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Biomedical subjects

J Ou

Publications and source records attributed to J Ou.

At least 19 recordsLinked to original sources
PubMed2006

MUTYH and the mismatch repair system: partners in crime?

Biallelic germline mutations of MUTYH-a gene encoding a base excision repair protein-are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations (P = 0.002) and the published controls (P = 0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Aerosol deposition of lipid complex amphotericin-B (Abelcet) in lung transplant recipients.

Lung transplant recipients exhibit a high incidence of invasive aspergillosis. The inhalation of lipid complex amphotericin-B (Abelcet; ABLC) offers a possible prophylactic strategy. The goals of this study were to select the optimal nebulizer delivery system for ABLC and to measure deposited aerosol dose in 12 lung transplant recipients. In vitro testing was performed to select a nebulizer delivery system, and an empirical model was used to estimate lung deposition. Estimated pulmonary doses varied by as much as 2-fold between different nebulizers. Aerosol deposition testing was performed in six single and six double lung recipients, each of whom received one 7 mL (35 mg) nebulized dose of Technetium-labeled ABLC using the selected nebulizer. In single lung recipients, the average deposited doses were 3.9 +/- 1.6 mg (mean +/- S.D.) in the allograft versus 2.1 +/- 1.1 mg in the native lung. Double lung recipients deposited on average 2.8 +/- 0.8 mg (left lung) and 4.0 +/- 1.3 mg (right lung). The drug was well distributed throughout the lungs, but delivery to the native lung was in some cases suboptimal. These studies provide an important precursor to studies of the efficacy of inhaled ABLC as a prophylaxis of invasive aspergillosis after lung transplant.

Aerosols↗
PubMed2004

Hereditary non-polyposis colorectal cancer: identification of mutation carriers and assessing pathogenicity of mutations.

Hereditary non-polyposis colorectal cancer (HNPCC), also referred to as Lynch syndrome, is an autosomal dominantly inherited disorder that is characterized by susceptibility to colorectal cancer and extracolonic malignancies, in particular endometrial cancer. HNPCC is caused by pathogenic mutations in the mismatch repair (MMR) genes, which play an important role in maintaining genomic stability during DNA replication. Identification of MMR gene mutation carriers is important as this enables them to enrol in surveillance programmes, thus reducing their risk of cancer and increasing survival. Clinical criteria as well as non-clinical criteria have been formulated to select patients for mutation analysis. In this paper we review the approaches used to select patients for mutation analysis. Mutation analysis in the MMR genes may yield mutations of which the pathogenic nature is unclear. Criteria to determine the pathogenicity of such variants are discussed, as well as differences in design of functional assays to assess pathogenicity.

Base Pair Mismatch↗
PubMed2001

Synthesis of a novel hepatitis C virus protein by ribosomal frameshift.

Hepatitis C virus (HCV) is an important human pathogen that affects approximately 100 million people worldwide. Its RNA genome codes for a polyprotein, which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products. We report here the discovery of a novel HCV protein synthesized by ribosomal frameshift. This protein, which we named the F protein, is synthesized from the initiation codon of the polyprotein sequence followed by ribosomal frameshift into the -2/+1 reading frame. This ribosomal frameshift requires only codons 8-14 of the core protein-coding sequence, and the shift junction is located at or near codon 11. An F protein analog synthesized in vitro reacted with the sera of HCV patients but not with the sera of hepatitis B patients, indicating the expression of the F protein during natural HCV infection. This unexpected finding may open new avenues for the development of anti-HCV drugs.

Amino Acid Sequence↗
PubMed2001

Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR.

