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Biomedical subjects

R Lu

Publications and source records attributed to R Lu.

At least 19 recordsLinked to original sources
PubMed2005

Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans.

The worm Caenorhabditis elegans is a model system for studying many aspects of biology, including host responses to bacterial pathogens, but it is not known to support replication of any virus. Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs and may act cooperatively. However, it is not known whether the single Dicer of worms and mammals is able to initiate the small RNA-guided RNA interference (RNAi) antiviral immunity as occurs in plants and insects. Here we show complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. We show that FHV replication in C. elegans triggers potent antiviral silencing that requires RDE-1, an Argonaute protein essential for RNAi mediated by small interfering RNAs (siRNAs) but not by microRNAs. This immunity system is capable of rapid virus clearance in the absence of FHV B2 protein, which acts as a broad-spectrum RNAi inhibitor upstream of rde-1 by targeting the siRNA precursor. This work establishes a C. elegans model for genetic studies of animal virus-host interactions and indicates that mammals might use a siRNA pathway as an antiviral response.

Animals↗
PubMed2004

Microsensor measurement of oxygen concentration in biofilms: from one dimension to three dimensions.

In this study, we measured oxygen concentration in biofilms in one dimension in field conditions and in three dimensions in laboratory conditions by using a robust oxygen microsensor in combination with an automation and data acquisition system. The biofilms were on the discs of rotating biological contactors treating domestic wastewater. The results of this study provide experimental evidence on oxygen distribution in wastewater biofilms and on biofilm structure. (1) The three dimensional measurements of oxygen concentration in biofilms revealed "pockets" of oxygen in deep sections of biofilms. In these isolated "pockets," located 600-760 microm from the biofilm surface, dissolved oxygen concentration was as high as 1 mg/L. This depth of oxygen diffusion is deeper than what was determined based on one dimensional measurements. (2) The heterogeneity of oxygen distribution was related to the surface structure of biofilms. The structure of the biofilm surface affected the diffusion boundary layer over the surface and, in turn, the oxygen diffusion and distribution inside biofilms. (3) Oxygen concentration in biofilms changed generally from a high degree of heterogeneity near the biofilm surface to a low degree of heterogeneity in deep sections of biofilms, indicating a cell-clusters-like structure near the surface and a more compact base layer close to the substratum.

Automation↗
PubMed2002

Endothelium-dependent vasorelaxation and the expression of calcitonin gene-related peptide in aged rats.

To explore the age-related change in endothelium-dependent vasorelaxation, plasma concentrations and the expression of calcitonin gene-related peptide (CGRP) as well as serum concentrations of asymmetry dimethylarginine (ADMA), male Sprague-Dawley rats, aged 6 and 24 months were tested. The level of ADMA and CGRP in blood, and CGRP mRNA in dorsal root ganglia (DRG) were determined by high-performance liquid chromatography (HPLC), radioimmunoassay or semi-quantitative reverse-transcription polymerase chain reaction, respectively. Vasodilator responses to cumulative concentrations of acetylcholine (ACh) (3 x 10(-9) - 3 x 10(-7)M) and CGRP (3 x 10(-10) - 3 x 10(-8)M) were tested at the plateau of contraction in aortas rings. Vasodilator responses to ACh or CGRP at 24 months of age were decreased compared with 6 months. The serum concentration of ADMA at 24 months of age was significantly increased compared with 6 months, while both plasma concentrations of CGRP and the expressions of alpha- and beta-CGRP mRNA were unchanged at two age groups. The results suggest that endothelium-dependent relaxation, but not concentrations and the expressions of CGRP, is decreased in aged rats, and the decrease in endothelium dependence of vasodilator response to CGRP may be related to the elevation of endogenous ADMA.

Aging↗
PubMed2001

Monocyte differentiation to macrophage requires interferon regulatory factor 7.

