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At least 523 records · Page 29Linked to original sources
PubMed2016

Matrix stiffness promotes cartilage endplate chondrocyte calcification in disc degeneration via miR-20a targeting ANKH expression.

The mechanical environment is crucial for intervertebral disc degeneration (IDD). However, the mechanisms underlying the regulation of cartilage endplate (CEP) calcification by altered matrix stiffness remain unclear. In this study, we found that matrix stiffness of CEP was positively correlated with the degree of IDD, and stiff matrix, which mimicked the severe degeneration of CEP, promoted inorganic phosphate-induced calcification in CEP chondrocytes. Co-expression analysis of the miRNA and mRNA profiles showed that increasing stiffness resulted in up-regulation of miR-20a and down-regulation of decreased ankylosis protein homolog (ANKH) during inorganic phosphate-induced calcification in CEP chondrocytes. Through a dual luciferase reporter assay, we confirmed that miR-20a directly targets 3'-untranslated regions of ANKH. The inhibition of miR-20a attenuated the calcium deposition and calcification-related gene expression, whereas the overexpression of miR-20a enhanced calcification in CEP chondrocytes on stiff matrix. The rescue of ANKH expression restored the decreased pyrophosphate efflux and inhibited calcification. In clinical samples, the levels of ANKH expression were inversely associated with the degeneration degree of CEP. Thus, our findings demonstrate that the miR-20a/ANKH axis mediates the stiff matrix- promoted CEP calcification, suggesting that miR-20a and ANKH are potential targets in restraining the progression of IDD.

3' Untranslated Regions↗
PubMed2005

Breast cancer metastasis suppressor 1 inhibits gene expression by targeting nuclear factor-kappaB activity.

Breast cancer metastasis suppressor 1 (BRMS1) functions as a metastasis suppressor gene in breast cancer and melanoma cell lines, but the mechanism of BRMS1 suppression remains unclear. We determined that BRMS1 expression was inversely correlated with that of urokinase-type plasminogen activator (uPA), a prometastatic gene that is regulated at least in part by nuclear factor-kappaB (NF-kappaB). To further investigate the role of NF-kappaB in BRMS1-regulated gene expression, we examined NF-kappaB binding activity and found an inverse correlation between BRMS1 expression and NF-kappaB binding activity in MDA-MB-231 breast cancer and C8161.9 melanoma cells stably expressing BRMS1. In contrast, BRMS1 expression had no effect on activation of the activator protein-1 transcription factor. Further, we showed that suppression of both constitutive and tumor necrosis factor-alpha-induced NF-kappaB activation by BRMS1 may be due to inhibition of IkappaBalpha phosphorylation and degradation. To examine the relationship between BRMS1 and uPA expression in primary breast tumors, we screened a breast cancer dot blot array of normalized cDNA from 50 breast tumors and corresponding normal breast tissues. There was a significant reduction in BRMS1 mRNA expression in breast tumors compared with matched normal breast tissues (paired t test, P < 0.0001) and a general inverse correlation with uPA gene expression (P < 0.01). These results suggest that at least one of the underlying mechanisms of BRMS1-dependent suppression of tumor metastasis includes inhibition of NF-kappaB activity and subsequent suppression of uPA expression in breast cancer and melanoma cells.

Animals↗
PubMed2004

A chimeric adenovirus vector encoding reovirus attachment protein sigma1 targets cells expressing junctional adhesion molecule 1.

The utility of adenovirus (Ad) vectors for gene transduction can be limited by receptor specificity. We developed a gene-delivery vehicle in which the potent Ad5 vector was genetically reengineered to display the mucosal-targeting sigma1 protein of reovirus type 3 Dearing (T3D). A sigma1 construct containing all but a small virion-anchoring domain was fused to the N-terminal 44 aa of Ad5 fiber. This chimeric attachment protein Fibtail-T3Dsigma1 forms trimers and assembles onto Ad virions. Fibtail-T3Dsigma1 was recombined into the Ad5 genome, replacing sequences encoding wild-type fiber. The resulting vector, Ad5-T3Dsigma1, expresses Fibtail-T3Dsigma1 and infects Chinese hamster ovary cells transfected with human or mouse homologs of the reovirus receptor, junctional adhesion molecule 1 (JAM1), but not the coxsackievirus and Ad receptor. Treatment of Caco-2 intestinal epithelial cells with either JAM1-specific antibody or neuraminidase reduced transduction by Ad5-T3Dsigma1, and their combined effect decreased transduction by 95%. Ad5-T3Dsigma1 transduces primary cultures of human dendritic cells substantially more efficiently than does Ad5, and this transduction depends on expression of JAM1. These data provide strong evidence that Ad5-T3Dsigma1 can be redirected to cells expressing JAM1 and sialic acid for application as a vaccine vector.