Sterol regulatory element-binding protein-1c (SREBP-1c) enhances transcription of genes encoding enzymes of unsaturated fatty acid biosynthesis in liver. SREBP-1c mRNA is known to increase when cells are treated with agonists of liver X receptor (LXR), a nuclear hormone receptor, and to decrease when cells are treated with unsaturated fatty acids, the end products of SREBP-1c action. Here we show that unsaturated fatty acids lower SREBP-1c mRNA levels in part by antagonizing the actions of LXR. In cultured rat hepatoma cells, arachidonic acid and other fatty acids competitively inhibited activation of the endogenous SREBP-1c gene by an LXR ligand. Arachidonate also blocked the activation of a synthetic LXR-dependent promoter in transfected human embryonic kidney-293 cells. In vitro, arachidonate and other unsaturated fatty acids competitively blocked activation of LXR, as reflected by a fluorescence polarization assay that measures ligand-dependent binding of LXR to a peptide derived from a coactivator. These data offer a potential mechanism that partially explains the long-known ability of dietary unsaturated fatty acids to decrease the synthesis and secretion of fatty acids and triglycerides in livers of humans and other animals.

Animals↗
PubMed2001

Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes.

Recent data indicate that sustained elevations in plasma insulin suppress the mRNA for IRS-2, a component of the insulin signaling pathway in liver, and that this deficiency contributes to hepatic insulin resistance and inappropriate gluconeogenesis. Here, we use nuclear run-on assays to show that insulin inhibits transcription of the IRS-2 gene in the livers of intact rats. Insulin also inhibited transcription of a reporter gene driven by the human IRS-2 promoter that was transfected into freshly isolated rat hepatocytes. The human promoter contains a heptanucleotide sequence, TGTTTTG, that is identical to the insulin response element (IRE) identified previously in the promoters of insulin-repressed genes. Single base pair substitutions in this IRE decreased transcription of the IRS-2-driven reporter in the absence of insulin and abolished insulin-mediated repression. We conclude that insulin represses transcription of the IRS-2 gene by blocking the action of a positive factor that binds to the IRE. Sustained repression of IRS-2, as occurs in chronic hyperinsulinemia, contributes to hepatic insulin resistance and accelerates the development of the diabetic state.

Animals↗
PubMed2001

Unique effects of zinc protoporphyrin on HO-1 induction and apoptosis.

Zinc protoporphyrin (ZnPP), a naturally occurring molecule, is increased in iron deficiency and lead intoxication. ZnPP can also induce heme oxygenase (HO-1), the enzyme it competitively inhibits. In cultured cells (HA-1), ZnPP was the strongest HO-1 inducer of any metalloporphyrin (MP) tested. This was not due to increased oxidative stress, enhanced binding at metal response element, nor increased binding at activator protein-1 (AP-1) or SP-1 sites on HO-1. Only ZnPP, however, increased binding of nuclear proteins to early growth response-1 (Egr-1) protein consensus sequence. Pretreatment of HA-1 with cycloheximide inhibited ZnPP-induced HO-1 messenger RNA (mRNA) by 55%. Incubation with antisense Egr-1 oligomers decreased ZnPP-induced HO-1 expression by 47%. Furthermore, the level of HO-1 mRNA induction by ZnPP was 2-fold less in Egr-1-deficient fibroblasts than in wild-type cells. Because no Egr-1 binding site was previously identified on the HO-1 promoter, HA-1 cells were transfected with HO-1 CAT constructs containing segments of a 12.5-kb enhancer region of HO-1. A 196-bp fragment (RH) located approximately 9.5 kb upstream of the transcription start site mediated HO-1 induction by ZnPP alone. DNase I footprinting analysis further revealed that nuclear proteins bound to a 50-bp sequence in the RH. Within this sequence, a novel 9-bp region with 78% homology to the Egr-1 consensus sequence was identified further suggesting that Egr-1 partially mediates HO-1 induction by ZnPP. Lastly, increased apoptosis and nuclear localization were only seen with ZnPP, suggesting that increased ZnPP in disease states may serve as a cellular signaling mechanism.

Active Transport, Cell Nucleus↗
PubMed2001

Expression of sterol regulatory element-binding protein 1c (SREBP-1c) mRNA in rat hepatoma cells requires endogenous LXR ligands.

