules of common oligosaccharides on the surface of mammalian cells, inhibits virus binding to the host cell and infection with the persistent BeAn virus but not the nonpersistent GDVII and chimera 39 viruses.

Amino Acid Sequence↗
PubMed1997

[The comparison of sensitivity of BGM cells, HeLa cells, HEL cells and A 549 cells to herpes simplex virus-1].

This study was undertaken to compare sensitivity of buffulo green monkey (BGM) cells, Hela cells, human embryo lung (HEL) cells and A549 cells to herpes simplex virus-1. The plague forming unit (PFU) technique was used to measure the four cell lines simultaneously. The results showed that BGM cells were superior to the Hela cells, HEL cells, A549 cells in terms of both sensitivity and rapidity of appearance of cytopathic effect (CPE). It indicates that BGM cells constitute a new, sensitive and valuable cell line used for the detection of HSV-1 in cell culture.

Animals↗
PubMed1997

[Auxiliary partial orthotopic liver transplantation in rats].

A rat model of auxiliary partial orthotopic liver transplantation (APOLT) was developed in this study. Under hemihepatic inflow and outflow occlusions, the recipient underwent 75% partial hepatectomy before the liver graft comprising 30% of the donor liver was implanted on the orthotopic site. Forty rat APOLT were successfully performed. The 5-day survival rate of the recipient was 87.5% and that of the graft was 75%. On the fifth postoperative day, the living graft weight increased by 100%, the liver cell showed an active proliferation with a diploid of deoxyribonuclease (DNA), and the liver cells in DNA-synthetic phase accounted for 22.6 +/- 2.75%, significantly exceeding 12.22% +/- 1.48% of the normal rat hepatocytes (P < 0.001). It may be concluded that the rat APOLT is a more ideal animal model.

Animals↗
PubMed1997

[Establishment of mutant strain from human lung cancer cell (A549) with characteristics of resistance to 8-AG and HGPRT deficiency].

A human lung cancer cell line (A549) mutant with hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficiency and resistance to 8-AG was established by treatment of the A549 cells with 3 micrograms.ml-1 N-methyl-N'-nitro-nitrosoguanidine (MNNG), and prescreened in 0.25% agarose DMEM semisolid medium containing 20 micrograms.ml-1 8-AG. The mutant cells are resistant to cytotoxic effect of 8-AG and sensitive to hypoxanthine-aminopterin thymidine (HAT) selective medium. The mutant cells can be used as a candidate parental cells to fuse with the somatic cells.

Antimetabolites, Antineoplastic↗
PubMed1996

Cloning of a cDNA encoding DNA topoisomerase I in Daucus carota and expression analysis in relation to cell proliferation.

DNA topoisomerase I is an enzyme involved in several processes related to DNA metabolism. Despite the physiological importance, the regulation of top1 gene expression has not yet been investigated in plants. In order to monitor the possible correlation between levels of top1 transcripts and the proliferative state of the cell, two partially overlapping cDNAs encoding DNA topoisomerase I from Daucus carota have been isolated from a poly(A)(+)-primed library, using an Arabidopsis thaliana probe, and from a cDNA library spanning the 5' region of the top1 transcript, which was constructed using an antisense specific oligonucleotide. The top1 nucleotide sequence encoded an open reading frame of 2370 bp, predicting a protein of 90 kDa. The deduced amino acid sequence showed a similarity of 51% with A. thaliana, 41% with S. cerevisiae, 40% with S. pombe and 31% with H. sapiens, respectively. Southern blot analysis, performed under moderate stringency conditions, showed the presence of a single-copy gene. Evaluation of the top1 mRNA steady-state level revealed, besides a constitutive expression in vegetative carrot tissues, an induced expression related to cell proliferation.

Amino Acid Sequence↗
PubMed1996

The structure of a highly virulent Theiler's murine encephalomyelitis virus (GDVII) and implications for determinants of viral persistence.

GDVII is a highly virulent Theiler's murine encephalomyelitis virus (TMEV) which causes acute encephalitis in mice, while the BeAn and DA strains are the less virulent TMEV which cause chronic demyelinating disease in the central nervous system as a result of persistent infection. Purified GDVII virus isolated from infected BHK-21 cells was crystallized and its structure was determined to 3.5-A resolution by X-ray crystallography. In contrast to other TMEV structures, the VP1 C-terminus of GDVII virus has an ordered conformation that forms a hook over the VP3 knob near the threefold axis. Comparisons with the atomic structures of the less virulent BeAn and DA viruses revealed significant structural variations in a major site (cluster B) on the protruding surface loop puff B of VP2. Puff B is located near the VP3 GH loop region which is structurally analogous to the host receptor attachment site of the major serogroup of human rhinoviruses. Mutations at residue 1101 in VP1 and residue 2141 in VP2, which are also near the VP3 GH loop and adjacent to cluster B, were previously shown to influence persistence of DA virus. These observations indicate that the characteristic interaction with the host receptor through these sites may potentially alter TMEV persistence.

Amino Acid Sequence↗