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Biomedical subjects

D A Mann

Publications and source records attributed to D A Mann.

At least 19 recordsLinked to original sources
PubMed2006

Pharmacological inhibitors of NF-kappaB accelerate apoptosis in chronic lymphocytic leukaemia cells.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a critical role in the inappropriate survival of various types of malignant cells. Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the Western world. Although overexpression and regulation of NF-kappaB has been described in CLL, its function remains unclear. Exposure of CLL cells to BAY117082 or Kamebakaurin, potent pharmacological inhibitors of the NF-kappaB pathway, accelerated apoptosis in approximately 70% of cases. Sensitivity to NF-kappaB pathway inhibitors was not related to the prognostic markers VH status, CD38 or Zap70 expression, or to the levels of nuclear NF-kappaB. Normal peripheral B cells were resistant to the apoptosis-inducing effects of these compounds. Cell death induced by the inhibitors was associated with activation of caspase-9 and -3, and loss of mitochondrial membrane polarization, but did not involve changes in the expression of Bcl-2 or Mcl-1. Inhibitors caused an increase in c-jun NH2-terminal kinase activity in CLL, but this did not appear to be important for apoptosis. Microarray analysis identified some potential novel NF-kappaB target genes, including interleukin-16- and the Bcl-2- related survival protein Bcl-w. These results demonstrate that a substantial proportion of CLL are dependent on NF-kappaB for enhanced survival and suggest that inhibition of NF-kappaB may have therapeutic potential.

ADP-ribosyl Cyclase 1↗
PubMed2006

Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis.

Myofibroblasts are critical cellular elements of wound healing generated at sites of injury by transdifferentiation of resident cells. A paradigm for this process is conversion of hepatic stellate cells (HSC) into hepatic myofibroblasts. Treatment of HSC with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC) blocked transdifferentiation. 5-azadC also prevented loss of IkappaBalpha and PPARgamma expression that occurs during transdifferentiation to allow acquisition of proinflammatory and profibrogenic characteristics. ChIP analysis revealed IkappaBalpha promoter is associated with transcriptionally repressed chromatin that converts to an active state with 5-azadC treatment. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. siRNA knockdown of MeCP2 elevated IkappaBalpha promoter activity, mRNA and protein expression in myofibroblasts. MeCP2 interacts with IkappaBalpha promoter via a methyl-CpG-dependent mechanism and recruitment into a CBF1 corepression complex. We conclude that MeCP2 and DNA methylation exert epigenetic control over hepatic wound healing and fibrogenesis.

Animals↗
PubMed2006

Naltrexone, an opioid receptor antagonist, attenuates liver fibrosis in bile duct ligated rats.

AIM: The aim of this study was to investigate the hypothesis that the opioid system is involved in the development of hepatic fibrosis. METHODS: The effect of naltrexone (an opioid receptor antagonist) on hepatic fibrosis in bile duct ligated (BDL) or sham rats was assessed by histology and hepatic hydroxyproline levels. Liver matrix metalloproteinase 2 (MMP-2) was measured by zymography, and alpha smooth muscle actin (alpha-SMA) and CD45 (leucocyte common antigen) by immunohistochemistry. The redox state of the liver was assessed by hepatic glutathione (GSH)/oxidised glutathione (GSSG) and S-nitrosothiol levels. Subtypes of opioid receptors in cultured hepatic stellate cells (HSCs) were characterised by reverse transcriptase-polymerase chain reaction, and the effects of selective delta opioid receptor agonists on cellular proliferation, tissue inhibitor of metalloproteinase 1 (TIMP-1), and procollagen I expression in HSCs determined. RESULTS: Naltrexone markedly attenuated the development of hepatic fibrosis as well as MMP-2 activity (p<0.01), and decreased the number of activated HSCs in BDL rats (p<0.05). The development of biliary cirrhosis altered the redox state with a decreased hepatic GSH/GSSG ratio and increased concentrations of hepatic S-nitrosothiols, which were partially or completely normalised by treatment with naltrexone, respectively. Activated rat HSCs exhibited expression of delta1 receptors, with increased procollagen I expression, and increased TIMP-1 expression in response to delta(1) and delta(2) agonists, respectively. CONCLUSIONS: This is the first study to demonstrate that administration of an opioid antagonist prevents the development of hepatic fibrosis in cirrhosis. Opioids can influence liver fibrogenesis directly via the effect on HSCs and regulation of the redox sensitive mechanisms in the liver.