The current paper describes a line of cultured rat hepatoma cells (McA-RH7777 cells) that mimics the behavior of rat liver by producing an excess of mRNA for sterol regulatory element-binding protein 1c (SREBP-1c) as opposed to SREBP-1a. These two transcripts are derived from a single gene by use of alternative promoters that are separated by many kilobases in the genome. The high level of SREBP-1c mRNA is abolished when cholesterol synthesis is blocked by compactin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase that inhibits cholesterol synthesis. Levels of SREBP-1c mRNA are restored by mevalonate, the product of the HMG CoA reductase reaction, and by ligands for the nuclear hormone receptor LXR, including 22(R)-hydroxycholesterol and T0901317. These data suggest that transcription of the SREBP-1c gene in hepatocytes requires tonic activation of LXR by an oxysterol intermediate in the cholesterol biosynthetic pathway. Reduction of this intermediate lowers SREBP-1c levels, and this in turn is predicted to lower the rates of fatty acid biosynthesis in liver.

Animals↗
PubMed2001

A genetic analysis of neural progenitor differentiation.

Genetic mechanisms regulating CNS progenitor function and differentiation are not well understood. We have used microarrays derived from a representational difference analysis (RDA) subtraction in a heterogeneous stem cell culture system to systematically study the gene expression patterns of CNS progenitors. This analysis identified both known and novel genes enriched in progenitor cultures. In situ hybridization in a subset of clones demonstrated that many of these genes were expressed preferentially in germinal zones, some showing distinct ventricular or subventricular zone labeling. Several genes were also enriched in hematopoietic stem cells, suggesting an overlap of gene expression in neural and hematopoietic progenitors. This combination of methods demonstrates the power of using custom microarrays derived from RDA-subtracted libraries for both gene discovery and gene expression analysis in the central nervous system.

Animals↗
PubMed2001

Change of visual acuity and related factors in idiopathic optic neuritis.

PURPOSE: To study the change of visual acuity and related factors in idiopathic optic neuritis. METHODS: Retrospectively studying 136 cases (210 eyes) of idiopathic optic neuritis hospitalized in our department during last 5 years. The data was analyzed with the methods of multivariate stepwise regression analysis by SPSS statistic package. RESULTS: The final visual acuity was very strongly related positively to the initial visual acuity at the time point of starting treatment and negatively to the time span beginning the treatment to the time showing improvement, age of patient and the duration from onset of the disorder to the time point of treatment beginning. CONCLUSIONS: Promptly right treatment can significantly change the natural course and the final vision of optic neuritis. Severe visual loss, old age, longer time without right therapy and slow improvement after the treatment may indicate an unfavorable final visual recovery. The different dosages of steroid applied in this study was not significant in view of the final visual acuity.

Adolescent↗
PubMed2001

[A case report of idiopathic polypoidal choroidal vasculopathy].

PURPOSE: To report a case of idiopathic polypoidal choroidal vasculopathy (IPCV) and its angiographic characteristics. METHODS: General ophthalmic check up, analyses of fundus picture, funds fluorescein angiography (FFA), indocyanine green angiography (ICGA) as well as literature review. RESULTS: The lack of typical choroidal neovascularization (CNV) on FFA and characteristic polypoidal choroidal vasculopathy on ICGA as well as its unsymmetrical manifestation and discrepancy with age-related macular degeneratio (AMD) on the fundus picture are typical signs for the diagnosis of IPCV. CONCLUSION: The angiographic features on FFA and ICGA are specific for the diagnosis of IPCV. Differentiating IPCV and AMD has clinical significance in prognosing the visual acuity, progress and management of the diseases.

Aged↗
PubMed2000

Overexpression of a novel zinc-finger protein induces apoptosis in NIH3T3 fibroblasts.

