Tumour biology. Weakening link to colorectal cancer?
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Animals↗
Biomedical subjects
B Lu
Publications and source records attributed to B Lu.
Ca(2+) binding protein frequenin mediates GDNF-induced potentiation of Ca(2+) channels and transmitter release.
Molecular mechanisms underlying long-term neurotrophic regulation of synaptic transmission and plasticity are unknown. We report here that long-term treatment of neuromuscular synapses with glial cell line-derived neurotrophic factor (GDNF) potentiates spontaneous and evoked transmitter release, in ways very similar to presynaptic expression of the Ca(2+) binding protein frequenin. GDNF enhances the expression of frequenin in motoneurons, and inhibition of frequenin expression or activity prevents the synaptic action of GDNF. GDNF also facilitates Ca(2+) influx into the nerve terminals during evoked transmission by enhancing Ca(2+) currents. The effect of GDNF on Ca(2+) currents is blocked by inhibition of frequenin expression, occluded by overexpression of frequenin, and is selective to N-type Ca(2+) channels. These results identify an important molecular target that mediates the long-term, synaptic action of a neurotrophic factor.
Animals↗
Protein synthesis-dependent and -independent regulation of hippocampal synapses by brain-derived neurotrophic factor.
A fundamental difference between short-term and long-term forms of synaptic plasticity is the dependence on transcription and translation of new genes. Using organotypic cultures of hippocampal slices, we have investigated whether the modulation of synapses by brain-derived neurotrophic factor (BDNF) also requires protein synthesis. Long-term treatment of hippocampal slice cultures with BDNF increased the number of docked vesicles, but not that of reserve pool vesicles, at CA1 excitatory synapses. BDNF also increased the levels of the vesicle proteins synaptophysin, synaptobrevin, and synaptotagmin, without affecting the presynaptic membrane proteins syntaxin and SNAP-25, or the vesicle-binding protein synapsin-I. The increase in synaptophysin and synaptobrevin expression was moderate (2-fold) and occurred within 6 h after BDNF application. In contrast, synaptotagmin expression took 24 h to reach maximum levels (5-fold). The delayed increase in synaptotagmin was blocked by protein synthesis inhibitors, while the early increase in synaptophysin and synaptobrevin was not. Moreover, the BDNF-induced increase of synaptotagmin was blocked by inhibiting the cAMP/protein kinase A (PKA) pathway. However, BDNF did not activate PKA, and application of a PKA activator did not mimic the BDNF effect. Taken together, these results suggest a novel, protein synthesis-dependent form of BDNF modulation that requires cAMP gating.
Animals↗
Enhanced sensitivity of insulin-resistant adipocytes to vanadate is associated with oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4).
Vanadate (sodium orthovanadate), an inhibitor of phosphotyrosine phosphatases (PTPs), mimics many of the metabolic actions of insulin in vitro and in vivo. The potential of vanadate to stimulate glucose transport independent of the early steps in insulin signaling prompted us to test its effectiveness in an in vitro model of insulin resistance. In primary rat adipocytes cultured for 18 h in the presence of high glucose (15 mm) and insulin (10(-7) m), sensitivity to insulin-stimulated glucose transport was decreased. In contrast, there was a paradoxical enhanced sensitivity to vanadate of the insulin-resistant cells (EC(50) for control, 325 +/- 7.5 microm; EC(50) for insulin-resistant, 171 +/- 32 microm; p < 0.002). Enhanced sensitivity was also present for vanadate stimulation of insulin receptor kinase activity and autophosphorylation and Akt/protein kinase B Ser-473 phosphorylation consistent with more effective PTP inhibition in the resistant cells. Investigation of this phenomenon revealed that 1) depletion of GSH with buthionine sulfoximine reproduced the enhanced sensitivity to vanadate while preincubation of resistant cells with N-acetylcysteine (NAC) prevented it, 2) intracellular GSH was decreased in resistant cells and normalized by NAC, 3) exposure to high glucose and insulin induced an increase in reactive oxygen species, which was prevented by NAC, 4) EPR (electron paramagnetic resonance) spectroscopy showed a decreased amount of vanadyl (+4) in resistant and buthionine sulfoximine-treated cells, which correlated with decreased GSH and increased vanadate sensitivity, while total vanadium uptake was not altered, and 5) inhibition of recombinant PTP1B in vitro was more sensitive to vanadate (+5) than vanadyl (+4). In conclusion, the paradoxical increased sensitivity to vanadate in hyperglycemia-induced insulin resistant adipocytes is due to oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4). Thus, sensitivity of PTP inhibition and glucose transport to vanadate is regulated by cellular redox state.
Acetylcysteine↗
Metabolic regulation of brain Abeta by neprilysin.
Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite in the brain. We examined the role of neprilysin, a candidate Abeta-degrading peptidase, in the metabolism using neprilysin gene-disrupted mice. Neprilysin deficiency resulted in defects both in the degradation of exogenously administered Abeta and in the metabolic suppression of the endogenous Abeta levels in a gene dose-dependent manner. The regional levels of Abeta in the neprilysin-deficient mouse brain were in the distinct order of hippocampus, cortex, thalamus/striatum, and cerebellum, where hippocampus has the highest level and cerebellum the lowest, correlating with the vulnerability to Abeta deposition in brains of humans with AD. Our observations suggest that even partial down-regulation of neprilysin activity, which could be caused by aging, can contribute to AD development by promoting Abeta accumulation.
Aging↗
Replacement of segmental bone defects using porous bioceramic cylinders: a biomechanical and X-ray diffraction study.
A porous ceramic material [hydroxyapatitetricalcium phosphate (HA-TCP)] was implanted in the femora of 30 dogs to investigate the possibility of using this material to repair segmental bone defects. A bone segment, 1.5 cm in length, was removed from the diaphysis of one femur in each dog to create the defect. Cylinders of corresponding size were inserted into the defects. The animals were divided into three groups with recovery times of 2 months, 4 months, and 6 months, respectively. The implants were harvested and subjected to biomechanic tests (bending strength) and X-ray diffraction analysis. The bending strengths of the implant construct increased gradually over time postoperatively. The values of strength for the three different time groups had significant variations (p < 0.05). The X-ray diffraction analysis indicated that the peaks of the TCP included in the cylinders decreased in intensity after implantation and tended to be similar to those of natural bone by 6 months after operation. Conversely, the peaks for the HA had fewer changes compared with preimplantation values. Based on the results of this experiment it was concluded that the porous HA-TCP ceramic cylinders have potential for repair of segmental bone defects if assisted by adequate stabilizing fixtures during the early postoperative period.
Animals↗
Adherens junctions inhibit asymmetric division in the Drosophila epithelium.
Asymmetric division is a fundamental mechanism for generating cellular diversity. In the central nervous system of Drosophila, neural progenitor cells called neuroblasts undergo asymmetric division along the apical-basal cellular axis. Neuroblasts originate from neuroepithelial cells, which are polarized along the apical-basal axis and divide symmetrically along the planar axis. The asymmetry of neuroblasts might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. Here we show that by disrupting adherens junctions we can convert the symmetric epithelial division into asymmetric division. We further confirm that the adenomatous polyposis coli (APC) tumour suppressor protein is recruited to adherens junctions, and demonstrate that both APC and microtubule-associated EB1 homologues are required for the symmetric epithelial division along the planar axis. Our results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions.
Adenomatous Polyposis Coli Protein↗
Drosophila par-1 is required for oocyte differentiation and microtubule organization.
BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.
Animals↗
Neutral endopeptidase terminates substance P-induced inflammation in allergic contact dermatitis.
Sensory nerve-derived neuropeptides such as substance P demonstrate a number of proinflammatory bioactivities, but less is known about their role in inflammatory skin disease. The cell surface metalloprotease neutral endopeptidase (NEP) is the principal proteolytic substance P-degrading enzyme. This study tests the hypothesis that the absence of NEP results in dysregulated inflammatory skin responses. The effector phase of allergic contact dermatitis (ACD) responses was examined in NEP(-/-) knockout and NEP(+/+) wild-type mice and compared with the irritant contact dermatitis response in these animals. NEP was found to be normally immunolocalized in epidermal keratinocytes and dermal blood vessels. The ACD ear swelling response was 2.5-fold higher in animals lacking NEP and was accompanied by a significant increase in plasma extravasation and infiltration of inflammatory leukocytes. The augmented ACD response in NEP(-/-) animals was abrogated by either administration of a neurokinin receptor 1 antagonist or by repeated pretreatment with topical capsaicin. Similar to NEP(-/-) mice, the acute inhibition of NEP in NEP(+/+) animals resulted in an augmented ACD response. In contrast to the ACD responses, little differences were observed in the irritant contact dermatitis response of NEP(-/-) compared with NEP(+/+) animals after epicutaneous application of the skin irritants croton oil or SDS. Thus, these results indicate that NEP and cutaneous neuropeptides have a significant role in the pathogenesis of ACD.
Administration, Cutaneous↗
Molecular cloning and functional characterization of the canine prostaglandin E2 receptor EP4 subtype.
Prostaglandin E2 (PGE2) is an important mediator of diverse biologic functions in many tissues and binds with high affinity to four cell surface, seven-transmembrane domain, G protein-coupled receptors (EP1-EP4). The EP4 receptor subtype has a long intracellular carboxy-terminal region and is functionally coupled to adenylate cyclase, resulting in elevated intracellular cyclic adenosine 5' monophosphate (cAMP) levels upon activation. To further study EP4 receptor subtype function, a canine kidney cDNA library was screened and three clones were isolated and sequenced. The longest clone was 3,103 bp and contained a single open reading frame of 1,476 bp, potentially encoding a protein of 492 amino acids with a predicted molecular weight of 53.4 kDa. Sequence analysis of this open reading frame reveals 89% identity to the human EP4 protein coding region at the nucleotide level and 90% identity when the putative canine and human protein sequences are compared. Northern blot analysis showed relatively high levels of canine EP4 expression in heart, lung and kidney, while Southern blot analysis of canine genomic DNA suggests the presence of a single copy gene. Following transfection of canine EP4 into CHO-KI cells, Scatchard analysis revealed a dissociation constant of 24 nM for PGE, while competition binding studies using 3H-PGE2 as ligand demonstrated specific displacement by PGE2 prostaglandin E, (PGE1), and prostaglandin A3 (PGA3). Treatment with PGE2 also resulted in increased levels of cAMP in transfected, but not in parental, CHO-KI cells. In contrast, butaprost, an EP2 selective ligand, and sulprostone, an EP1/EP3 selective ligand, did not bind to this receptor at the maximal concentration used (320 nM). To further investigate secondary signaling, the canine EP4 cDNA was truncated to produce an 1,117 bp fragment encoding a 356 amino acid protein lacking the intracellular carboxy-terminus. When transfected, this truncated cDNA produced a protein with a dissociation constant of 11 nM for PGE2 and a binding and cAMP accumulation profile similar to that of the full-length protein. Both full-length and truncated canine EP4 underwent short term PGE2-induced desensitization as shown by a lack of continuing cAMP accumulation after the initial PGE2 stimulation, suggesting no involvement of the C-terminal intracellular tail. This result is in contrast to that reported for the human EP4 receptor, where residues within the C-terminal intracellular tail were shown to mediate short term, ligand induced desensitization.
Amino Acid Sequence↗
Screening patients with chest pain in the emergency department using electron beam tomography: a follow-up study.
OBJECTIVES: The high sensitivity of electron beam tomography (EBT) in the detection of coronary artery calcium (CAC) and obstructive coronary artery disease prompted us to investigate the association between CAC detection and future cardiac events in patients with acute chest pain syndromes requiring hospitalization. BACKGROUND: Three studies have documented that EBT is a rapid and efficient screening tool for patients admitted to the emergency department (ED) with chest pain, but there is a paucity of long-term follow-up data on these chest pain patients. METHODS: We conducted a prospective observational study of 192 patients admitted to the ED of a large tertiary care hospital for chest pain syndromes. Upon admission, patients underwent EBT scanning in addition to the usual care for chest pain syndromes. During the 17-month enrollment period, 221 patients were scanned (54% men with a mean age of 53 +/- 9 years). Average follow-up was 50 +/- 10 months using chart review. RESULTS: Fifty-eight patients had coronary events confirmed by a blinded medical record review. The presence of CAC (a total calcium score >0) and increasing score quartiles were strongly related to the occurrence of hard cardiac events including myocardial infarction and death (p < 0.001) and all cardiovascular events (p < 0.001). Stratification by age- and gender-matching further increased the prognostic ability of EBT (for scores above vs. below the age- and gender-matched CAC scores; odds ratio: 13.1, 95% confidence intervals: 5.62, 35.9). CONCLUSIONS: These data support previous reports demonstrating that the presence of CAC in a symptomatic cohort is a strong predictor of future cardiac events. This study supports the use of EBT in a symptomatic cohort with prompt discharge of those patients with negative scans. Furthermore, the absence of CAC is associated with a very low risk of future cardiac risk events in this population over the subsequent seven years (annual event rate <1%).
Adult↗
GADD45gamma mediates the activation of the p38 and JNK MAP kinase pathways and cytokine production in effector TH1 cells.
The p38 and JNK stress-activated MAPK signal transduction pathways are activated by T cell receptor (TCR) signaling and are required for IFN-gamma production by TH1 effector cells. Here, we show that the expression of GADD45gamma is induced during T cell activation and that the level of expression is higher in TH1 cells than in TH2 cells. TH1 cells from GADD45gamma(-/-) mice are severely compromised in their abilities to activate p38 and JNK in response to TCR signaling, produce much less IFN-gamma upon restimulation, and are deficient in activation-induced cell death (AICD). Additionally, GADD45gamma deficiencies caused reduced contact hypersensitivity in mice. Thus, GADD45gamma mediates activation of the p38 and JNK pathways and effector function of TH1 cells.
Animals↗
Medial axis reformation: a new visualization method for CT angiography.
RATIONALE AND OBJECTIVES: The authors performed this study to evaluate a new method (medial axis reformation [MAR]) for visualizing three-dimensional vascular data at electron-beam computed tomographic (CT) angiography. MATERIALS AND METHODS: MAR was performed automatically with a personal computer-based workstation. After the region of interest was edited, voxels were divided into groups according to their path lengths. Centroids of groups were connected to form the medial axis. Then, the medial axis was refined with multiscale medial response. Bifurcations were also detected and refined. Finally, curved sections were generated through the branches and laid out onto a single image by using a splitting method. The authors performed MAR during electron-beam CT angiography of coronary arteries, common carotid arteries, and iliac arteries. RESULTS: MAR displayed curved sections of branched vessels on one image, cut through the axis of vessels to show the vessel diameter objectively, and allowed the viewing direction to be altered arbitrarily. CONCLUSION: Results of preliminary applications demonstrate that MAR is a valuable new visualization method for CT angiography.
Adult↗
Effects of window and threshold levels on the accuracy of three-dimensional rendering techniques in coronary artery electron-beam CT angiography.
RATIONALE AND OBJECTIVES: The authors performed this study to evaluate the effect of window level and gray-scale threshold on the demonstration of coronary artery lumina at three-dimensional electron-beam computed tomographic (CT) angiography. MATERIALS AND METHODS: Forty-four coronary artery branches in postmortem pigs were evaluated with electron-beam CT angiography, and the findings were compared with those from conventional angiography. Images from electron-beam CT angiography were reconstructed with maximal intensity projection (MIP), multiplanar reformation (MPR), and shaded-surface display (SSD). Four categories of window level and gray-scale threshold were evaluated. RESULTS: Three-dimensional electron-beam CT angiography accurately depicted the luminal diameters of the coronary arteries compared with conventional angiography (r = 0.83-0.90, P < .0001). The length of lumina visualized at electron-beam CT angiography was significantly shorter than that visualized with conventional angiography (P < .001). The use of MPR enabled visualization of longer segments of coronary arteries than did the use of MIP or SSD (P < .05). The higher the window level and gray-scale threshold used, the smaller the coronary luminal diameters measured (P < .05). The most accurate window level and gray-scale threshold (82.6 HU +/- 29.8 and 89.5 HU +/- 29.7, respectively) were found to correspond to the attenuation of the lumina (275.8 HU +/- 58.8). Results of simple linear regression showed a strong correlation between luminal attenuation and window level (r = 0.89, P < .0001) or gray-scale threshold (r = 0.95, P < .0001). CONCLUSION: Electron-beam CT angiography shows promise in the visualization of coronary artery lumina. For accurate display of lumina, a proper window level and gray-scale threshold for three-dimensional rendering techniques should be determined and used on the basis of the attenuation of the target vessel.
Animals↗
PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling.
Wnt signalling regulates beta-catenin-dependent developmental processes through the Dishevelled protein (Dsh). Dsh regulates two distinct pathways, one mediated by beta-catenin and the other by Jun kinase (JNK). We have purified a Dsh-associated kinase from Drosophila that encodes a homologue of Caenorhabditis elegans PAR-1, a known determinant of polarity during asymmetric cell divisions. Treating cells with Wnt increases endogenous PAR-1 activity coincident with Dsh phosphorylation. PAR-1 potentiates Wnt activation of the beta-catenin pathway but blocks the JNK pathway. Suppressing endogenous PAR-1 function inhibits Wnt signalling through beta-catenin in mammalian cells, and Xenopus and Drosophila embryos. PAR-1 seems to be a positive regulator of the beta-catenin pathway and an inhibitor of the JNK pathway. These findings show that PAR-1, a regulator of polarity, is also a modulator of Wnt-beta-catenin signalling, indicating a link between two important developmental pathways.
Adaptor Proteins, Signal Transducing↗
PI-3 kinase and IP3 are both necessary and sufficient to mediate NT3-induced synaptic potentiation.
Signaling mechanisms underlying neurotrophic regulation of synaptic transmission are not fully understood. Here we show that neurotrophin-3 (NT3)-induced potentiation of synaptic transmission at the neuromuscular synapses is blocked by inhibition of phosphoinositide-3 kinase, phospholipase C-gamma or the downstream IP3 receptors of phospholipase C-gamma, but not by inhibition of MAP kinase. However, neither stimulation of Ca2+ release from intracellular stores by photolysis of caged IP3, nor expression of a constitutively active phosphoinositide-3 kinase (PI3K*) in presynaptic motoneurons alone is sufficient to enhance transmission. Photo-uncaging of IP3 in neurons expressing PI3K* elicits a marked synaptic potentiation, mimicking the NT3 effect. These results reveal an involvement of PI3 kinase in transmitter release, and suggest that concomitant activation of PI3 kinase and IP3 receptors is both necessary and sufficient to mediate the NT3-induced synaptic potentiation.
Animals↗
Defective T cell activation and autoimmune disorder in Stra13-deficient mice.
Stra13, a basic helix-loop-helix transcription factor, is up-regulated upon activation of CD4+ T cells. Here we show that Stra13-deficient mice exhibit defects in several phases of CD4+ T cell activation. In vivo, Stra13 deficiency results in ineffective elimination of activated T and B cells, which accumulate progressively, leading to lymphoid organ hyperplasia. Consequently, aging Stra13-/- mice develop autoimmune disease characterized by accumulation of spontaneously activated T and B cells, circulating autoantibodies, infiltration of T and B lymphocytes in several organs and immune complex deposition in glomeruli. Our studies identify Stra13 as a key regulator of lymphocyte activation that is vital for maintenance of self-tolerance and for constraint of autoimmunity.
Aging↗
GDNF acutely modulates excitability and A-type K(+) channels in midbrain dopaminergic neurons.
Glial cell line-derived neurotrophic factor (GDNF) prevents lesion-induced death of midbrain dopaminergic neurons, but its function in normal brain remains uncertain. Here we show that GDNF acutely and reversibly potentiated the excitability of cultured midbrain neurons by inhibiting transient A-type K(+) channels. The effects of GDNF were limited to large, tyrosine hydroxylase (TH)-positive dopaminergic neurons, and were mediated by mitogen associated protein (MAP) kinase. Application of GDNF also elicited a MAP kinase-dependent enhancement of the excitability in dopaminergic neurons in midbrain slice. These results demonstrate an acute regulation of GDNF on ion channels and its underlying signaling mechanism, and reveal an unexpected role of GDNF in normal midbrain dopaminergic neurons.
4-Aminopyridine↗