Interferon regulatory factors are a growing family of transcription factor that have been implicated in cellular events such as cell-growth regulation, antiviral defense, and development of the immune system. Interferon regulatory factor 7 (IRF-7) is expressed predominantly in lymphoid tissues and has been studied extensively in the context of viral infection and the induction of interferon and cytokine gene expression. In this paper, the involvement of IRF-7 in monocyte differentiation was examined in U937, HL60, and human primary macrophages. We report the induction of IRF-7 expression by 12-O-tetradecanoylphorbol-13-acetate in U937 and HL60 cells and demonstrate that this induction is essential for the monocyte differentiation to macrophages. We show that the monocyte differentiation is inhibited in cells expressing a dominant negative IRF-7 mutant, as evidenced by decreased expression of two macrophage-differentiation markers, CD11b and CD11c, and impaired phagocytic activity. In addition, we demonstrate that overexpression of IRF-7 is sufficient to trigger monocyte differentiation and to induce cell cycle arrest. The identification of IRF-7 as a key regulator in monocyte differentiation suggests a novel function of IRF-7 in innate immunity.

Blotting, Northern↗
PubMed2001

Olig bHLH proteins interact with homeodomain proteins to regulate cell fate acquisition in progenitors of the ventral neural tube.

BACKGROUND: Organizing signals such as Sonic hedgehog are thought to specify neuronal subtype identity by regulating the expression of homeodomain proteins in progenitors of the embryonic neural tube. One of these, Nkx2.2, is necessary and sufficient for the development of V3 interneurons. RESULTS: We report that Olig genes, encoding basic helix-loop-helix (bHLH) proteins, are expressed in a subset of Nkx2.2 progenitors before the establishment of interneurons and oligodendroglial precursors. Gain-of-function analysis in transgenic mouse embryos indicates that Olig genes specifically inhibit the establishment of Sim1-expressing V3 interneurons. Moreover, coexpression of Olig2 with Nkx2.2 in the chick neural tube generated cells expressing Sox10, a marker of oligodendroglial precursors. Colocalization of Olig and Nkx2.2 proteins at the dorsal extent of the Nkx2.2 expression domain is consistent with regulatory interactions that define the potential of progenitor cells in the border region. CONCLUSIONS: Interactions between homeodomain and Olig bHLH proteins evidently regulate neural cell fate acquisition and diversification in the ventral neural tube. In particular, interactions between Olig and Nkx2.2 proteins inhibit V3 interneuron development and promote the formation of alternate cell types, including those expressing Sox10.

Animals↗
PubMed2001

Delayed cardioprotection by intestinal preconditioning is mediated by calcitonin gene-related peptide.

Previous studies have shown that nitric oxide and calcitonin gene-related peptide (CGRP) are involved in mediation of the delayed cardioprotection of ischemic or pharmacological preconditioning, and nitric oxide can evoke the release of CGRP. In the present study, we examined the role of CGRP in nitric oxide-mediated delayed cardioprotection by brief intestinal ischemia in rats. The serum concentration of creatine kinase and infarct size were measured after 45-min coronary artery occlusion and 180-min reperfusion. Ischemic preconditioning was induced by six cycles of 4-min ischemia and 4-min reperfusion of the small intestine. Pretreatment with intestinal ischemic preconditioning for 24, 48, or 72 h significantly reduced infarct size and creatine kinase release, and the effects of ischemic preconditioning were completely abolished by L-nitroarginine methyl ester (L-NAME, 10 mg/kg, i.p.), an inhibitor of nitric oxide synthase, or by pretreatment with capsaicin (50 mg/kg, s.c.), which selectively depletes transmitters in capsaicin-sensitive sensory nerves. Intestinal preconditioning caused a significant increase in plasma concentrations of CGRP, and the effect was also abolished by L-NAME or capsaicin. These results suggest that the delayed cardioprotection afforded by intestinal ischemic preconditioning is mediated by endogenous CGRP via the nitric oxide pathway.

Animals↗
PubMed2001

Calcitonin gene-related peptide-mediated ischemic preconditioning in the rat heart: influence of age.

In the present study, we examined whether age-related reduction of ischemic preconditioning is related to calcitonin gene-related peptide (CGRP) release in the rat heart. Thirty minutes of global ischemia and 40 min of reperfusion caused a significant decrease of cardiac function and a marked increase of creatine kinase (CK) release at 2, 6 and 20 months of age. Ischemic preconditioning and pretreatment with CGRP for 5 min significantly improved cardiac function and reduced CK release during reperfusion at 2 and 6 months of age but not at 20 months of age. The content of CGRP in the coronary effluent during ischemic preconditioning was significantly increased in the first cycle at 2, 6 months of age but not at 20 months of age. These results suggest that the protection afforded by ischemic preconditioning is decreased in aging hearts, and the age-related change may be related to reduction of the release and effect of CGRP in the rat heart.