Adenoviridae↗
PubMed2004

[Soluble expression and target study to brain of anti-TfR ScFv].

The single-chain antibody gene (ox26-scFv) to transferrin receptor (TfR) was synthesized and amplified by three-step PCR. After sequencing, the gene was cloned into prokaryotic expression vector pTIG-Trx which carried thioredoxin (Trx) gene and a C-terminal His.tag. The Ox26-scFv proteins achieved 31% yields of total bacteria proteins at 20 degrees C, after 0.02mM IPTG induction using the strain E. coli BL21 (DE3). The soluble scFv proteins in cytoplasm suspension were about 35% and the inclusion bodies were about 65%. The soluble products were purified by immobilized metal chelation affinity chromatography (Ni-NTA), a single band with molecular weight 29 kD appeared on SDS-PAGE gel. Rat GH3 cell immunocytochemistry staining showed that Ox26-scFv protein could recognize and bind to transferrin receptor. Injected SD rats with Ox26-scFv proteins by tail veins, the antibodies were detected from brain tissues specially on the brain capillaries 4 h later which indicate that Ox26-scFv proteins have a good target function to brain capillaries and can permeate the blood-brain barrier mediated by the transferrin receptors.

Animals↗
PubMed2000

Suppression of gene expression by targeted disruption of messenger RNA: available options and current strategies.

At least three different approaches may be used for gene targeting including: A) gene knockout by homologous recombination; B) employment of synthetic oligonucleotides capable of hybridizing with DNA or RNA, and C) use of polyamides and other natural DNA-bonding molecules called lexitropsins. Targeting mRNA is attractive because mRNA is more accessible than the corresponding gene. Three basic strategies have emerged for this purpose, the most familiar being to introduce antisense nucleic acids into a cell in the hopes that they will form Watson-Crick base pairs with the targeted gene's mRNA. Duplexed mRNA cannot be translated, and almost certainly initiates processes which lead to its destruction. The antisense nucleic acid can take the form of RNA expressed from a vector which has been transfected into the cell, or take the form of a DNA or RNA oligonucleotide which can be introduced into cells through a variety of means. DNA and RNA oligonucleotides can be modified for stability as well as engineered to contain inherent cleaving activity. It has also been proven that because RNA and DNA are very similar chemical compounds, DNA molecules with enzymatic activity could also be developed. This assumption proved correct and led to the development of a "general-purpose" RNA-cleaving DNA enzyme. The attraction of DNAzymes over ribozymes is that they are very inexpensive to make and that because they are composed of DNA and not RNA, they are inherently more stable than ribozymes. Although mRNA targeting is impeccable in theory, many additional considerations must be taken into account in applying these strategies in living cells including mRNA site selection, drug delivery and intracellular localization of the antisense agent. Nevertheless, the ongoing revolution in cell and molecular biology, combined with advances in the emerging disciplines of genomics and informatics, has made the concept of nontoxic, cancer-specific therapies more viable then ever and continues to drive interest in this field.

Animals↗
PubMed2003

Target cell expression of suppressor of cytokine signaling-1 prevents diabetes in the NOD mouse.

Although lymphocyte infiltration and islet destruction are hallmarks of diabetes, the mechanisms of beta-cell destruction are not fully understood. One issue that remains unresolved is whether cytokines play a direct role in beta-cell death. We investigated whether beta-cell cytokine signaling contributes to autoimmune type 1 diabetes. We demonstrated that NOD mice harboring beta-cells expressing the suppressor of cytokine signaling-1 (SOCS-1), an inhibitor of Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, have a markedly reduced incidence of diabetes. Similar to their non-transgenic (Tg) littermates, SOCS-1-Tg mice develop insulitis and their splenocytes transfer disease to NODscid recipients. Disease protection correlates with suppression of cytokine-induced STAT-1 phosphorylation in SOCS-1-expressing beta-cells and with a reduced sensitivity of these cells to destruction by diabetogenic cells in vivo. Interestingly, lymphocytes recruited to the pancreas of SOCS-1-Tg mice transferred diabetes to NODscid recipients with a reduced efficiency, suggesting that the pancreatic environment in SOCS-1-Tg mice does not support the maintenance of functionally differentiated T-cells. These results suggest that cytokines contribute to the development of type 1 diabetes by acting directly on the target beta-cell. Importantly, given that the SOCS-1-expressing mouse maintain normal blood glucose levels throughout life, this study also showed that SOCS-1 expression by beta-cells can represent a promising strategy to prevent type 1 diabetes.

