Immunoblot Bands Research Articles

Methods for detection of cardiac glycogen-autophagy.

Glycogen-autophagy ('glycophagy') is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N-terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.

Characterization of Relevant Bovine Dander Allergen Components.

Diagnostic tests in occupational allergic diseases are highly dependent on the quality of available allergen extracts and specific IgE tests. To enhance diagnostic testing in cattle-related occupational rhinitis, asthma, and urticaria, we produced an in- house cow dander extract, assessed its allergen profile and performance in clinical tests, and compared it with commercial bovine dander extracts. One hundred patients with a suspected cattle-related occupational disease underwent skin prick tests (SPTs) with in-house and 1 or 2 commercial bovine dander extracts. Nasal allergen provocation tests were performed on 31 patients with suspected occupational rhinitis. We used Western blot to study the specific IgE-protein reactions from the sera of the patients with positive provocation test results and identified allergens from immunoblot bands using tandem mass spectrometry. The odorant-binding protein Bos d OBP, bovine serum albumin (Bos d 6), and the lipocalin (Bos d 2) were identified as the major allergens. We found a total of 24 bovine dander allergens, of which several were formerly unknown. The sensitivity and specificity of the in-house extract in SPTs were 100% and 94%, respectively, in 87 patients. The SPT results were negative in 20 healthy controls. Nasal allergen provocation tests with in-house extract detected occupational rhinitis with 100% sensitivity in 21 patients. The provocation results remained negative in 5 healthy controls. Three major and several minor allergens in bovine dander caused occupational rhinitis. Diagnosis of bovine allergen-related occupational diseases requires a sufficient concentration and variety of tested allergens.

Aqueous humor TGFβ and fibrillin-1 in Tsk mice reveal clues to POAG pathogenesis

Aqueous humor (AH) and blood levels of transforming growth factor β (TGFβ) are elevated in idiopathic primary open angle glaucoma (POAG) representing a disease biomarker of unclear status and function. Tsk mice display a POAG phenotype and harbor a mutation of fibrillin-1, an important regulator of TGFβ bioavailability. AH TGFβ2 was higher in Tsk than wild-type (WT) mice (by 34%; p = 0.002; ELISA); similarly, AH TGFβ2 was higher in human POAG than controls (2.7-fold; p = 0.00005). As in POAG, TGFβ1 was elevated in Tsk serum (p = 0.01). Fibrillin-1 was detected in AH from POAG subjects and Tsk mice where both had similar levels relative to controls (p = 0.45). 350 kDa immunoblot bands representing WT full-length fibrillin-1 were present in human and mouse AH. A 418 kDa band representing mutant full-length fibrillin-1 was present only in Tsk mice. Lower molecular weight fibrillin-1 antibody-reactive bands were present in similar patterns in humans and mice. Certain bands (130 and 32 kDa) were elevated only in human POAG and Tsk mice (p ≤ 0.04 relative to controls) indicating discrete isoforms relevant to disease. In addition to sharing a phenotype, Tsk mice and human POAG subjects had common TGFβ and fibrillin-1 features in AH and also blood that are pertinent to understanding glaucoma pathogenesis.

Potential Role for the RASD1 Glucocorticoid-Responsive Gene in Corticotroph Tumorigenesis

Introduction: Originally identified due to its dexamethasone inducibility in mouse corticotropinoma AtT20 cells, RASD1 is a receptor-independent activator of G-proteins, via guanine nucleotide exchange factor (GEF) activity. It remains unclear, however, whether, and if so, how RASD1 mediates the effects of glucocorticoids on corticotroph cells. We identified a rare germline RASD1 variant and investigated its functional effects in vitro. Methods: We screened 209 CD patients (94.3% pediatric) studied at the at the National Institutes of Health Clinical Research Center between 1997 and 2018 by germline whole-exome sequencing (WES) only (n=157), germline and tumor WES (n=27), and/or RASD1 droplet digital PCR germline copy number variant (CNV) analysis (n=201). Corticotropinoma DNA was available in 72 patients to screen for USP8 hotspot variants by Sanger sequencing. A RASD1 variant was identified and functionally characterized. Results: We studied 119 female (56.9%) and 90 (43.1%) male CD cases, including 197 pediatric (≤18 years at disease onset) and 12 adult patients. USP8 defects were present in 19.4% (14/72) of cases. No RASD1 CNVs were found. A rare (with a minor allele frequency of 0.0022% in gnomAD v3) heterozygous germline missense RASD1 variant, c.580A>C, p.M194L was detected in one male sporadic case. Neither USP8 variants nor loss of heterozygosity at the RASD1 variant position were observed in the patient’s microadenoma. The wild type and p.M194L RASD1 transiently overexpressed proteins displayed similar short half-lives (<1 h) by cycloheximide chase in HEK293 cells, as well as cytoplasmic localization by immunocytofluorescence in AtT20 cells. A CRISPR/Cas9 Rasd1 knockout AtT20 cell line displayed reduced Pomc expression compared with the parental cell line at the mRNA level (Actb-normalized absolute quantification 5.80±0.92 vs 9.62±0.7, P=0.005). Viability of the cell lines did not differ significantly by MTT assay. Overexpression of p.M194L resulted in increased accumulation of phospho-CREB S133 (1.83±0.8 vs 1±0.2 in empty vector control, P=0.0390) as well as a non-significant increase in Pomc expression in wild type, but not in Rasd1 knockout AtT20 cells by immunoblot band densitometry. Conclusions: We found an infrequent RASD1 variant in one CD patient. Rasd1 seems to have a role within the intracellular signaling pathways controlling Pomc expression. Overexpression of the p.M194L variant caused phospho-CREB S133 activation, suggesting increased GEF activity for this variant. Interestingly, another variant at the same position, p.M194I, was found in the COSMIC database (COSS2121715) as a somatic change in cutaneous malignant melanoma. Further studies are required to better define the role of RASD1 in corticotroph physiology and its possible involvement in tumorigenesis.