Aging↗
PubMed2001

Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain.

Zonula occludens toxin (Zot) is an enterotoxin elaborated by Vibrio cholerae that increases intestinal permeability by interacting with a mammalian cell receptor with subsequent activation of intracellular signaling leading to the disassembly of the intercellular tight junctions. Zot localizes in the bacterial outer membrane of V. cholerae with subsequent cleavage and secretion of a carboxyl-terminal fragment in the host intestinal milieu. To identify the Zot domain(s) directly involved in the protein permeating effect, several zot gene deletion mutants were constructed and tested for their biological activity in the Ussing chamber assay and their ability to bind to the target receptor on intestinal epithelial cell cultures. The Zot biologically active domain was localized toward the carboxyl terminus of the protein and coincided with the predicted cleavage product generated by V. cholerae. This domain shared a putative receptor-binding motif with zonulin, the Zot mammalian analogue involved in tight junction modulation. Amino acid comparison between the Zot active fragment and zonulin, combined with site-directed mutagenesis experiments, confirmed the presence of an octapeptide receptor-binding domain toward the amino terminus of the processed Zot.

Amino Acid Sequence↗
PubMed2001

Epiplakin, a novel member of the Plakin family originally identified as a 450-kDa human epidermal autoantigen. Structure and tissue localization.

A 450-kDa human epidermal autoantigen was originally identified as a protein that reacted with the serum from an individual with a subepidermal blistering disease. Molecular cloning of this protein has now shown that it contains 5065 amino acids and has a molecular mass of 552 kDa. As reported previously this protein, which we call epiplakin, belongs to the plakin family, but it has some very unusual features. Epiplakin has 13 domains that are homologous to the B domain in the COOH-terminal region of desmoplakin. The last five of these B domains, together with their associated linker regions, are particularly strongly conserved. However, epiplakin lacks a coiled-coil rod domain and an amino-terminal domain, both of which are found in all other known members of the plakin family. Furthermore, no dimerization motif was found in the sequence. Thus, it is likely that epiplakin exists in vivo as a single-chain structure. Epitope mapping experiments showed that the original patient's serum recognized a sequence unique to epiplakin, which was not found in plectin. Immunofluorescence staining revealed the presence of epiplakin in whole sheets of epidermis and esophagus, in glandular cells of eccrine sweat and parotid glands and in mucous epithelial cells in the stomach and colon.

Amino Acid Sequence↗
PubMed2001

Mediation of estrogen mitogenic effect in human breast cancer MCF-7 cells by PC-cell-derived growth factor (PCDGF/granulin precursor).

PC-cell-derived growth factor (PCDGF) is an 88-kDa glycoprotein corresponding to the granulin precursor. We have reported that PCDGF was expressed in human breast cancer cells. In estrogen-receptor positive cells, 17-beta-estradiol (E(2)) transcriptionally stimulated PCDGF expression in a dose- and time-dependent fashion. We demonstrate here that PCDGF mediates the mitogenic effect of E(2) in MCF-7 cells. PCDGF substituted for E(2) to stimulate DNA synthesis. The E(2) mitogenic effect was inhibited in a dose-dependent fashion by anti-PCDGF neutralizing antibody. Inhibition of PCDGF expression by antisense transfection also inhibited the E(2) mitogenic effect. In contrast, overexpression of PCDGF in MCF-7 cells resulted in cells that were able to proliferate in the absence of estrogen and were tamoxifen resistant. The PCDGF signaling pathway was examined. Like E(2), PCDGF stimulated mitogen-activated protein kinase activity. PCDGF could substitute for E(2) in stimulating cyclin D1 expression. The cyclin D1 stimulation by E(2) was 50% inhibited by anti-PCDGF antibody. In contrast, PCDGF did not stimulate c-myc expression, another molecular target of E(2). We conclude that autocrine PCDGF mediates the E(2) mitogenic effect via stimulation of cyclin D1. These studies provide information on estrogen action and identify an autocrine molecular target in human breast cancer cells.

Antibodies↗
PubMed2001

Purification and preliminary characterization of the zonula occludens toxin receptor from human (CaCo2) and murine (IEC6) intestinal cell lines.

In the present study, we report the preliminary characterization of the epithelial cell receptor for Vibrio cholerae zonula occludens toxin (Zot). Zot receptor was purified by ligand-affinity chromatography. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis revealed a protein of ca. 66 kDa. Partial N-terminal sequence obtained from purified murine and human Zot receptor revealed homology between the two proteins and with human alpha-1-chimaerin. Zot protein domain(s) involved in receptor binding were also analyzed by constructing several in frame deletion derivatives of a recombinant fusion Zot protein tagged with maltose binding protein. Our results suggest that Zot binding to its cellular membrane receptor requires a sequence that spans between amino acids 118 and 299.

Amino Acid Sequence↗
PubMed2001

Glutamine ameliorates mechanical obstruction-induced intestinal injury.

BACKGROUND: Glutamine has been shown to be an important dietary component for the maintenance of gut integrity. Although considered a nonessential amino acid in normal circumstances, glutamine may become conditionally essential for the bowel during episodes of severe illness and malnutrition. In this study, we employed an animal model simulating mechanical intestinal obstruction to explore the beneficial effects of glutamine on the intestine in response to obstruction-induced injury. MATERIALS AND METHODS: Rats were on three feeding regimens-standard diet and water (control group), diet and water containing 2% glutamine (glutamine group), or diet and water containing 2% arginine (arginine group)-for 3 days prior to surgical preparation of intestinal obstruction. The bowel distension, fluid accumulation, and histological alterations in the intestinal mucosa were measured 40 h after ileal ligation. RESULTS: After 3 days of drinking water intervention, the plasma glutamine levels in the glutamine group (677 +/- 12 microM) were higher than those in the control (451 +/- 27 microM) and arginine (379 +/- 25 microM) groups. The distension ratio measured 40 h after ileal ligation was significantly lower in the glutamine group (30.9 +/- 4.2%) than in the control and arginine groups (45.9 +/- 1.7 and 46.1 +/- 3.4%, respectively). Also, glutamine markedly decreased the fluid accumulation in the obstructed bowel segment (control group, 178.41 +/- 18.60 mg/cm; glutamine group, 104.97 +/- 13.17 mg/cm; arginine group, 141.4 +/- 12.85 mg/cm). Furthermore, the obstruction-induced mucosal injury was substantially improved in glutamine-fed rats. CONCLUSIONS: Our findings indicate that glutamine can significantly reduce the degree of those physiological derangements induced by mechanical intestinal obstruction.

Administration, Oral↗
PubMed2001

Alterations of telomerase activity and terminal restriction fragment in gastric cancer and its premalignant lesions.

AIMS: In order to explore the role of alterations of telomerase activity and terminal restriction fragment (TRF) length in the development and progression of gastric cancer. METHODS: Telomerase activity was detected in 176 specimens of gastric mucosa obtained through an operation or endoscopical biopsy by using the telomeric repeat amplification protocol (TRAP) assay. Meanwhile, the mean length of TRF was measured with the use of a Southern blot in part of those samples. RESULTS: Telomerase activity was detected in 14 of 57 (24.6%) chronic atrophy gastritis patients, six of 18 (33.3%) intestinal metaplasia patients, three of eight (37.5%) dysplasia patients and 60 of 65 (92.3%) gastric cancer patients, respectively. Normal gastric mucosa revealed no telomerase activity. No association was found between telomerase activity and any clinicopathological parameters. The mean TRF length was decreased gradually with age in normal mucosa and in gastric cancer tissue. Regression analysis demonstrated that the reduction rate in these tissues was 41 +/- 12 base pairs/year. Among 35 gastric cancers, TRF length was shown to be shorter in 20 cases (57.1%), similar in 12 cases (34.3%) and elongated in three cases (7.6%), compared to the corresponding adjacent tissues. The mean TRF length tended to decrease as the mucosa underwent chronic atrophy gastritis, intestinal metaplasia, dysplasia and into gastric cancer. The mean TRF length in gastric cancer was not statistically correlated with clinicopathological parameters and telomerase activity. CONCLUSIONS: Our results suggest that telomerase is expressed during the early stage of gastric carcinogenesis, and that the clinical significance of TRF length appears to be limited in gastric cancer.