Animals↗
PubMed2005

The role and regulation of hepatic stellate cell apoptosis in reversal of liver fibrosis.

Liver fibrosis and its end-stage disease cirrhosis are major world health problems arising from chronic injury of the liver by a variety of etiological factors including viruses, alcohol and drug abuse, the metabolic syndrome, autoimmune disease and hereditary disorders of metabolism. Fibrosis is a progressive pathological process in which wound-healing myofibroblasts of the liver respond to injury by promoting replacement of the normal hepatic tissue with a scar-like matrix composed of cross-linked collagen. Until recently it was believed that this process was irreversible. However emerging experimental and clinical evidence is starting to show that even cirrhosis is potentially reversible. Key to this is the discovery that reversion of fibrosis is accompanied by clearance of hepatic stellate cells (HSC) by apoptosis. Furthermore, proof-of-concept studies in rodents have demonstrated that experimental augmentation of HSC apoptosis will promote the resolution of fibrosis. Consequently there is now considerable interest in determining the molecular events that regulate HSC apoptosis and the discovery of drugs that will stimulate HSC apoptosis in a selective manner. This review will consider the regulatory role played by growth factors (e.g. NGF, IGF-1, TGFbeta), death receptor ligands (TRAIL, FAS), components and regulators of extracellular matrix (integrins, collagen, matrix metalloproteinases and their tissue inhibitors) and signal transduction proteins and transcription factors (Rho/Rho kinase, Jun N-terminal Kinase (JNK), IkappaKinase (IKK), NF-kappa B). The potential for known pharmacological agents such as gliotoxin, sulfasalazine, benzodiazepine ligands, curcumin and tanshinone I to induce HSC apoptosis and therefore to be used therapeutically will be explored.

Animals↗
PubMed2005

Novel sulfasalazine analogues with enhanced NF-kB inhibitory and apoptosis promoting activity.

The NF-kB transcription factor plays a key role in the regulation of apoptosis by modulating expression of a wide range of cell death control molecules. NF-kB also plays an important role in human diseases by promoting inappropriate cell survival. Small molecule inhibitors of NF-kB are therefore likely to provide novel therapeutic opportunities. Sulfasalazine (SFZ) is a synthetic anti-inflammatory comprising an aminosalicylate, 5-amino salicylic acid (5-ASA), linked to an antibiotic, sulfapyridine (SPY). SFZ, but not 5-ASA or SPY, inhibits activation of NF-kB. We synthesised a small number of SFZ analogues and determined their ability to inhibit NF-kB activity and promote apoptosis in chronic lymphocytic leukaemia and hepatic stellate cells, where NF-kB plays an important role in cell survival. Remarkably, 3 of the 6 analogues synthesised were significantly more effective (up to 8-fold) inhibitors of NF-kB dependent transcription and this increased activity was associated with enhanced apoptosis. Therefore, it is possible to readily improve the NF-kB inhibiting activity of SFZ and analogues of SFZ may be attractive therapeutic agents for malignancies and chronic liver disease where NF-kB is thought to play a significant role.

Animals↗
PubMed2005

Herpesvirus saimiri-based vector biodistribution using noninvasive optical imaging.

Herpesvirus saimiri (HVS) is capable of infecting a range of human cell types with high efficiency and the viral genome persists as high copy number, circular, nonintegrated episomes which segregate to progeny upon cell division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Here we assess the dissemination of HVS-based vectors in vivo following intravenous and intraperitoneal administration. Bioluminescence imaging of an HVS-based vector expressing luciferase demonstrates that the virus can infect and establish a persistent latent infection in a variety of mouse tissues. Moreover, the long-term in vivo maintenance of the HVS genome as a nonintegrated circular episome provided sustained expression of luciferase over a 10-week period. A particularly high level of transgene expression in the liver and the ability of HVS to infect and persist in hepatic stellate cells suggest that HVS-based vectors may have potential for the treatment of inherited and acquired liver diseases.