Animals↗
PubMedDate not supplied

Ets-2 interacts with co-repressor BS69 to repress target gene expression.

BACKGROUND: The ETS-family of proteins consists of over 30 members that regulate the growth, differentiation and survival of both normal and tumor cells. How specificity is achieved within this family remains largely unresolved. One mechanism for attaining specificity is through the action of signaling pathways on specific family members. For example, Ets-2 is an activator modulated by ras-dependent phosphorylation of a single residue in the conserved pointed domain of this factor. We hypothesized that phosphorylation of the pointed domain regulates the proteins that can interact with ets-2 in the cell nucleus, resulting in regulation of target genes. MATERIALS AND METHODS: We used a combination of biochemical assays, yeast two-hybrid screens and transfection assays to identify and characterize proteins interacting with the pointed domain. RESULTS: BS69, a known co-repressor, was identified in a yeast two hybrid screen as an ets-2 interacting partner. BS69 can interact with ets-2 in vivo and phosphorylation of the ets-2 pointed domain decreased the interaction with BS69 in vitro. In transfection assays, co-expression of ets-2 and BS69 resulted in repression of defined ets-2 target genes. CONCLUSION: These results support a role for ets-2 as a repressor and indicate that BS69 is required as co-repressor. Phosphorylation of ets-2 may switch its activity from repressor to activator by interfering with formation of the BS69 complex.

3T3 Cells↗
PubMed1997

Cross-clade human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte responses in HIV-infected Zambians.

We have examined cross-clade HIV-specific cytotoxic T-lymphocyte (CTL) activity in peripheral blood of eight Zambian individuals infected with non-B-clade human immunodeficiency virus type 1 (HIV-1). Heteroduplex mobility assay and partial sequence analysis of env and gag genes strongly suggests that all the HIV-infected subjects were infected with clade C HIV-1. Six of eight C-clade HIV-infected individuals elicited CTL activity specific for recombinant vaccinia virus-infected autologous targets expressing HIV gag-pol-env derived from B-clade HIV-1 (IIIB). Recognition of individual recombinant HIV-1 B-clade vaccinia virus-infected targets expressing gag, pol, or env was variable among the patients tested, indicating that cross-clade CTL activity is not limited to a single HIV protein. These data demonstrate that HIV clade C-infected individuals can mount vigorous HIV clade B-reactive CTL responses.

Cross Reactions↗
PubMed1999

Genetic dissection of behavior: modulation of locomotion by light in the Drosophila melanogaster larva requires genetically distinct visual system functions.

The Drosophila larva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results were obtained when these responses were measured in strains where the larval photoreceptor neurons were ablated by mutations in the glass (gl) gene or by the targeted expression of the cell death gene head involution defective (hid). A mutation in the homeobox gene sine oculis (so) that ablates the larval visual system, or the targeted expression of the reaper (rpr) cell death gene, abolishes all responses to light detected as a change of direction. We propose the existence of an extraocular light perception that does not use the same phototransduction cascade as the adult photoreceptors. Our results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by the so gene.

Animals↗
PubMed2005

Effects of a mosquitocidal toxin on a mammalian epithelial cell line expressing its target receptor.

The spread of diseases transmitted by Anopheles and Culex mosquitoes, such as malaria and West Nile fever, is a growing concern for human health. Bacillus sphaericus binary toxin (Bin) is one of the few available bioinsecticides able to control populations of these mosquitoes efficiently. We previously showed that Bin binds to Cpm1, an alpha-glucosidase located on the apical side of Culex larval midgut epithelium. We analysed the effects of Bin by expressing a construct encoding Cpm1 in the mammalian epithelial MDCK cell line. Cpm1 is targeted to the apical side of polarized MDCK, where it is anchored by glycosylphosphatidylinositol (GPI) and displays alpha-glucosidase activity. Bin bound to transfected cells and induced a non-specific current presumably related to the opening of pores. The formation of these pores may be related to the location of the toxin/receptor complex in lipid raft microdomains. Finally, Bin promoted the time-dependent appearance of intracytoplasmic vacuoles but did not drive cell lysis. Thus, the dual functionality (enzyme/toxin receptor) of Cpm1 is fully conserved in MDCK cells and Cpm1 is an essential target protein for Bin cytotoxicity in Culex mosquitoes.