Adult↗
PubMed2001

Effect of resveratrol on the expression of autocrine growth modulators in human breast cancer cells.

The effect of resveratrol on the growth of human breast cancer cells was examined. Resveratrol inhibited the growth of estrogen receptor-positive MCF-7 cells cultivated in the presence of estradiol in a dose-dependent fashion. At 10(-5) M, resveratrol maximally inhibited the growth stimulatory effect mediated by 10(-9) M estradiol without affecting cell viability. At the molecular level, resveratrol in a dose-dependent fashion antagonized the stimulation by estradiol of an estrogen response element reporter gene construct and of progesterone receptor gene expression in MCF-7 cells. Resveratrol also inhibited the proliferation of the estrogen-receptor negative human breast carcinoma cell line MDA-MB-468. These later data suggest that resveratrol can also inhibit breast cancer cell proliferation by another mechanism besides estrogen receptor antagonism. We show here that resveratrol altered the expression of several autocrine growth modulators and their receptors in MCF-7 cells. Resveratrol at 10(-5) M inhibited the expression of the autocrine growth stimulators transforming growth factor-alpha (TGF-alpha), PC cell-derived growth factor, and insulin-like growth factor I receptor mRNA. In addition, resveratrol significantly elevated the expression of the growth inhibitor TGF-beta2 mRNA without changes in TGF-beta1 and TGF-beta3 expression. These data suggest that resveratrol inhibits proliferation by altering autocrine growth modulator pathways in breast cancer cells.

Antineoplastic Agents, Phytogenic↗
PubMed2001

Human immunodeficiency virus type 1 replication in the absence of integrase-mediated dna recombination: definition of permissive and nonpermissive T-cell lines.

Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

CD4-Positive T-Lymphocytes↗
PubMed2001

Genome segment S8 of grass carp hemorrhage virus encodes a virion protein.

The complete nucleotide sequence of the genome segment S8 of grass carp hemorrhage virus (GCHV) was determined from cDNA corresponding to the viral genomic RNA. It is 1,287 nucleotides in length and contains a large open reading frame that could encode a protein of 409 amino acids with a predicted molecular mass of 44 kD. The S8 was expressed using the pET fusion protein vector and detected by Western blotting analysis using the chicken egg IgY against intact GCHV particles, indicating that S8 encodes a virion protein. Amino acid sequence comparisons revealed that the protein encoded by S8 is closely related to protein sigma2 of mammalian reovirus, suggesting that the deduced protein of S8 is an inner capsid protein.

Amino Acid Sequence↗
PubMed2001

Transgenic mice overexpressing leptin accumulate adipose mass at an older, but not younger, age.

Sensitivity to leptin is associated with a normal regulation of the adipose mass, whereas decreased leptin sensitivity results in elevated adipose tissue stores. To address whether the effects of chronic hyperleptinemia are sustained with age, we generated transgenic mice that overexpress leptin under the control of the fat specific aP2 promoter/enhancer. At 6-9 weeks of age, transgenic mice overexpressed 5-fold more human leptin than endogenous mouse levels and had consistently low body weights, with reduced brown and white fat depots characterized by adipocytes either devoid of or containing minute lipid droplets. However, at 33-37 weeks, despite continuous secretion of human leptin, the transgenic mice showed a rebound effect characterized by an increase in body weight, accumulation of adipose mass, and lipid-filled adipocytes. Thus, this mouse model exhibits a two-stage phenotype, with respect to fat accumulation. In addition, plasma glucose, triglycerides, and cholesterol levels were markedly depressed in young, but not older, transgenic mice. A detrimental consequence of early hyperleptinemia was a failure of the transgenic mice to acclimatize to the cold, as a result of depleted fat stores within their brown adipocytes. Cold exposure was tolerated after a 2-week high-fat diet or at an older age when fat depots had naturally accumulated. Treatment of the older transgenic mice with large doses of leptin stimulated weight loss, demonstrating that the leptin pathway still responds to pharmacological levels of leptin. Overall, these studies show that moderate hyperleptinemia in normal mice results in a sensitivity of the adipose mass to leptin at a younger (but not older) age. The mechanisms that lead to the accumulation of fat at an older age remain largely unknown, and this hyperleptinemic mouse model will allow the uncovering of at least some of these mechanisms.

Adipocytes↗