Role of the Rel-family of transcription factors in the regulation of c-myc gene transcription and apoptosis of WEHI 231 murine B-cells.
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Animals↗
Biomedical subjects
M Wu
Publications and source records attributed to M Wu.
Potential role of roxithromycin against the Mycobacterium avium complex.
Until the recent experience with azithromycin and clarithromycin, macrolides were not considered to be important agents against mycobacteria. Clinical evidence is now growing that the newer 14 and 15 membered macrolide compounds have therapeutic activity against Mycobacterium avium, Mycobacterium chelonae and Mycobacterium leprae. Several years ago, when evaluating the activity of roxithromycin using one of the more virulent M. avium in our collection, the authors found that roxithromycin exerted a bacteriostatic effect in cultured human macrophages. However, in combination with tumour necrosis factor, which induces macrophage activation, roxithromycin caused enhanced intracellular killing. The significance of this finding is that tumour necrosis factor can be elaborated by activated macrophages during the course of infection. The roxithromycin doses that were chosen for these studies were less than achievable blood levels. More recently, the in vitro effect of roxithromycin against a panel of isolates from AIDS patients has been assessed and it was found that some (but not all) of the inhibitory concentrations, by the T-100 method of Inderlied, are within achievable serum levels. This, however, may not be the basis for anticipating in vivo activity since macrolide compounds are known to be concentrated within cells and particularly within phagolysosomes. Demonstration of effect in an in vitro test system is encouraging, but should be considered only as a preliminary step to careful assessments in experimental animals, such as the beige mouse, and studies in humans.
AIDS-Related Opportunistic Infections↗
A novel method employing polymerase chain reaction to disrupt genes lacking convenient restriction enzyme sites in yeast.
A novel method employing polymerase chain reaction was developed for the disruption of yeast genes lacking convenient restriction enzyme sites. The method was found to be easy and effective. Using this method, a yeast YKE2 gene (a yeast homolog of murine k-region expressed genes) were successfully disrupted by replacement of HIS3 marker gene.
Cloning, Molecular↗
Genome organization of ageratum yellow vein virus, a monopartite whitefly-transmitted geminivirus isolated from a common weed.
A full-length copy of a single genomic component of the whitefly-transmitted geminivirus ageratum yellow vein virus (AYVV) has been cloned from an extract of infected Ageratum conyzoides originating from Singapore. Sequence analysis shows that the genomic component encodes two virion-sense (V1 and V2) and four complementary-sense open reading frames (C1-C4), typical of DNA A of whitefly-transmitted geminiviruses from the Eastern hemisphere. A genomic component equivalent to DNA B was not detected in extracts of infected A. conyzoides. The cloned genomic component produced a systemic infection in Nicotiana benthamiana, Phaseolus vulgaris and Lycopersicon esculentum when introduced into plants by agroinoculation, and symptoms were identical to those produced by wild-type virus introduced into these hosts using viruliferous whiteflies. However, attempts to re-establish a systemic infection in A. conyzoides either by agroinoculation or by whitefly transmission of the cloned progeny were unsuccessful, suggesting that additional factors are required for infection of the natural host. The significance of A. conyzoides as a reservoir host for the economically important geminivirus diseases is discussed.
Animals↗
Numerical solution of the Kardar-Parisi-Zhang equation with a long-range spatially correlated noise.
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Journal Article↗
Cloning and nucleotide sequence of a cDNA encoding S-adenosyl-L-methionine synthetase from mustard (Brassica juncea [L.] Czern & Coss).
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Brassica↗
Ultraviolet radiation activates the human elastin promoter in transgenic mice: a novel in vivo and in vitro model of cutaneous photoaging.