Animals↗
PubMed1996

Emotion-specific processing deficit in focal brain-damaged patients.

Focal brain-damaged patients (left hemisphere damage, right hemisphere damage) and hospitalized general medical patients were asked to sort test photographs into target expressions of four facial emotions, happy, sad, fear and anger. In a second task, patients were asked to match neutral photographs with these target emotion expressions in a forced-choice format. Patients were also asked to rate their mood state on a two-dimensional affect grid. Right hemisphere-damaged patients were significantly inaccurate in matching the test-target expressions of facial emotions in comparison to left hemisphere-damaged or general medical patients. Analysis of error scores indicated a bias toward negative emotions by left hemisphere-damaged patients. Congruent to their mood state, left hemisphere-damaged patients also attributed "sadness" on neutral state of expression significantly more often than in right hemisphere-damaged patients.

Adult↗
PubMed2000

Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia.

In preparation for being shed into the environment as infectious cysts, trophozoites of Giardia spp. synthesize and deposit large amounts of extracellular matrix into a resistant extracellular cyst wall. Functional aspects of this developmentally regulated process were investigated by expressing a series of chimeric cyst wall protein 1 (CWP1)-green fluorescent protein (GFP) reporter proteins. It was demonstrated that a short 110 bp 5' flanking region of the CWP1 gene harbors all necessary cis-DNA elements for strictly encystation-specific expression of a reporter during in vitro encystation, whereas sequences in the 3' flanking region are involved in modulation of steady-state levels of its mRNA during encystation. Encysting Giardia expressing CWP1-GFP chimeras showed formation and maturation of labeled dense granule-like vesicles and subsequent incorporation of GFP-tagged protein into the cyst wall, dependent on which domains of CWP1 were included. The N-terminal domain of CWP1 was required for targeting GFP to regulated compartments of the secretory apparatus, whereas a central domain containing leucine-rich repeats mediated association of the chimera with the extracellular cyst wall. We show that analysis of protein transport using GFP-tagged molecules is feasible in an anaerobic organism and provides a useful tool for investigating the organization of primitive eukaryotic vesicular transport.

3' Untranslated Regions↗
PubMed2005

Presynaptic D1 dopamine receptors in primate prefrontal cortex: target-specific expression in the glutamatergic synapse.

Dopaminergic modulation of glutamate neurotransmission in prefrontal cortex (PFC) microcircuits is commonly perceived as a basis for cognitive operations. Yet it appears that although the control of recurrent excitation between deep-layer prefrontal pyramids may involve presynaptic and postsynaptic D1 receptor (D1R) mechanisms, pyramid-to-interneuron communication will engage a postsynaptic D1R component. The substrate underlying such target-specific neuromodulatory patterns was investigated in the infragranular PFC with immunoelectron microscopy for D1R and parvalbumin, a marker for fast-spiking interneurons. In addition to their proverbial postsynaptic expression, gold-labeled D1Rs were distinctly distributed on perisynaptic/extrasynaptic membranes and the axoplasm of 13% of excitatory-like, presumably glutamatergic varicosities. Most importantly, presynaptic D1Rs were highly specific with regard to the cellular compartment and neurochemical identity of the postsynaptic neuron, being present in spine-targeting varicosities but distinctly absent from those synapsing with parvalbumin profiles often coexpressing D1Rs. We define therein an axonal D1 heteroreceptor component, apparently mediating volume neurotransmission, yet strategically positioned to convey target cell-specific modulation of the glutamatergic drive. We also indicate that presynaptic D1R mechanisms may indeed be associated with recurrent excitation in prefrontal microcircuits, consistent with physiological evidence for a role of these receptors in modulating the persistent activity-profile of neurons essential for working memory.

Animals↗
PubMed2004

Inhibition of cyclooxygenase-1 and -2 expression by targeting the endothelin a receptor in human ovarian carcinoma cells.

