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Biomedical subjects

G Luo

Publications and source records attributed to G Luo.

At least 163 records · Page 9Linked to original sources
PubMed1994

Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation.

The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to study the role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reverse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560 can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change from cysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail. This interpretation is further supported by experiments in which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted in infectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position 560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate a biological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, we cannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA--rather than altered palmitylation--are the determining factors for infectious-particle formation.

Amino Acid Sequence↗
PubMed1994

Expression of MUC2 gene is down-regulated by vitamin A at the transcriptional level in vitro in tracheobronchial epithelial cells.

The functional role of airway mucin in the respiratory system is well recognized. The isolation of mucin cDNA clones, MUC genes, introduces new information regarding the structure of the mucin core protein; however, the nature of the authentic core protein of airway mucin is still unresolved. In this communication, the effects of vitamin A on the regulation of MUC2 gene expression in primary tracheobronchial epithelial (TBE) cells of human and nonhuman primates were examined. Vitamin A has been recognized as one of the most important nutrients in the regulation of airway mucous cell differentiation. The expression of the MUC2 gene has been demonstrated in both rat and human tracheal tissues. The monkey cDNA clone MT80 was isolated from a cDNA library derived from vitamin A-depleted cultures of monkey TBE cells using a synthetic oligonucleotide probe corresponding to the 69 nucleotides of a tandemly repeated sequence in human MUC2 cDNA. DNA sequencing revealed a similar tandemly repeated sequence, except that 72 oligonucleotide repeats were observed in the monkey cDNA clone. Using the MT80 cDNA as a probe, the expression of the MUC2 gene was studied in vitro. The corresponding MUC2 message level in primary cultures of monkey TBE cells was down-regulated by vitamin A. This result was consistently demonstrated in primary human and hamster TBE cultures. The down-regulation was both time- and dosage-dependent on vitamin A. A nuclear run-on assay demonstrated a decrease in the transcriptional rate of the MUC2 gene in nuclei isolated from vitamin A-treated cultures. These results suggest that MUC2 gene expression in TBE cells is transcriptionally down-regulated by vitamin A.

Amino Acid Sequence↗
PubMed1994

A PCR differential screening method for rapid isolation of clones from a cDNA library.

We have developed a new two-step differential screening method that allows the rapid isolation of induced or suppressed pure gene clones from a lambda phage cDNA library. This method involves a primary differential screening step and a PCR differential screening step. From the primary screening step, impure pools of positive cDNA clones are obtained. Each pool of clones is then amplified directly by PCR using two primers flanking the cloning site in the vector. The PCR products are run on two duplicate agarose gels and blotted onto two filter strips. These two filters are then subjected to differential Southern hybridization with different cDNA probes. Each pure positive cDNA band on the gel is identified and selected for further subcloning. This method has three advantages over the traditional differential screening method. First, several rounds of plaque rescreening are replaced by a single PCR screening. Second, plaque hybridization is replaced by a more reliable and accurate PCR DNA Southern hybridization. Third, the time-consuming and tedious phage DNA isolation step is eliminated, and subcloning is facilitated by direct cloning of the pure PCR product to a plasmid vector. We have successfully used this method to isolate ozone-responsive genes from a cDNA library of monkey respiratory airways.

Animals↗
PubMed1994

Direct observation of the effect of autoreceptors on stimulated release of catecholamines from adrenal cells.