Animals↗
PubMed2004

Oxidant regulation of the bivalent cation transporter Nramp1.

Nramp1 (murine natural resistance-associated macrophage protein 1 gene)/Slc11a1 (solute carrier family 11 member a1 gene) encodes a bivalent-metal/iron transporter that is expressed within late endosomes/lysosomes of macrophages. A functionally null Nramp1 allele that exhibits impaired bivalent cation transport enables excessive growth of intracellular pathogens. Iron is important for many cellular activities, including defence against pathogens; however, redox-active/free iron can participate in Fenton chemistry that generates reactive oxygen species. Using Raw264.7 cells, non-functional for Nramp1, and stable Nramp1 transfectants, we have examined the effects of impaired bivalent cation transport on macrophage function using glutathione depletion as OS (oxidant stress). Our results demonstrate that OS itself is a signal for increasing Nramp1 transcription and that Nramp1 expression protects against OS. We suggest that OS-mediated protection by Nramp1 function may arise from direct removal of redox-active bivalent cations from a cytosolic pool. We show that OS transcriptional responses are probably mediated by the Sp1 transcription factor.

Actins↗
PubMed2002

c-Myc represses the murine Nramp1 promoter.

The Nramp1 (natural resistance-associated macrophage protein 1) gene modulates the growth of intracellular pathogens and encodes a divalent cation transporter within lysosomes/late endosomes of macrophages. Nramp1 modulates the cytoplasmic iron pool. Wu, Polack and Dalla-Favera [(1999) Science 283, 676-679] showed reciprocal control of H-ferritin and IRP2 by c-Myc, and suggest that c-Myc regulates genes to increase cytoplasmic iron. A role for c-Myc in Nramp1 regulation was evaluated. Co-transfection studies show that c-Myc represses Nramp1 promoter function. Five non-canonical Myc-max binding sites (E-box) identified within the Nramp1 5'-flanking sequence are not responsible for the inhibitory effects of c-Myc on Nramp1 expression. An initiator(s) adjacent to the transcription-initiation site is a candidate for the inhibition observed. Results are consistent with a role for Nramp1 removing iron from the cytosol and antagonizing c-Myc function.

Animals↗
PubMed2002

Transcriptional regulation of hepatic stellate cell activation.

The hepatic stellate cell (HSC) is now well established as the key cellular element involved in the development of hepatic fibrosis and because of this there is considerable interest in establishing the molecular events that trigger and perpetuate HSC activation. HSC activation at the level of gene transcription requires the coordinated activity of several key transcriptional regulators of the HSC genome. The considerable advances that have been made in the past five years into the mechanisms by which specific families of transcription factors regulate the profibrogenic characteristics of the activated HSC are reviewed.

CCAAT-Enhancer-Binding Proteins↗
PubMed2001

JunD regulates transcription of the tissue inhibitor of metalloproteinases-1 and interleukin-6 genes in activated hepatic stellate cells.

Activation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in hepatic wound healing and fibrosis. Rat HSCs activated in vitro express JunD, Fra2, and FosB as the predominant AP-1 DNA-binding proteins, and all three associate with an AP-1 sequence that is essential for activity of the tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter. In this study, we used expression vectors for wild-type, dominant-negative, and forced homodimeric (Jun/eb1 chimeric factors) forms of JunD and other Fos and Jun proteins to determine the requirement for JunD in the transcriptional regulation of the TIMP-1 and interleukin-6 (IL-6) genes. JunD activity was required for TIMP-1 gene promoter activity, whereas overexpression of Fra2 or FosB caused a repression of promoter activity. The ability of homodimeric JunD/eb1 to elevate TIMP-1 promoter activity supports a role for JunD homodimers as the major AP-1-dependent transactivators of the TIMP-1 gene. IL-6 promoter activity was induced upon activation of HSCs and also required JunD activity; however, expression of JunD/eb1 homodimers resulted in transcriptional repression. Mutagenesis of the IL-6 promoter showed that an AP-1 DNA-binding site previously reported to be an activator of transcription in fibroblasts functions as a suppressor of promoter activity in HSCs. We conclude that JunD activates IL-6 gene transcription as a heterodimer and operates at an alternative DNA-binding site in the promoter. The relevance of these findings to events occurring in the injured liver was addressed by showing that AP-1 DNA-binding complexes are induced during HSC activation and contain JunD as the predominant Jun family protein. JunD is therefore an important transcriptional regulator of genes responsive to Jun homo- and heterodimers in activated HSCs.

