Low-Background gamma counting at the Kimballton Underground Research Facility

The next generation of low-background physics experiments will require the use of materials with unprecedented radio-purity. A gamma-counting facility at the Kimballton Underground Research Facility (KURF) has been commissioned to perform initial screening of materials for radioactivity primarily from nuclides in the 238U and 232Th decay chains, 40K and cosmic-ray induced isotopes. The facility consists of two commercial low-background high purity germanium (HPGe) detectors. A continuum background reduction better than a factor of 10 was achieved by going underground. This paper describes the facility, detector systems, analysis techniques and selected assay results.Comment: 7 pages, 7 figures. Submitted to NIM

A Novel Function of Noc2 in Agonist-Induced Intracellular Ca2+ Increase during Zymogen-Granule Exocytosis in Pancreatic Acinar Cells

Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca2+-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 µM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca2+]i-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca2+]i increases

Constitutive activation of glycogen synthase kinase-3β correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer

Background: Aberrant regulation of glycogen synthase kinase-3 beta (GSK-3 beta) has been implicated in several human cancers; however, it has not been reported in the gastric cancer tissues to date. The present study was performed to determine the expression status of active form of GSK-3 beta phosphorylated at Tyr(216) (pGSK-3 beta) and its relationship with other tumor-associated proteins in human gastric cancers. Methods: Immunohistochemistry was performed on tissue array slides containing 281 human gastric carcinoma specimens. In addition, gastric cancer cells were cultured and treated with a GSK-3 beta inhibitor lithium chloride (LiCl) for immunoblot analysis. Results: We found that pGSK-3 beta was expressed in 129 (46%) of 281 cases examined, and was higher in the early-stages of pathologic tumor-node-metastasis (P < 0.001). The expression of pGSK-3 beta inversely correlated with lymphatic invasion (P < 0.001) and lymph node metastasis (P < 0.001) and correlated with a longer patient survival (P < 0.001). In addition, pGSK-3 beta expression positively correlated with that of p16, p21, p27, p53, APC, PTEN, MGMT, SMAD4, or KAl1 (P < 0.05), but not with that of cyclin D1. This was confirmed by immunoblot analysis using SNU-668 gastric cancer cells treated with LiCl. Conclusions: GSK-3 beta activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis. Thus, these findings suggest that GSK-3 beta activation is a useful prognostic marker for the early-stage gastric cancer.Hirakawa H, 2009, ONCOL REP, V22, P481, DOI 10.3892/or_00000460Dar AA, 2009, ONCOGENE, V28, P866, DOI 10.1038/onc.2008.434Holmes T, 2008, STEM CELLS, V26, P1288, DOI 10.1634/stemcells.2007-0600Wang Q, 2008, CELL DEATH DIFFER, V15, P908, DOI 10.1038/cdd.2008.2Takahashi-Yanaga F, 2008, CELL SIGNAL, V20, P581, DOI 10.1016/j.cellsig.2007.10.018Pan MH, 2007, J AGR FOOD CHEM, V55, P7777, DOI 10.1021/jf071520hShakoori A, 2007, CANCER SCI, V98, P1388, DOI 10.1111/j.1349-7006.2007.00545.xZheng HC, 2007, ANTICANCER RES, V27, P3561Saegusa M, 2007, J PATHOL, V213, P35, DOI 10.1002/path.2198Ma C, 2007, CANCER RES, V67, P7756, DOI 10.1158/0008-5472.CAN-06-4665Forde JE, 2007, CELL MOL LIFE SCI, V64, P1930, DOI 10.1007/s00018-007-7045-7Li YW, 2007, J BIOL CHEM, V282, P21542, DOI 10.1074/jbc.M701978200Ding QQ, 2007, CANCER RES, V67, P4564, DOI 10.1158/0008-5472.CAN-06-1788Kunnimalaiyaan M, 2007, MOL CANCER THER, V6, P1151, DOI 10.1158/1535-7163.MCT-06-0665Soto-Cerrato V, 2007, MOL CANCER THER, V6, P362, DOI 10.1158/1535-7163.MCT-06-0266Cao Q, 2006, CELL RES, V16, P671, DOI 10.1038/sj.cr.7310078Yang CH, 2006, PRECIS AGRIC, V7, P33, DOI 10.1007/s11119-005-6788-0Crew KD, 2006, WORLD J GASTROENTERO, V12, P354Mai W, 2007, ONCOLOGY-BASEL, V71, P297, DOI 10.1159/000106429Tan J, 2005, CANCER RES, V65, P9012, DOI 10.1158/0008-5472.CAN-05-1226Shakoori A, 2005, BIOCHEM BIOPH RES CO, V334, P1365, DOI 10.1016/j.bbrc.2005.07.041Farago M, 2005, CANCER RES, V65, P5792Ghosh JC, 2005, CLIN CANCER RES, V11, P4580Liao XB, 2003, MOL CANCER THER, V2, P1215Lee HS, 2003, J PATHOL, V200, P39, DOI 10.1002/path.1288Doble BW, 2003, J CELL SCI, V116, P1175, DOI 10.1242/jcs.00384Gotoh J, 2003, CARCINOGENESIS, V24, P435Goto H, 2002, ORAL ONCOL, V38, P549Lee HS, 2001, INT J CANCER, V91, P619D`Amico M, 2000, J BIOL CHEM, V275, P32649, DOI 10.1074/jbc.M000643200Endoh Y, 2000, J PATHOL, V191, P257Wu LY, 1998, J NATL MED ASSOC, V90, P410WOODGETT JR, 1984, BIOCHIM BIOPHYS ACTA, V788, P339

Integrin α5β1 Function Is Regulated by XGIPC/kermit2 Mediated Endocytosis during Xenopus laevis Gastrulation

During Xenopus gastrulation α5β1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the α5 subunit and regulates the activity of α5β1 integrin. The interaction of kermit2 with α5β1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates α5β1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the α5β1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the α5β1 integrin through receptor endocytosis

Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells

IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PSF is required for meningitic E. coli K1 penetration and leukocyte transmigration across the blood-brain barrier (BBB), which are the hallmarks of bacterial meningitis. However, it is unknown how vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB, which are required for bacteria-mediated pathogenicities.IbeA-induced E. coli K1 invasion, polymorphonuclear leukocyte (PMN) transmigration and IKK/NF-κB activation are blocked by Caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB. IKKα/β phosphorylation is blocked by ERK inhibitors. Co-immunoprecipitation analysis shows that vimentin forms a complex with IκB, NF-κB and tubulins in the resting cells. A dissociation of this complex and a simultaneous association of PSF with NF-κB could be induced by IbeA in a time-dependent manner. The head domain of vimentin is required for the complex formation. Two cytoskeletal components, vimentin filaments and microtubules, contribute to the regulation of NF-κB. SiRNA-mediated knockdown studies demonstrate that IKKα/β phosphorylation is completely abolished in HBMECs lacking vimentin and PSF. Phosphorylation of ERK and nuclear translocation of NF-κB are entirely dependent on PSF. These findings suggest that vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB activation. PSF is essential for translocation of NF-κB and ERK to the nucleus.These findings reveal previously unappreciated facets of the IbeA-binding proteins. Cooperative contributions of vimentin and PSF to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB may represent a new paradigm in pathogen-induced signal transduction and lead to the development of novel strategies for the prevention and treatment of bacterial meningitis

Establishment of a Transgenic Zebrafish Line for Superficial Skin Ablation and Functional Validation of Apoptosis Modulators In Vivo

BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)(cy17) (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+) signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+) fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling. CONCLUSION/SIGNIFICANCE: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo