Norrie disease (ND), a rare X-linked recessive disorder, is characterized by congenital blindness and, occasionally, mental retardation and hearing loss. ND is caused by the Norrie Disease Protein gene (NDP), which codes for norrin, a cysteine-rich protein involved in ocular vascular development. Here, we report a novel mutation of NDP that was identified in a Chinese family in which three members displayed typical ND symptoms and other complex phenotypes, such as cerebellar atrophy, motor disorders, and mental disorders.
We conducted an extensive clinical examination of the proband and performed a computed tomography (CT) scan of his brain. Additionally, we performed ophthalmic examinations, haplotype analyses, and NDP DNA sequencing for 26 individuals from the proband’s extended family.
The proband’s computed tomography scan, in which the fifth ventricle could be observed, indicated cerebellar atrophy. Genome scans and haplotype analyses traced the disease to chromosome Xp21.1-p11.22. Mutation screening of the NDP gene identified a novel nonsense mutation, c.343C>T, in this region.
Although recent research has shown that multiple different mutations can be responsible for the ND phenotype, additional research is needed to understand the mechanism responsible for the diverse phenotypes caused by mutations in the NDP gene.
Norrin/Frizzled4 (Fz4) signaling activates the canonical Wnt pathway to control retinal vascular development. Using genetically engineered mice, we show that precocious Norrin production leads to premature retinal vascular invasion and delayed Norrin production leads to characteristic defects in intra-retinal vascular architecture. In genetic mosaics, wild type endothelial cells (ECs) instruct neighboring Fz4−/− ECs to produce an architecturally normal mosaic vasculature, a cell non-autonomous effect. However, over the ensuing weeks, Fz4−/− ECs are selectively eliminated from the mosaic vasculature, implying the existence of a quality control program that targets defective ECs. In the adult retina and cerebellum, gain or loss of Norrin/Fz4 signaling results in a cell-autonomous gain or loss, respectively, of blood retina barrier (BRB) and blood brain barrier (BBB) function, indicating an ongoing requirement for Frizzled signaling in barrier maintenance and substantial plasticity in mature CNS vascular structure.
Xenopus maternal Norrin, which activates Wnt signaling but inhibits TGF-β family molecules, is essential for neuroectoderm formation. Loss of TGF-β inhibition in Norrin may contribute to the development of Norrie disease.
Dorsal–ventral specification in the amphibian embryo is controlled by β-catenin, whose activation in all dorsal cells is dependent on maternal Wnt11. However, it remains unknown whether other maternally secreted factors contribute to β-catenin activation in the dorsal ectoderm. Here, we show that maternal Xenopus Norrin (xNorrin) promotes anterior neural tissue formation in ventralized embryos. Conversely, when xNorrin function is inhibited, early canonical Wnt signaling in the dorsal ectoderm and the early expression of the zygotic neural inducers Chordin, Noggin, and Xnr3 are severely suppressed, causing the loss of anterior structures. In addition, xNorrin potently inhibits BMP- and Nodal/Activin-related functions through direct binding to the ligands. Moreover, a subset of Norrin mutants identified in humans with Norrie disease retain Wnt activation but show defective inhibition of Nodal/Activin-related signaling in mesoderm induction, suggesting that this disinhibition causes Norrie disease. Thus, xNorrin is an unusual molecule that acts on two major signaling pathways, Wnt and TGF-β, in opposite ways and is essential for early neuroectoderm specification.
A key step during early embryogenesis is the generation of neural precursors, which later form the central nervous system. In vertebrates, this process requires proper dorsal–ventral axis specification, and we know that the canonical Wnt and BMP signaling pathways help pattern the dorsal ectoderm. In this study, we examine other factors that are involved in neuroectoderm development in the frog species Xenopus laevis. We find that maternal Xenopus Norrin (xNorrin) is required for canonical Wnt signaling in the dorsal ectoderm, functions upstream of neural inducers, and is required for neural formation. We also find that xNorrin not only activates Wnt signaling, but also inhibits BMP/Nodal-related signaling. In humans, mutations in Norrin cause Norrie disease. Using Norrin mutants identified in patients with Norrie disease, we find that some Norrin mutants fail to inhibit BMP/Nodal-related signaling (specifically, TGF-β) but retain the ability to activate the Wnt pathway, suggesting that loss of TGF-β inhibition may contribute to Norrie disease development.
Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled4 (Fz4) receptor, Lrp5 co-receptor, or Norrin ligand cause retinal hypovascularization, but the role of Norrin/Fz4/Lrp signaling in vascular development has not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, while excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is up-regulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.
Nuclear dots containing PML and Sp100 proteins (NDs) play a role in the development of acute
promyelocytic leukemia, are modified after infection
with various viruses, and are autoimmunogenic in patients with primary biliary cirrhosis (PBC). PML and
Sp100 gene expression is strongly enhanced by interferons (IFN). Based on immunostaining with a monoclonal
antibody (mAb C8A2), a third protein, nuclear dot protein 52 (NDP52), was recently localized in NDs. Here
we analyzed the cellular localization, expression, and
structure of NDP52 in more detail. Our NDP52-specific
sera revealed mainly cytoplasmic staining but no ND
pattern, neither in untreated nor in IFN-treated cells.
Cells transfected with NDP52 expression vectors
showed exclusively cytoplasmic staining. In subcellular
fractionation experiments, NDP52 was found in cytoplasmic and nuclear fractions. Unlike as described for
Sp100 and PML, NDP52 mRNA and protein levels
were only marginally enhanced by IFN γ and not enhanced at all by IFN β. NDP52 homodimerization but
no heterodimerization with Sp100 or PML could be
demonstrated. None of the 93 PBC sera tested contained autoantibodies against NDP52. Finally, mAb
C8A2 reacted not only with NDP52 but also with a
conformation-dependent epitope on the Sp100 protein.
These data imply that NDP52 forms homodimers but
no heterodimers with Sp100 and PML, lacks autoantigenicity in PBC, localizes mainly in the cytoplasm, and
is associated with the nucleus, but not with NDs. Finally, unlike Sp100 and PML, NDP52 expression is neither markedly enhanced nor localization detectably altered by type I and II IFNs.
The melanocortin system is crucial to regulation of energy homeostasis. The melanocortin receptor type 4 (MC4R) modulates insulin signaling via effects on c-Jun N-terminal kinase (JNK). The melanocortin agonist NDP-MSH dose-dependently inhibited JNK activity in HEK293 cells stably expressing the human MC4R; effects were reversed by melanocortin receptor antagonist. NDP-MSH time- and dose-dependently inhibited IRS-1ser307 phosphorylation, effects also reversed by a specific melanocortin receptor antagonist. NDP-MSH augmented insulin-stimulated AKT phosphorylation in vitro. The melanocortin agonist melanotan II increased insulin-stimulated AKT phosphorylation in the rat hypothalamus in vivo. NDP-MSH increased insulin-stimulated glucose uptake in hypothalamic GT1-1 cells. The current study shows that the melanocortinergic system interacts with insulin signaling via novel effects on JNK activity.
c-Jun N-terminal kinase (JNK); melanocortin receptor; AKT; insulin; insulin receptor substrate 1 (IRS-1); MT II; NDP-MSH; hypothalamus
Levels of nm23 gene product/nucleoside diphosphate kinase (NDP kinase) expression have been demonstrated to correlate inversely with metastatic potential in several tumours, indicating that this could be a useful tool as a prognostic indicator. Using an antibody to NDP kinase, levels of nm23 gene product/NDP kinase expression in pulmonary adenocarcinoma were examined immunohistochemically. Of 88 patients tested, 39 (44%; Group B) showed strong immunoreactivity for NDP kinase in most of cancer cells within the tumour tissues, while 49 (56%; Group A) contained few or no NDP kinase-positive cancer cells. Nm23 gene product/NDP kinase was expressed independently of clinicopathological factors, and unexpectedly, no correlation of survival rates between both Groups could be demonstrated. Thus, in pulmonary adenocarcinoma, levels of nm23 gene product/NDP kinase expression may lack prognostic value.
The leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4, also called GPR48) plays a key role in multiple developmental processes, and mice lacking Lgr4 display anterior segment dysgenesis leading to early-onset glaucomatous retinal ganglion cell loss as well as defective eyelid formation. This paper will review Lgr4 signaling and its regulation of the Axenfeld-Rieger syndrome gene Pitx2, a crucial developmental transcription factor. In addition, Wnt signaling plays an important role in eye development, with Norrin functioning to activate the Wnt receptor Frizzled 4 required for proper retinal vascularization. Recent discoveries identifying Lgr4 as a receptor for Norrin highlight the potential for Lgr4 function in retinal vascularization. Finally, several unanswered questions impeding a full understanding of Lgr4 in glaucoma are considered as avenues for further research.
Norrin is a potent Wnt pathway ligand. Aberrant activation of this signaling pathway can result in colon tumors but the role of norrin-based signaling in the genesis of colon cancer, and its relationship to activation of the pathway by traditional Wnt ligands, is not defined.
Fresh normal human colon tissue and all the cell lines studied expressed mRNA for Fz4, LRP5 and norrin, except Colo205 which lacked Fz4 expression. Canonical Wnt pathway throughput was increased slightly in NCM460 following treatment with Wnt3a CM but was inhibited by Wnt2 and Wnt1. The colon cancer cell line, RKO, responded to Wnt3a CM, Wnt2 and Wnt1 by increasing canonical Wnt pathway throughput. Wnt5a did not affect Wnt pathway throughput in either cell line. Wnt2, but not Wnt3a, abrogated Fz4 expression in NCM460, but not in RKO or another colon cancer cell line, HCT116. This effect on Fz4 was confirmed at both the RNA and protein levels via RT-PCR and a norrin binding assay. The expression of all others 9 Fz receptors did not change after treatment of NCM460 cells with Wnt2.
The data suggests that colonic mucosa and colon tumors may possess two autoregulatory positive Wnt feedback loops, one through canonical signals induced by Wnt:Fz interactions and one through signals resulting from norrin:Fz4 interactions. The latter interactions may be modulated via regulation of Fz4 expression by Wnt2. Retention of Fz4 by cancers, in contrast to the loss of Fz4 by normal mucosal cells, could provide a selective advantage to the tumor cells. Fz4 expression may play a critical role in responses to Wnt signaling in the tumor microenvironment.
Expression of nucleoside diphosphate(NDP) kinase, which is homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential. However, the relationship remains controversial according to the tumor cell types and experimental system, with conflicting results from different research groups. In order to determine whether NDP kinase expression serves as a marker for metastatic potential in human skin cancer, we assessed the levels of NDP kinase expression in 9 keratoacanthomas (KAs), 26 squamous cell carcinomas (SCCs), and 25 basal cell carcinomas (BCCs) using immunohistochemistry. The expression of NDP kinase was intense in KA and SCC compared with BCC. However, the difference of NDP kinase expression between KA and SCC was not statistically significant. And there was no statistically significant difference in NDP kinase expression between SCC with metastasis and SCC without metastasis. Our results contradict the hypothesis concerning the possible role of nm23 gene as a metastatic suppressor gene in human skin cancer. The mechanism of overexpression in various tumor cell types and its biological significance in cutaneous carcinogenesis remain to be determined.
To develop melanoma-targeted hollow gold nanospheres (HAuNS) and evaluate their potential utility for selective photothermal ablation (PTA) in melanoma.
A new class of photothermal coupling agents based on HAuNS was synthesized. HAuNS were stabilized with poly(ethylene-glycol) coating and attached with α-melanocyte-stimulating hormone analog, [Nle4,D-Phe7]α-MSH (NDP-MSH), which is a potent agonist of melanocortin type-1 receptor overexpressed in melanoma. The intracellular uptake of the NDP-MSH-conjugated PEGylated HAuNS (NDP-MSH-PEG-HAuNS) and the distribution of β-arrestin were examined in murine B16/F10 melanoma cells. The biodistribution of NDP-MSH-PEG-HAuNs was assessed at 4 h post intravenous injection in tumor-bearing nude mice. PTA effect of the nanoparticles was evaluated both histologically using excised tissue and functionally by [18F]fluorodeoxyglucose positron emission tomography ([18F]FDG-PET).
NDP-MSH-PEG-HAuNS consist only of a thin gold wall with hollow interior (outer diameter, 43.5±2.3 nm; shell thickness, 3–4 nm), which display strong and tunable resonance absorption in near-infrared region (NIR, peak 808 nm). The nanoparticles were specifically taken up by melanoma cells, which initiated the recruitment of β-arrestins, the adapters to link the activated G-protein-coupled receptors to clathrin, indicating the involvement of receptor-mediated endocytosis. This resulted in enhanced extravasation of NDP-MSH-PEG-HAuNS from tumor blood vessels and their dispersion into tumor matrix compared with non-specific PEGylated HAuNS. Successful selective PTA of B16/F10 melanoma with targeted HAuNS was confirmed by histological and [18F]FDG-PET evaluation at 24 h post NIR laser irradiation at a low dose energy of 30 J/cm2.
NDP-MSH-PEG-HAuNS have the potentials to mediate targeted photothermal ablation of melanoma.
hollow gold nanospheres; melanocyte-stimulating hormone; photothermal ablation; melanoma; targeting
The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) can induce marked nephrotoxicity in rats following a single intraperitoneal (ip) administration of 0.4 mmol/kg or greater. Although NDPS induces direct renal proximal tubular toxicity, a role for renal vascular effects may also be present. The purpose of this study was to examine the possible role of vasoconstrictor leukotrienes in NDPS and NDPS metabolite nephrotoxicity. Male Fischer 344 rats (4 rats/group) were administered diethylcarbamazine (DEC; 250 or 500 mg/kg, ip), an inhibitor of LTA4 synthesis, 1h before NDPS (0.4 mmol/kg, ip), N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, 0.1, 0.2, or 0.4 mmol/kg, ip), or N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, 0.1 mmol/kg, ip) or vehicle. In a separate set of experiments, the LTD4 receptor antagonist LY171883 (100 mg/kg, po) was administered 0.5 h before and again 6 h after NDHS (0.1 mmol/kg, ip) or 2-NDHSA (0.1 mmol/kg, ip) or vehicle. Renal function was monitored for 48 h post-NDPS or NDPS metabolite. DEC markedly reduced the nephrotoxicity induced by NDPS and its metabolites, while LY171883 treatments provided only partial attenuation of NDHS and 2-NDHSA nephrotoxicity. These results suggest that leukotrienes contribute to the mechanisms of NDPS nephrotoxicity.
Leukotrienes; Kidney; Rats; Nephrotoxicity; N-(3, 5-Dichlorophenyl)succinimide
Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting ‘autophagy receptors’ in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF–TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-011-0819-y) contains supplementary material, which is available to authorized users.
Autophagy; A20; NDP52; Signal transduction; Toll-like receptor (TLR)
The melanocortin signaling system is integral in regulating energy homeostasis. The melanocortin-4 receptor (MC4R) activates several signaling pathways in performance of this function. The effect of MC4R on insulin-stimulated mammalian target of rapamycin (mTOR), a cellular energy sensor, signaling was investigated. The GT1-1 cell line which expresses MC4R expression was utilized. mTOR signaling was measured by Western blotting analysis using Phospho-mTOR (Ser2448) antibody. NDP-MSH dose-dependently enhanced insulin-stimulated mTOR phosphorylation. The MC4R antagonist SHU9119 blocked this effect, demonstrating specificity. The protein kinase A - cyclic AMP pathway and the MAP kinase pathway were not involved in NDP-MSH actions on insulin-stimulated mTOR phosphorylation. In contrast, the AMP-activated protein kinase agonist, AICAR, attenuated this effect. MC4R activation potentiates insulin-stimulated mTOR signaling via the AMPK pathway.
Melanocortin-4 receptor; NDP-MSH; mammalian target of rapamycin; AMPK
Plasmid pLNBIV was used to overexpress the biosynthetic pathway of nucleoside-diphosphate (NDP)-activatedl-digitoxose in the mithramycin producer Streptomyces argillaceus. This led to a “flooding” of the biosynthetic pathway of the antitumor drug mithramycin (MTM) with NDP-activated deoxysugars, which do not normally occur in the pathway, and consequently to the production of the four new mithramycin derivatives 1-4 with altered saccharide patterns. Their structures reflect that NDP sugars produced by pLNBIV, namely, l-digitoxose and its biosynthetic intermediates, influenced the glycosyl transfer to positions B, D, and E, while positions A and C remained unaffected. All four new structures have unique, previously not found sugar decoration patterns, which arise from either overcoming the substrate specificity or inhibition of certain glycosyltransferases (GTs) of the MTM pathway with the foreign NDP sugars expressed by pLNBIV. An apoptosis TUNEL (=terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay revealed that compounds 1 (demycarosyl-3D-β-d-digitoxosyl-MTM) and 3 (deoliosyl-3C-β-d-mycarosyl-MTM) show improved activity (64.8 ± 2% and 50.3 ± 2.5% induction of apoptosis, respectively) against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 compared with the parent drug MTM (37.8 ± 2.5% induction of apoptosis). In addition, compounds 1 and 4 (3A-deolivosyl-MTM) show significant effects on the ER-negative human breast cancer cell line MDA-231 (63.6 ± 2% and 12.6 ± 2.5% induction of apoptosis, respectively), which is not inhibited by the parent drug MTM itself (2.6 ± 1.5% induction of apoptosis), but for which chemotherapeutic agents are urgently needed.
The development of a general 1-Zn(II) NDP sensor assay for rapid evaluation of GT activity is described. The 1-Zn(II) NDP sensor assay offers submicromolar sensitivity, compatibility with both purified enzymes and crude cell extracts, and exquisite selectivity for nucleoside diphosphates over the corresponding NDP-sugars. Thus, the 1-Zn(II) NDP sensor assay is anticipated to offer broad applicability in the context of GT engineering and characterization.
glycosyltransferase; enzyme; evolution; engineering; carbohydrate; sugar nucleotide
Monoamine oxidases (MAO-A and MAO-B) have a key role in the degradation of amine neurotransmitters, such as dopamine, norepinephrine and serotonin. We identified an inherited 240 kb deletion on Xp11.3–p11.4, which encompasses both monoamine oxidase genes but, unlike other published reports, does not affect the adjacent Norrie disease gene (NDP). The brothers who inherited the deletion, and thus have no monoamine oxidase function, presented with severe developmental delay, intermittent hypotonia and stereotypical hand movements. The clinical features accord with published reports of larger microdeletions and selective MAO-A and MAO-B deficiencies in humans and mouse models and suggest considerable functional compensation between MAO-A and MAO-B under normal conditions.
monoamine oxidase; MAOA and MAOB; array CGH; X chromosome; abnormal hand movement
The nuclear domain (ND)10 also described as POD or Kr bodies is involved in the development of acute promyelocytic leukemia and virus- host interactions. Immunofluorescence analysis using a variety of human autoimmune sera and monoclonal antibodies showed a typical dot like nuclear staining for ND10, suggesting that this structure consists of several proteins. Two of the ND10 proteins, Sp100 and PML are genetically characterized and show homology with several transcription factors. Here we describe NDP52, an additional novel protein of the ND10. We raised a new mAb C8A2, that specifically recognizes NDP52. Immunofluorescence analysis using this mAb showed a typical nuclear dot staining as it was described for ND10. Isolation and sequencing of the corresponding cDNA revealed that NDP52 has a predicted molecular mass of 52 kD. The deduced amino acid sequence exhibits an extended central coiled coil domain containing a leucine zipper motif. The COOH terminus of NDP52 shows homology with LIM domains, that have recently been described to mediate protein interactions, which let NDP52 appear as a suitable candidate for mediating interactions between ND10 proteins. In vivo, NDP52 is transcribed in all human tissues analyzed. Furthermore, we show that NDP52 colocalizes with the ND10 protein PML and can be redistributed upon viral infection and interferon treatment. These data suggest that ND10 proteins play an important role in the viral life cycle.
AIM: To increase satisfaction and diminish anxiety and shame during colonoscopy, we developed novel double pants (NDP) which consist of doubled fabrics with an inner hole. The aim of study was to compare satisfaction, anxiety and shame between NDP and conventional single pants (CSP).