Genes coding for zinc-finger proteins constitute about 1% of the mammalian genome. Here we report the cloning of a novel mouse gene (Zfp319) encoding a nuclear protein with 11 zinc-finger motifs of the C2H2 type. Zfp319 consists of two exons, the second of which contains the entire coding sequence. Preliminary evidence suggests that the primary transcript undergoes alternative splicing with the potential of producing Zfp319 isoforms that contain different numbers of zinc fingers or none. The Zfp319 gene maps to chromosome 8, in a region of conserved synteny with the human counterpart on chromosome 16. Finally, overexpression of the Zfp319 protein in stably transfected fibroblasts results in significant reduction of viable cells due to induction of programmed cell death.

3T3 Cells↗
PubMed2000

Synergistic cooperation between Sp1 and Smad3/Smad4 mediates transforming growth factor beta1 stimulation of alpha 2(I)-collagen (COL1A2) transcription.

Transforming growth factor-beta1 (TGFbeta) is a strong activator of extracellular matrix accumulation. TGFbeta stimulates the gene coding for human alpha2(I)-collagen (COL1A2) by inducing binding of an Sp1-containing complex to an upstream promoter element (TGFbeta responsive element or TbRE) that contains a CAGA box. Here we report that the CAGA box of the TbRE is the binding site of the Smad3/Smad4 complex, and that the binding of the complex is required for TGFbeta-induced COL1A2 up-regulation. Recombinant Smad3 and Smad4 bind in vitro to the CAGA box of COL1A2; TGFbeta treatment of cultured fibroblasts induces Smad3/Smad4 binding to the TbRE; transient overexpression of Smad3 and Smad4 in fibroblasts transactivates TbRE-driven transcription; and COL1A2 gene up-regulation by TGFbeta is abolished in cells stably transfected with plasmids that express dominant negative forms of Smad3 or Smad4. In Sp1-deficient Drosophila Schneider cells, there was cooperative synergy between Smad3/Smad4 and Sp1 at the TbRE site. The analysis also emphasized the requirement of both Sp1- and Smad-binding sites for optimal promoter transactivation. In cells stably transfected with a plasmid expressing a dominant negative form of Sp1, the synergy was shown to be promoter-specific and dependent on the binding of Sp1 to the TbRE. Interestingly, overexpression of dominant negative Sp1 was found to block the antagonistic signal of tumor necrosis factor-alpha on COL1A2 transcription, as well. These results provide the first linkage between the Smad3 and Smad4 proteins and TGFbeta stimulation of type I collagen biosynthesis.

3T3 Cells↗
PubMed2000

Unsaturated fatty acids down-regulate srebp isoforms 1a and 1c by two mechanisms in HEK-293 cells.

Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that increase the synthesis of fatty acids as well as cholesterol in animal cells. All three SREBP isoforms (SREBP-1a, -1c, and -2) are subject to feedback regulation by cholesterol, which blocks their proteolytic release from membranes. Previous data indicate that the SREBPs are also negatively regulated by unsaturated fatty acids, but the mechanism is uncertain. In the current experiments, unsaturated fatty acids decreased the nuclear content of SREBP-1, but not SREBP-2, in cultured human embryonic kidney (HEK)-293 cells. The potency of unsaturated fatty acids increased with increasing chain length and degree of unsaturation. Oleate, linoleate, and arachidonate were all effective, but the saturated fatty acids palmitate and stearate were not effective. Down-regulation occurred at two levels. The mRNAs encoding SREBP-1a and SREBP-1c were markedly reduced, and the proteolytic processing of these SREBPs was inhibited. When SREBP-1a was produced by a cDNA expressed from an independent promoter, unsaturated fatty acids reduced nuclear SREBP-1a without affecting the mRNA level. There was no effect when the cDNA encoded a truncated version that was not membrane-bound. When administered together, sterols and unsaturated fatty acids potentiated each other in reducing nuclear SREBP-1. In the absence of fatty acids, sterols did not cause a sustained reduction of nuclear SREBP-1, but they did reduce nuclear SREBP-2. We conclude that unsaturated fatty acids, as well as sterols, can down-regulate nuclear SREBPs and that unsaturated fatty acids have their greatest inhibitory effects on SREBP-1a and SREBP-1c, whereas sterols have their greatest inhibitory effects on SREBP-2.