Animals↗
PubMed2001

Structure of the complete extracellular domain of the common beta subunit of the human GM-CSF, IL-3, and IL-5 receptors reveals a novel dimer configuration.

The receptor systems for the hemopoietic cytokines GM-CSF, IL-3, and IL-5 consist of ligand-specific alpha receptor subunits that play an essential role in the activation of the shared betac subunit, the major signaling entity. Here, we report the structure of the complete betac extracellular domain. It has a structure unlike any class I cytokine receptor described thus far, forming a stable interlocking dimer in the absence of ligand in which the G strand of domain 1 hydrogen bonds into the corresponding beta sheet of domain 3 of the dimer-related molecule. The G strand of domain 3 similarly partners with the dimer-related domain 1. The structure provides new insights into receptor activation by the respective alpha receptor:ligand complexes.

Amino Acid Sequence↗
PubMed2001

Expression, crystallization and derivatization of the complete extracellular domain of the beta(c) subunit of the human IL-5, IL-3 and GM-CSF receptors.

The major signalling entity of the receptors for the haemopoietic cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is the shared beta(c) receptor, which is activated by ligand-specific alpha receptors. The beta(c) subunit is a stable homodimer whose extracellular region consists of four fibronectin domains and appears to be a duplication of the cytokine receptor homology module. No four domain structure has been determined for this receptor family and the structure of the beta(c) subunit remains unknown. We have expressed the extracellular domain in insect cells using the baculovirus system, purified it to homogeneity and determined its N-terminal sequence. N-glycosylation at two sites was demonstrated. Crystals of the complete domain have been obtained that are suitable for X-ray crystallographic studies, following mutagenesis to remove one of the N-glycosylation sites. The rhombohedral crystals of space group R3, with unit cell dimensions 186.1 A and 103.5 A, diffracted to a resolution of 2.9 A using synchrotron radiation. Mutagenesis was also used to engineer cysteine substitution mutants which formed isomorphous Hg derivatives in order to solve the crystallographic phase problem. The crystal structure will help to elucidate how the beta(c) receptor is activated by heterodimerization with the respective alpha/ligand complexes.

Alternative Splicing↗
PubMed2001

Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis and stimulating their apoptosis could be an effective treatment for liver fibrosis. METHODS: Activated HSCs, hepatocytes, and rats with liver fibrosis were treated with gliotoxin. RESULTS: Addition of gliotoxin to activated (alpha-smooth muscle actin positive) rat and human HSCs resulted in morphologic alterations typical of apoptosis. Within 2-3 hours of incubation, caspase 3 activity was markedly induced and caspase inhibitor 1 (Z-VAD-FMK)-sensitive oligonucleosome-length DNA fragments were detectable by gel electrophoresis of low molecular weight DNA. Apoptosis was widespread as judged by fluorescence-activated cell sorter analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining in both rat and human HSCs at concentrations that had no effect on the viability of rat hepatocytes. Gliotoxin treatment significantly reduced the number of activated stellate cells and mean thickness of bridging fibrotic septae in livers from rats treated with carbon tetrachloride. CONCLUSIONS: These data demonstrate proof-of-concept that by up-regulating HSC apoptosis, the extent of fibrosis can be decreased in inflammatory liver injury.

Animals↗
PubMed2001

Ultrasound detection by clupeiform fishes.

It has previously been shown that at least one species of fish (the American shad) in the order clupeiforms (herrings, shads, and relatives) is able to detect sounds up to 180 kHz. However, it has not been clear whether other members of this order are also able to detect ultrasound. It is now demonstrated, using auditory brainstem response (ABR), that at least one additional species, the gulf menhaden (Brevoortia patronus), is able to detect ultrasound, while several other species including the bay anchovy (Anchoa mitchilli), scaled sardine (Harengula jaguana), and Spanish sardine (Sardinella aurita) only detect sounds to about 4 kHz. ABR is used to confirm ultrasonic hearing in the American shad. The results suggest that ultrasound detection may be limited to one subfamily of clupeiforms, the Alosinae. It is suggested that ultrasound detection involves the utricle of the inner ear and speculate as to why, despite having similar ear structures, only one group may detect ultrasound.

Animals↗
PubMed2001

Regulation of E-box DNA binding during in vivo and in vitro activation of rat and human hepatic stellate cells.

BACKGROUND: Activation of hepatic stellate cells (HSCs) to a myofibroblastic phenotype is a key event in liver fibrosis. Identification of transcription factors with activities that are modulated during HSC activation will improve our understanding of the molecular events controlling HSC activation. AIMS: To determine if changes in E-box DNA binding activity occur during in vitro and in vivo activation of rat and human HSCs and to investigate mechanisms underlying any observed changes. METHODS: Nuclear extracts were prepared from rat HSCs isolated and cultured from normal and carbon tetrachloride injured rat livers and from HSCs isolated from human liver. EMSA analysis of E-box DNA binding activity was performed on nuclear extracts to determine changes during HSC activation. Western and northern blot analysis of MyoD and Id1 basic helix-loop-helix (bHLH) proteins was performed to confirm expression in HSC. RESULTS: HSC activation was associated with inducible expression of two low mobility E-box binding complexes that were immunoreactive with an anti-MyoD antibody. MyoD mRNA expression was found at similar levels in freshly isolated and activated HSCs; in contrast, MyoD protein expression was elevated in activated HSCs. Activation of rat HSCs was accompanied by reduced expression of the inhibitory bHLH protein Id1. CONCLUSIONS: In vitro and in vivo activation of rat and human HSCs is accompanied by induction of MyoD binding to E-box DNA sequences which appears to be mechanistically associated with elevated MyoD protein expression and reduced expression of the inhibitory Id1 protein. Clarification of the role of MyoD and Id1 proteins in HSC activation and liver fibrogenesis is now required.

Adult↗
PubMed2000

Upstream tissue inhibitor of metalloproteinases-1 (TIMP-1) element-1, a novel and essential regulatory DNA motif in the human TIMP-1 gene promoter, directly interacts with a 30-kDa nuclear protein.

Elevated expression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein and mRNA has been reported in human diseases including cancers and tissue fibrosis. Regulation of TIMP-1 gene expression is mainly mediated at the level of gene transcription and involves the activation of several well known transcription factors including those belonging to the AP-1, STAT, and Pea3/Ets families. In the current study, we have used DNase-1 footprinting to identify a new regulatory element (5'-TGTGGTTTCCG-3') present in the human TIMP-1 gene promoter. Mutagenesis and transfection studies in culture-activated rat hepatic stellate cells and the human Jurkat T cell line demonstrated that the new element named upstream TIMP-1 element-1 (UTE-1) is essential for transcriptional activity of the human TIMP-1 promoter. Electrophoretic mobility shift assay studies revealed that UTE-1 can form protein-DNA complexes of distinct mobilities with nuclear extracts from a variety of mammalian cell types and showed that induction of a high mobility UTE-1 complex is associated with culture activation of freshly isolated rat hepatic stellate cells. A combination of UV-cross-linking and Southwestern blotting techniques demonstrated that UTE-1 directly interacts with a 30-kDa nuclear protein that appears to be present in all cell types tested. We conclude that UTE-1 is a novel regulatory element that in combination with its cellular binding proteins may be an important component of the mechanisms controlling TIMP-1 expression in normal and pathological states.

Animals↗