METHODS: Total 160 consecutive examinees were randomly divided into NDP and CSP group. Before colonoscopy, questionnaires identifying state and trait anxiety were completed. After colonoscopy, questionnaires for overall satisfaction (Group Health Association of America 9) and pants-specific satisfaction (5-20), state anxiety (20-80), and shame (6-24) were interviewed.
RESULTS: Pants-specific satisfaction scores regarding willingness to repeat colonoscopy using same pants (3.3 ± 0.8 vs 2.1 ± 0.9, P < 0.001) and recommendation of same pants to other people (3.3 ± 0.7 vs 2.0 ± 1.0, P < 0.001) were significantly higher in NDP than CSP groups. State anxiety (33.0 ± 7.0 vs 35.4 ± 6.9, P = 0.028) and shame (6.6 ± 1.5 vs 8.1 ± 3.2, P = 0.001) after colonoscopy was lower in NDP group compared with CSP group.
CONCLUSION: The NDP contribute to increase satisfaction and decrease anxiety and shame after colonoscopy.
Pants; Colonoscopy; Satisfaction; Shame; Anxiety
In human cells, three proteins are currently known to colocalize in di screte nuclear domains (designated nuclear dots): Sp100, a transcription-activating protein autoantigenic primarily in patients with primary biliary cirrhosis; PML, a tumor suppressor protein involved in development of acute promyelocytic leukemia; and NDP52, a protein of unknown function. Here we report sequence similarities between the Sp100 protein and a putative protein encoded by a highly amplified mouse gene which is visible as an inherited homogeneously staining region (HSR) on chromosome 1 of some mouse populations. By in situ hybridization, the Sp100 gene was mapped to locus 2q37, the syntenic region of the HSR on mouse chromosome 1. Unlike the highly amplified mouse gene, Sp100 was found to be a single-copy gene and showed no restriction fragment length polymorphisms. Sequence similarities in the promoter regions and similar exon-intron organizations of the two genes were revealed. As for Sp100, steady-state levels of the mRNAs of the HSR-encoded genes could be greatly increased by interferon (IFN) treatment. As in human cells, IFN treatment led to an enlargement in both size and number of nuclear dots in mouse cells as visualized by immunofluorescence staining with autoimmune sera from patients with primary biliary cirrhosis. These data indicate that a gene located in the inherited HSR of mice, designated mSp100, is homologous to the human Sp100 gene, has a similar gene organization, and responds similarly to IFN treatment.
A major step in the pathogenesis of Mycobacterium tuberculosis is the ability to survive inside macrophages, where it is exposed to a number of DNA damaging agents. The alternative sigma factor SigG has been shown to be upregulated by DNA damaging agents and by macrophage infection, but not to regulate genes of the DNA repair pathway. Here we show that SigG is expressed from at least two promoters, the most dominant of these being the DNA damage inducible RecA_Ndp promoter. This promoter is located within the annotated coding region of SigG and so the correct translational start site was determined experimentally and found to be 114 bp downstream of the annotated start site. Examining the gene expression profile of a SigG over-expression strain found a small number of genes to up-regulated, two of these encoded proteins containing glyoxylase-like domains.
Mycobacterium tuberculosis; Sigma factors; SigG; DNA damage response
K-Ras dependent non-small cell lung cancer (NSCLC) cells are ‘addicted’ to basal autophagy that reprograms cellular metabolism in a lysosomal-sensitive manner. Here we demonstrate that the xenophagy-associated kinase TBK1 drives basal autophagy, consistent with its known requirement in K-Ras-dependent NSCLC proliferation. Furthermore, basal autophagy in this context is characterised by sequestration of the xenophagy cargo receptor Ndp52 and its paralogue Tax1bp1, which we demonstrate here to be a bona fide cargo receptor. Autophagy of these cargo receptors promotes non-canonical NF-κB signalling. We propose that this TBK1-dependent mechanism for NF-κB signalling contributes to autophagy addiction in K-Ras driven NSCLC.