Base Sequence↗
PubMed2000

Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta.

The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily that are bound and activated by oxysterols. These receptors serve as sterol sensors to regulate the transcription of gene products that control intracellular cholesterol homeostasis through catabolism and transport. In this report, we describe a novel LXR target, the sterol regulatory element-binding protein-1c gene (SREBP-1c), which encodes a membrane-bound transcription factor of the basic helix-loop-helix-leucine zipper family. SREBP-1c expression was markedly increased in mouse tissues in an LXR-dependent manner by dietary cholesterol and synthetic agonists for both LXR and its heterodimer partner, the retinoid X receptor (RXR). Expression of the related gene products, SREBP-1a and SREBP-2, were not increased. Analysis of the mouse SREBP-1c gene promoter revealed an RXR/LXR DNA-binding site that is essential for this regulation. The transcriptional increase in SREBP-1c mRNA by RXR/LXR was accompanied by a similar increase in the level of the nuclear, active form of the SREBP-1c protein and an increase in fatty acid synthesis. Because this active form of SREBP-1c controls the transcription of genes involved in fatty acid biosynthesis, our results reveal a unique regulatory interplay between cholesterol and fatty acid metabolism.

Animals↗
PubMed2000

A high-performance liquid chromatographic method to measure sphingosine 1-phosphate and related compounds from sphingosine kinase assays and other biological samples.

Sphingosine 1-phosphate is an intermediate of sphingosine catabolism as well as a potent signaling compound. Conditions were established for the extraction and analysis of sphingosine 1-phosphate and other sphingoid base 1-phosphates from in vitro sphingosine kinase assays and other biological samples. The sphingoid base 1-phosphates were extracted in high yield (85%) using small C-18 reverse-phase columns (LiChroprep RP-18). After the extracts were treated with 0.1 N KOH to remove glycerolipids, the sphingoid base 1-phosphates were converted to fluorescent o-phthalaldehyde derivatives that were separated by HPLC using C-18 columns with a mobile phase of methanol:10 mM potassium phosphate (pH 7.2):1 M tetrabutylammonium dihydrogen phosphate (in water) (83:16:1, v/v/v). The o-phthalaldehyde derivative of sphingosine 1-phosphate was reasonably stable (t(1/2) > or = 18 h) when EDTA was present and could be detected in picomole amounts. The HPLC retention time of the sphingoid base 1-phosphates could be shifted by adjusting the mobile phase to pH 5.5, which is useful in separating overlapping compounds (such as sphingosine 1-phosphate and 4-D-hydroxysphinganine) and in confirming the identity of sphingoid base 1-phosphates in biological samples. The extraction procedure and HPLC method facilitated assays of sphingosine kinase with different sphingoid bases as substrates and/or inhibitors and enabled the quantitation of sphingoid base 1-phosphates in human plasma, serum, and platelets as well as in strains of Saccharomyces cerevisae with mutations in sphingolipid metabolism.

Blood Platelets↗
PubMed2000

Simulation modeling of nuclear steam generator water level process--a case study

Simulation modeling of the nuclear steam generator (SG) water level process in Qinshan Nuclear Power Plant (QNPP) is described in this paper. A practical methodology was adopted so that the model is both simple and accurate for control engineering implementation. The structure of the model is in the form of a transfer function, which was determined based on first-principles analysis and expert experience. The parameters of the model were obtained by taking advantage of the recorded historical response curves under the existing closed-loop control system. The results of process dimensional data verification and experimental tests demonstrate that the simulation model depicts the main dynamic characteristics of the SG water level process and is in accordance with the field recorded response curves. The model has been successfully applied to the design and test of an advanced digital feedwater control system in QNPP.

Journal Article↗