PURPOSE AND EXPERIMENTAL DESIGN: New therapies against cancer are based on targeting cyclooxygenase (COX)-2. Activation of the endothelin A receptor (ET(A)R) by endothelin (ET)-1 is biologically relevant in several malignancies, including ovarian carcinoma. In this tumor, the ET-1/ET(A)R autocrine pathway promotes mitogenesis, apoptosis protection, invasion, and neoangiogenesis. Because COX-1 and COX-2 are involved in ovarian carcinoma progression, we investigated whether ET-1 induced COX-1 and COX-2 expression through the ET(A)R at the mRNA and protein level in HEY and OVCA 433 ovarian carcinoma cell lines by Northern blot, reverse transcription-PCR, Western blot, and immunohistochemistry; we also investigated the activity of the COX-2 promoter by luciferase assay and the release of prostaglandin (PG) E(2) by ELISA. RESULTS: ET-1 significantly increases the expression of COX-1 and COX-2, COX-2 promoter activity, and PGE(2) production. These effects depend on ET(A)R activation and involve multiple mitogen-activated protein kinase (MAPK) signaling pathways, including p42/44 MAPK, p38 MAPK, and transactivation of the epidermal growth factor receptor. COX-2 inhibitors and, in part, COX-1 inhibitor blocked ET-1-induced PGE(2) and vascular endothelial growth factor release, indicating that both enzymes participate in PGE(2) production to a different extent. Moreover, inhibition of human ovarian tumor growth in nude mice after treatment with the potent ET(A)R-selective antagonist ABT-627 is associated with reduced COX-2 and vascular endothelial growth factor expression. CONCLUSIONS: These results indicate that impairing COX-1 and COX-2 and their downstream effect by targeting ET(A)R can be therapeutically advantageous in ovarian carcinoma treatment. Pharmacological blockade of the ET(A)R is an attractive strategy to control COX-2 induction, which has been associated with ovarian carcinoma progression and chemoresistance.

Animals↗
PubMed2006

Recombinant Escherichia coli expressing invasin targets the Peyer's patches: the basis for a bacterial formulation for oral vaccination.

We have investigated the tropism of nonpathogenic recombinant invasive Escherichia coli in the gastrointestinal tract and the efficacy of this invasive E. coli as an oral vaccine for cancer immunotherapy. E. coli expressing invasin from Yersinia pseudotuberculosis selectively invade nonphagocytic cells in which beta(1)-integrin is expressed and accessible. Following internalization the E. coli are degraded in the phagosome. Coexpression of listeriolysin O (LLO) mediates release of the content of the bacteria into the cytosol of the invaded cell. In vitro and in vivo experiments demonstrated that gut epithelial cells failed to be invaded by invasive E. coli, due to a basolateral localization of beta(1)-integrin. By contrast, selective uptake of invasive bacteria from the intestinal lumen into Peyer's patches was observed ex vivo. Once in this structure, invasive E. coli colocalized with dendritic cells and possibly B cells. Oral administration of invasive E. coli coexpressing the model antigen ovalbumin and LLO from Listeria monocytogenes was able to elicit systemic protection against a lethal challenge of B16 tumor cells expressing ovalbumin. These data demonstrate the selectivity of invasin-mediated invasion to the Peyer's patches and indicate the potential of nonpathogenic, invasive E. coli as an oral vaccine with applications in immunotherapy.

Adhesins, Bacterial↗
PubMed2006

Regulation of p53 tumour suppressor target gene expression by the p52 NF-kappaB subunit.

The p52/p100 nuclear factor kappa B (NF-kappaB) subunit (NF-kappaB2) is aberrantly expressed in many tumour types and has been implicated as a regulator of cell proliferation. Here, we demonstrate that endogenous p52 is a direct regulator of Cyclin D1 expression. However, stimulation of Cyclin D1 expression alone cannot account for all the cell cycle effects of p52/p100 and we also find that p52 represses expression of the Cyclin-dependent kinase inhibitor p21(WAF/CIP1). Significantly, this latter effect is dependent upon basal levels of the tumour suppressor p53. By contrast, p52 cooperates with p53 to regulate other known p53 target genes such as PUMA, DR5, Gadd45alpha and Chk1. p52 associates directly with these p53-regulated promoters where it regulates coactivator and corepressor binding. Moreover, recruitment of p52 is p53 dependent and does not require p52-DNA-binding activity. These results reveal a complex role for p52 as regulator of cell proliferation and p53 transcriptional activity. Furthermore, they imply that in some cell types, p52 can regulate p53 function and influence p53-regulated decision-making following DNA damage and oncogene activation.

Apoptosis Regulatory Proteins↗