Emerging evidence points to reactive glia as a pivotal factor in Parkinson’s disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of basal ganglia injury, but whether astrocytes and microglia activation may exacerbate dopaminergic (DAergic) neuron demise and/or contribute to DAergic repair is presently the subject of much debate. Here, we have correlated the loss and recovery of the nigrostriatal DAergic functionality upon acute MPTP exposure with extensive gene expression analysis at the level of the ventral midbrain (VM) and striata (Str) and found a major upregulation of pro-inflammatory chemokines and wingless-type MMTV integration site1 (Wnt1), a key transcript involved in midbrain DAergic neurodevelopment. Wnt signaling components (including Frizzled-1 [Fzd-1] and β-catenin) were dynamically regulated during MPTP-induced DAergic degeneration and reactive glial activation. Activated astrocytes of the ventral midbrain were identified as candidate source of Wnt1 by in situ hybridization and real-time PCR in vitro. Blocking Wnt/Fzd signaling with Dickkopf-1 (Dkk1) counteracted astrocyte-induced neuroprotection against MPP+ toxicity in primary mesencephalic astrocyte–neuron cultures, in vitro. Moreover, astroglial-derived factors, including Wnt1, promoted neurogenesis and DAergic neurogenesis from adult midbrain stem/neuroprogenitor cells, in vitro. Conversely, lack of Wnt1 transcription in response to MPTP in middleaged mice and failure of DAergic neurons to recover were reversed by pharmacological activation of Wnt/β-catenin signaling, in vivo, thus suggesting MPTP-reactive astrocytes in situ and Wnt1 as candidate components of neuroprotective/neurorescue pathways in MPTP-induced nigrostriatal DAergic plasticity.
Astroglia; Neurodegeneration; Neuroinflammation; Neuroprotection; Parkinson disease
Disorders of retinal vascular growth and function are responsible for vision loss in a variety of diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and retinal artery or vein occlusion. Over the past decade, a new signaling pathway that controls retinal vascular development has emerged from the study of inherited disorders - in both humans and mice - that are characterized by retinal hypovascularization. This pathway utilizes a glial-derived extracellular ligand, Norrin, that acts on a transmembrane receptor, Frizzled4, a coreceptor, Lrp5, and an auxiliary membrane protein, Tspan12, on the surface of developing endothelial cells. The resulting signal controls a transcriptional program that regulates endothelial growth and maturation. It will be of great interest to determine whether modulating this pathway could represent a therapeutic approach to human retinal vascular disease.
The ability to distinguish nonself from self is a fundamental characteristic of biological systems. In the filamentous fungus Neurospora crassa, multiple incompatibility genes mediate nonself recognition during vegetative growth. One of these genes, un-24, encodes both nonself recognition function and the large subunit of a type I ribonucleotide reductase, an evolutionarily conserved enzyme that is essential for the conversion of NDP precursors into dNDPs for use in DNA synthesis. Previous studies have shown that co-expression of the two allelic forms of un-24, Oakridge (OR) and Panama (PA), in the same cell results in cell death.
We identify a 135 amino acid nonself recognition domain in the C-terminus region of UN-24 that confers an incompatibility-like phenotype when expressed in the yeast, Saccharomyces cerevisiae. Low-level expression of this domain results in several cytological and phenotypic characteristics consistent with an incompatibility reaction in filamentous fungi. These incompatibility phenotypes are correlated with the presence of a non-reducible complex consisting of the PA incompatibility domain and Rnr1p, a large subunit of ribonucleotide reductase in yeast. When the PA incompatibility domain is switched to high-level expression, the incompatibility phenotype transitions to wild-type concomitant with the appearance of a complex containing the PA incompatibility domain and Ssa1p, an Hsp70 homolog.
Results from this study provide insights into the mechanism and control of vegetative nonself recognition mediated by ribonucleotide reductase in N. crassa, thus establishing the yeast system as a powerful tool to study fungal nonself recognition. Our work shows that heat shock proteins may function to deactivate vegetative incompatibility systems, as required for entry into the sexual cycle. Finally, our results suggest that variations on the PA incompatibility domain may serve as novel and specific antimicrobial peptides.
Nonself recognition; Heterokaryon incompatibility; Ribonucleotide reductase; Heat shock protein; Sexual incompatibility.