The direct effect of alpha 2-autoreceptors was studied by measuring the effects of piperoxan, an alpha 2-autoreceptor antagonist, and clonidine, an agonist on catecholamine exocytosis, from single bovine chromaffin cells in culture. Catecholamine release was elicited by stimulation with 100 microM nicotine and was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. These electrodes allowed the number of exocytotic release events to be monitored and reported as total charge for release following a specific stimulus. Repeated stimulation with 100 microM nicotine showed that total release caused by the second exposure to nicotine was 32% of the first, and release caused by the third exposure to nicotine was 80% of the second. Total release of catecholamine increased significantly after application of 20 microM piperoxan relative to a control application of balanced salt solution. Application of 20 microM piperoxan alone did not cause release. After the cells were incubated in culture medium containing 20 microM clonidine, a significant decrease in nicotine-stimulated catecholamine release was observed. These results confirm that there are autoreceptors on chromaffin cells and, when relatively high levels of catecholamine are released, the catecholamine stimulates the alpha 2-autoreceptors, which inhibits subsequent release through a negative feedback mechanism. In addition to piperoxan, the sympathomimetic drug amphetamine also increases quantal release after application of nicotine. Amphetamine increases the extracellular concentration of catecholamine, and these data appear to indicate that at least part of the pharmacology of amphetamine might involve blocking catecholamine autoreceptors.

Adrenal Medulla↗
PubMed1993

Continuous electrophoretic separations in narrow channels coupled to small-bore capillaries.

Continuous zone electrophoretic separations in channels have been demonstrated. This new technique has the potential to continuously sample and separate analytes from volume-limited microenvironments. A small-bore capillary is used to electrophoretically sample, but not separate, a mixture of dansylated amino acids. The capillary is coupled to a quartz channel structure in a manner which allows continuous injection of the sampled material into the channel. The channel functions to continuously separate the sampled material via electrophoresis. A laser-induced fluorescence detection scheme, which involves two fiber optic arrays situated at the channel exit, monitors eluting analytes. A continuous separation of dansylated amino acids on the time scale of a few minutes demonstrates the utility of the technique. Sampling has been performed continuously up to 400 s, and initial detection limits are approximately 30 microM.

Dansyl Compounds↗
PubMed1993

Immunization of mice with Yersinia enterocolitica leads to the induction of antithyrotropin receptor antibodies.

It is well established that autoimmune Graves' disease, which is characterized by hyperthyroidism, is mediated by autoantibodies to the thyrotropin receptor (TSHr). Although what initially triggers this autoantibody response is not known, a number of studies have suggested that Yersinia enterocolitica, an enterobacteria, could initiate the immune response against the TSHr. In this study, we produced antibodies against purified extracellular domain of human TSHr (ETSHr) and showed that anti-ETSHr antibodies reacted with envelope preparations from Y. enterocolitica. This reactivity was specifically blocked by preincubating sera with purified ETSHr. Moreover, antibodies reactive with ETSHr were induced by immunizing mice with Y. enterocolitica but not with Shigella flexneri SA100, Salmonella typhimurium TML, and Listeria monocytogenes. Anti-Y. enterocolitica antisera specifically reacted with the ETSHr protein and the reactivity could be blocked both by ETSHr and Y. enterocolitica envelope proteins. Our studies provide the first direct evidence that immunization with Y. enterocolitica can lead to the production of antibodies capable of reacting with TSHr and might provide the initial stimulus necessary for breakdown of self-tolerance to TSHr, eventually leading to the development of autoimmunity to TSHr.

Animals↗
PubMed1993

Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions.

The neuraminidase (NA) of influenza viruses cleaves sialic acids from receptors, prevents self-aggregation and facilitates release of virus during budding from host cells. Although the structure and function of the globular head of the influenza virus NA has been well studied, much less is known about the stalk of the NA, the region between the viral membrane and the globular head. Applying a reverse genetics system, we altered the stalk of the influenza A/WSN/33 virus NA by making deletions, insertions and mutations in this region of the gene. Our data show that the length of the NA stalk can be variable. Deletions of up to 28 amino acids and insertions of up to 41 amino acids in the stalk region did not abolish formation of infectious progeny virus. The data also indicate that the cysteine at position 76 is essential for formation of infectious virus, and that deletions beyond the cysteine did not result in infectious virus. Interestingly, shortening of the length of the stalk region by 28 amino acids resulted in a virus with a markedly reduced growth rate in MDCK cells as compared to that in MDBK cells. An insertion of 41 extra amino acids into the stalk did not significantly interfere with viral growth in MDCK or MDBK cells, which suggests that the stalk region would tolerate the introduction of long foreign sequences.

Amino Acid Sequence↗
PubMed1993

Tumor necrosis factor alpha binding to bacteria: evidence for a high-affinity receptor and alteration of bacterial virulence properties.

Human and murine receptors for tumor necrosis factor alpha (TNF-alpha) are present on most somatic cells and have been characterized and cloned. In contrast, very little is currently known about whether TNF-alpha can bind to pathogens and whether such binding results in important biological consequences for the infected host. We now report that a number of gram-negative bacteria have receptors for TNF-alpha. Using 125I-labeled TNF-alpha, we show that Shigella flexneri has 276 receptors for TNF-alpha, with a Kd of 2.5 nM. The binding of labeled TNF-alpha to these bacterial receptors can be inhibited by cold TNF-alpha but not by cold TNF-beta. Binding of 125I-TNF-alpha to S. flexneri was inhibited by trypsin treatment of bacterial cells or incubation at 52 degrees C for 3 min. Monoclonal antibody to either the 55-kDa or the 75-kDa TNF-alpha receptor, which are present on different eukaryotic cells, had no effect on 125I-TNF-alpha binding to bacteria. A number of gram-negative bacteria were capable of binding 125I-TNF-alpha. Gram-positive bacteria bound significantly less 125I-TNF-alpha than gram-negative bacteria. Pretreatment of S. flexneri with TNF-alpha resulted in enhanced bacterial invasion of HeLa cells and enhanced uptake by human and murine macrophages. Pretreatment of HeLa cells with antibody to the 55-kDa TNF-alpha receptor abrogated enhanced invasion of HeLa cells by TNF-alpha-bacterium complexes. These results suggest that TNF-alpha-bacterium complexes can interact with TNF-alpha receptors present on eukaryotic cells. This report shows that gram-negative bacteria have receptors for TNF-alpha and that a virulence property of a bacterium is altered as a consequence of cytokine binding.

Bacterial Proteins↗
PubMed1992

Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.

RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.

Bacterial Proteins↗
PubMed1992

Genetic analysis of influenza virus.

The newly developed ribonucleoprotein reconstitution and transfection systems have facilitated the characterization of cis and trans functions required for transcription and replication of the influenza virus genome. For the first time, the genome of a negative-strand RNA virus can be manipulated using recombinant DNA techniques.

Base Sequence↗
PubMed1992

Mechanism of attenuation of a chimeric influenza A/B transfectant virus.

The ribonucleoprotein transfection system for influenza virus allowed us to construct an influenza A virus containing a chimeric neuraminidase (NA) gene in which the noncoding sequence is derived from the NS gene of influenza B virus (T. Muster, E. K. Subbarao, M. Enami, B. P. Murphy, and P. Palese, Proc. Natl. Acad. Sci. USA 88:5177-5181, 1991). This transfectant virus is attenuated in mice and grows to lower titers in tissue culture than wild-type virus. Since such a virus has characteristics desirable for a live attenuated vaccine strain, attempts were made to characterize this virus at the molecular level. Our analysis suggests that the attenuation of the virus is due to changes in the cis signal sequences, which resulted in a reduction of transcription and replication of the chimeric NA gene. The major finding concerns a sixfold reduction in NA-specific viral RNA in the virion, causing a reduction in the ratio of infectious particles to physical particles compared with the ratio in wild-type virus. Although the NA-specific mRNA level is also reduced in transfectant virus-infected cells, it does not appear to contribute to the attenuation characteristics of the virus. The levels of the other RNAs and their expression appear to be unchanged for the transfectant virus. It is suggested that downregulation of the synthesis of one viral RNA segment leads to the generation of defective viruses during each replication cycle. We believe that this represents a general principle for attenuation which may be applied to other segmented viruses containing either single-stranded or double-stranded RNA.

Animals↗
PubMed1992

[Isolation and identification of yibeissine].

A new steroidal alkaloid, yibeissine (II), with a know alkaloid (I) was isolated from the bulb of Fritillaria pallioiflora Schrenk by column chromatographic techniques. Their structures have been determined based on spectral and chemical data.

Alkaloids↗