The major alteration in photoaged skin is the deposition of massive amounts of abnormal elastic material, termed solar elastosis. In previous work, it has been shown that solar elastosis is accompanied by increased abundance of elastin and fibrillin mRNAs and upregulation of elastin promoter activity. Using a transgenic mouse line, which expresses the human elastin promoter, linked to a chloramphenicol acetyltransferase reporter gene, in a tissue-specific and developmentally regulated manner, we investigated the effects of ultraviolet A radiation and ultraviolet B radiation on human elastin promoter activity in vivo and in vitro. Irradiation of mice with a single dose of ultraviolet B radiation (491.4 mJ/cm2) resulted in an increase up to 8.5-fold in promoter activity, whereas a more modest increase of 1.8-fold was measured with ultraviolet A radiation (38.2 J/cm2). In addition, in vitro studies revealed over a thirtyfold increase in elastin promoter activity in response to ultraviolet B radiation (5.5 mJ/cm2), whereas no change was measured in response to ultraviolet A radiation (2.2 J/cm2). These results confirm the role of ultraviolet B radiation in elastin promoter activation in photoaging, and identify ultraviolet A radiation as a contributing factor. This system should serve as a useful in vivo and in vitro model to study cutaneous photoaging, and for testing compounds that may protect against cutaneous photodamage.
Animals↗
Interleukin-12-stimulated natural killer cells can activate human macrophages to inhibit growth of Mycobacterium avium.
Interleukin-12 (IL-12) is a critical cytokine that affects many of the biological functions of NK cells and T cells. We have previously shown that both human and murine NK cells are important in host defense against Mycobacterium avium complex and act by secreting cytokines that induce macrophages to inhibit the growth of intracellular M. avium. To define the role of IL-12 in M. avium complex infection, we stimulated human NK cells with recombinant human IL-12 at 0.01 to 1 ng/ml for 24 h and used the tissue culture supernatant to treat human monocyte-derived macrophage monolayers infected with M. avium. IL-12 had no direct effect on M. avium-infected macrophages, but culture supernatant from IL-12-treated NK cells activated macrophages to inhibit the growth of intracellular M. avium in a dose-dependent manner. Stimulation of NK cells with IL-12 in combination with tumor necrosis factor alpha (TNF-alpha) or IL-1 increased the ability of supernatant from NK-cell culture to limit M. avium growth within macrophages, compared with that of culture supernatant from IL-12-treated NK cells. Results with supernatant from nonstimulated NK cells were similar to those with supernatant from untreated controls. Treatment of supernatant from IL-12-stimulated NK cells with anti-TNF-alpha, anti-granulocyte-macrophage colony-stimulating factor, but not anti-gamma interferon antibodies decreased the ability of NK-cell supernatant to induce anti-M. avium activity in infected macrophages. Treatment of macrophage monolayers with anti-transforming growth factor beta antibody before adding supernatant from IL-12-stimulated NK cells was associated with an increase of anti-M. avium activity compared with that of supernatant from IL-12-treated NK cells. These results suggest that IL-12 has a role in host defense against M. avium and that the effect of IL-12 is dependent chiefly on TNF-alpha and granulocyte-macrophage colony-stimulating factor.
Animals↗
Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.
The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.
Amino Acid Sequence↗
Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion.
To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N omega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2- by > 300% but decreases LDL-treated EC (LDL-EC) release by > 95%. L-Arginine inhibits the release of O2- from LDL-ECs by > 95% but did not effect C-EC release of O2-. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2- production by approximately 50% and 30%, respectively. Thus, n-LDL increases O2- and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO-, and tyrosine by approximately 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Oxidoreductases↗
A transgenic mouse model provides a novel biological assay of topical glucocorticosteroid potency.
BACKGROUND AND DESIGN: A homozygous line of transgenic mice that expresses the human elastin promoter/CAT (chloramphenicol acetyltransferase) reporter gene construct in a tissue-specific and developmentally regulated manner is presented. Previous studies have shown that subcutaneous injections of various glucocorticosteroids up-regulate the human transgene in the mouse skin potentially through their interaction with three putative glucocorticosteroid-responsive elements contained within the human elastin promoter. In this study, we propose the use of these transgenic mice as a model system for assaying the potency of various topical glucocorticosteroid preparations. RESULTS: In the first set of experiments, three different commercially available topical glucocorticosteroid creams, 2.5% Hytone (2.5% hydrocortisone) (Dermik Laboratories, Fort Washington, Pa), Cutivate (0.05% fluticasone propionate) (Glaxo Inc, Research Triangle Park, NC), and Temovate (0.05% clobetasol propionate) (Glaxo Inc) (being classified into class VII, V, and I steroids, respectively) were applied to the skin of transgenic mice, with Eucerin (Beiersdorf Inc, Lindenhurst, NY) as the control cream. In a series of six experiments, Hytone 2.5% cream caused a 3.1-fold increase on the average, with Cutivate and Temovate creams resulting in 2.2-fold and 12.4-fold increases in CAT activity over control, respectively. Next, two different preparations of diflorasone diacetate 0.05% cream (Florone [class III] and Psorcon [class II], both from Dermik Laboratories), formulated with different vehicles, were compared. Psorcon caused a 22.8-fold increase in CAT activity over the control compared with a 4.4-fold increase for Florone. However, an assay comparing Psorcon ointment (class I) and Psorcon cream (class II) showed no statistically significant difference in their potencies. CONCLUSIONS: These preliminary findings suggest the usefulness of these transgenic mice as a model system for assaying the potency of topical glucocorticosteroid preparations. Discrepancies between our data and the published classification of some topical steroids may result from anatomic differences between human and murine skin, with mouse skin much thinner, Alternatively, the discrepancies may reflect the fact that our assay measures the biological activity of these steroids on gene transcription, while previous ranking is based on their vasoconstrictive activity.
Administration, Topical↗
Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA.
We introduced plasmid pCmVCAT containing a chloramphenicol acetyltransferase (CAT) gene, flanked by the cauliflower mosaic virus 35S promoter and nopaline synthase polyadenylation sequences, into Chlamydomonas reinhardtii by electroporation; chloramphenicol (CAM) resistance was used for selection. Several mutants with aberrant response to cadmium (Cd) toxicity were obtained by screening CAM resistant transformants. Southern blot analysis showed random integration of pCmVCAT sequence into the nuclear genome. Expression of CAT gene was confirmed by the detection of CAT gene transcript in Northern blot analysis and the detection of CAT enzyme by ELISA assay. This study demonstrated the feasibility of transforming Chlamydomonas reinhardtii with heterologous DNA by electroporation, and the expression of heterologous gene, in this alga.
Animals↗
Abrupt reduction of c-myc expression by antisense RNA inducing terminal differentiation and apoptosis of a human esophageal cancer cell line.
A human esophageal cancer cell line (EC8712) expressing high-level Myc protein was infected with recombinant retroviral particles (pA-BD9) at a multiplicity of infection (MOI) 1:1. This viral particle contains a neomycin-resistant gene and a 1.53-kb antisense RNA spanning the 2nd exon and its flanking sequences of the human c-myc oncogene. The G418-resistant EC8712 clones showed an 86% inhibition of growth rate and morphological changes characteristic of terminal differentiation and apoptosis. A decrease of about 80% of Myc protein was also observed in these infected cells by ABC-ELISA assay. 12-24 h after the infection of EC8712 cells with pA-BD9 at a high viral particle concentration (MOI = 1:10), the integration of the extrinsic 1.53-kb antisense c-myc fragment into the cancer cell genome was evidenced by the Southern blot analysis. Northern blot analyses showed the expression of this antisense fragment and a decrease of the intrinsic c-myc expression by 74% in comparison with that of the parental EC8712 cells. Heterotransplants of the infected EC8712 cells into the nude mice revealed a substantial decrease in tumorigenicity and morphological changes characteristic of terminal differentiation and apoptosis. Primary monolayer cell cultures of normal epithelia derived from the fetal and adult esophagus mucosa were set as controls. No noticeable increase in c-myc expression was found in these cultures. Infection of these cells with the same recombinant viral particles neither affected the growth rate of the cells nor their normal morphology. Our experiments indicate that the drastic decrease of the over-expressed Myc protein in cancer cells may also be an entrance to one of many pathways leading to the terminal differentiation and programmed cell death.
Animals↗
Is the relationship between supine blood pressure and postural changes in blood pressure artefactual? Results from the Kuopio Ischemic Heart Disease Risk Factor Study.
OBJECTIVE: To investigate whether there is an association between initial supine blood pressure and postural changes in blood pressure (standing minus supine blood pressure). METHODS: Using data from the Kuopio (Finland) Ischemic Heart Disease Risk Factor Study (KIHD), we simulated the problem and found the suggested solution based on the work of Blomqvist. We then applied the Blomqvist correction to the KIHD data with real measurement errors. RESULTS: The observed regression slope was substantially reduced, indicating that there is no relationship between the initial blood pressure and the postural change in blood pressure. CONCLUSION: Only the broad application of the method of Blomqvist to other data sets will determine the generalizability of the present finding that initial blood pressure is unrelated to the postural change in blood pressure.
Adult↗
[Radioimmunoimaging (RII) and radioimmunotherapy (RIT) of human primary hepatic cancer (hPHC) with anti-PHC monoclonal antibody in tumor bearing nude mice].
Tumor xenografts of human PHC were grown in nude mice subcutaneously. The studies were performed in nude mice with tumor base aear of 0.8cm-1.0cm. For the study of RII, the mice were injected intravenously with 1.11 x 10(7)Bq/mouse of 131I-hepama-1. All tumours were clearly visualied on the 4th day after injection and remained the same until the 7th day. No tumor image was found in 131I-mIgG. The tumor to liver ratio of radioactivity increased from 1.3 to 3.03 in 131I-Hepama-1 group, but decreased from 13.6 to 6.88% in 131I-mIgG group. For the study of RIT 131I-hepama-1 (1.85 x 10(10)Bq) was administered intraperitoneally. A tumor growth inhibition rate at the 4th week after the infusion of 70.5% was obtained. The median survival time of the nude mice in 131I-hepama-1 group was much longer than in control groups in mice.
Animals↗
[Changes of ABH substances amount in human tissues after treatment with physico-chemical factors].
The forensic samples for identify testing are always affected by the environmental or the physico-chemical influence. The aim of the present study is to study whether the physico-chemical or biological factors affect the quantity of ABH substances in human tissues or not. The quantity of the ABH substances was determined by immunohistochemical ABC method. The tissues were treated by a series of methods, including immersing in water, treatment with heat, acid, alkali as well as putrefaction and mold growing. The results revealed that although the ABH substances slightly decreased in some tissues pretreated, no effect of ABO typing was observed. The correct ABO typing was performed with tissues under the following condition: the tongue and skin immersed in water for 21 days; the tongue treated with heat (100 degrees C) for 40 min; the tongue and skin treated with 0.2mol/L HCl and 0.2 mol/L NaOH for 48 h and the skin treated with 12mol/L HCl and 12mol/L NaOH for 30 min. The ABO typing of pancreas undergoing putrefaction and mold growing 21 days was carried out correctly. It is concluded that the ABC immunohistochemical technique is a good method for ABO typing of human tissues.
ABO Blood-Group System↗
An approximate generalized linear model with random effects for informative missing data.
This paper develops a class of models to deal with missing data from longitudinal studies. We assume that separate models for the primary response and missingness (e.g., number of missed visits) are linked by a common random parameter. Such models have been developed in the econometrics (Heckman, 1979, Econometrica 47, 153-161) and biostatistics (Wu and Carroll, 1988, Biometrics 44, 175-188) literature for a Gaussian primary response. We allow the primary response, conditional on the random parameter, to follow a generalized linear model and approximate the generalized linear model by conditioning on the data that describes missingness. The resultant approximation is a mixed generalized linear model with possibly heterogeneous random effects. An example is given to illustrate the approximate approach, and simulations are performed to critique the adequacy of the approximation for repeated binary data.
Bayes Theorem↗
[Study and clinical application of tumor-infiltrating lymphocytes isolated from hepatocarcinoma].
Tumor-infiltrating lymphocytes isolated from freshly resected tumors of 12 patients with hepatocarcinoma were propagated in vitro and infused to patients. The average weight of tumor for culture of TIL was 4.8g, number of cells 5.8 x 10(7), time for in vitro expansion was 31.8 days. The maximal expansion achieved 1000 fold, with a mean amplification rate of 198.5 fold. LAK cell culture supernatant stimulated TIL growth. 10 of 12 patients received clinical treatment, the number of cells infused reached 4 x 10(8)-1.1 x 10(10). TIL was infused by hepatic artery. TIL isolated from hepatocarcinoma demonstrated cytotoxic activity to tumor targets.
Adult↗