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

Aminoglycosides are efficient reagents to induce readthrough of premature termination codon in mutant B4GALNT1 genes found in families of hereditary spastic paraplegia.

The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1, in which gradual neurological disorders emerged with aging.

Discrete localization patterns of Arf6, and its activators EFA6A and BRAG2, and its effector PIP5kinaseγ on myofibrils of myotubes and plasma membranes of myoblasts in developing skeletal muscles of mice

Arf6 (ADP ribosylation factor 6), activated by Arf-GEF (guanine nucleoside exchange factor), is involved in the membrane trafficking and actin-remodeling which are critical for maintenance of cell organization and activity and for fusion of myoblasts to form myotubes/myofibers. EFA6A (exchange factor for Arf6 type A) and BRAG2 (brefeldin A-resistant Arf-GEF 2) represent members of discrete subfamilies of Arf-GEF, while PIP5Kγ (phosphatidylinositol4-phosphate5-kinase γ) produces PI 4,5-bisphosphate (PIP2) and it is target for Arf6. In the present study, immunoreactive bands for Arf6, EFA6A, BRAG2 and PIP5Kγ were detected in immunoblots of skeletal muscle homogenates of mice at E18D (embryonic day 18), while the bands for Arf6, EFA6A and PIP5Kγ were reduced in density and no significant bands for BRAG2 were discerned at P1D (postnatal 1 day). No immunoblot bands for any of the molecules were eventually detected in skeletal fibers of adult mice. Immunoreactivities for endogenous Arf6, EFA6A and PIP5Kγ were visualized using immuno-light microscopy localized as periodic striations running perpendicular to the longitudinal axes of skeletal muscle fibers of mice at E18D and P1D. All the striations were co-immunoreactive for β-actin in double immunofluorescence microscopy, and the immunoreactivities were confined to thin myofilaments at sarcomeric I-domains in immuno-electron microscopy. On the other hand, immunoreactivities for Arf6, BRAG2 and PIP5Kγ were conspicuous on plasmalemma of myoblasts at E14D, while immunoreactivity for EFA6A was already distinct in striations perpendicular to myofibrils in myotubes at E14D. The present findings suggest three possibilities: involvement of EFA6A-activated Arf6 together with PIP5Kγ in maturation of myofibrils, movement of Arf6 and PIP5Kγ from the plasmalemma of myoblasts to myofibrils of myotubes, and that of BRAG2 to the cytoplasm of myotubes; and further a function of EFA6A independent of the activation of Arf6 in immature myofibrils. In addition, the involvement of Arf6, BRAG2 and PIP5Kγ in the fusion of myoblasts into myotubes was supported by the present finding.

Deletion of major porins from meningococcal outer membrane vesicle vaccines enhances reactivity against heterologous serogroup B Neisseria meningitidis strains

Detergent-extracted detoxified outer membrane vesicle (dOMV) vaccines are effective at preventing invasive serogroup B meningococcal (MenB) disease caused by the homologous Neisseria meningitidis strain from which they are produced, but offer limited protection from heterologous strains. Differences in vaccine efficacy are partially due to strain-specific variations in the antigenic sequence types and expression levels of outer membrane proteins (OMPs), including the immunodominant OMP PorA. In this study, dOMV vaccines deficient in major OMPs, including PorA, PorB, and RmpM were isolated and used to immunize rabbits and mice. Serum samples were obtained from each animal and tested for antibody responses against five MenB strains. Immunization with wild type dOMVs elicited antibodies to major antigens including PorA, PorB, RmpM, and lipooligosaccharide (LOS), and demonstrated limited bactericidal activity against heterologous strains. In contrast, OMP-deficient dOMV vaccines elicited broadly cross-reactive bactericidal antibodies, with PorA/PorB-dual deficient dOMVs inducing antibodies exhibiting the greatest cross-reactivity. Enhanced killing of heterologous strains correlated with binding to unique protein bands in immunoblots, suggestive of improved immunogenicity of antigens under-represented in the wild type vaccine.

Expression and localization of endogenous phospholipase Cβ3 confined to basal cells in situ of immature ducts and adult excretory ducts of submandibular gland of mice

Our previous study demonstrated that, different from the parotid and sublingual glands, the submandibular glands of adult mice did not show an immunoblot band for PLCβ3 which is critical in the secretion mechanism by muscarinic cholinergic signaling. Therefore, the submandibular glands of mice at various stages of postnatal development were examined for this enzyme molecule in immunoblot and immunohistochemistry. In immunoblot, a weak band for PLCβ3-expression was detected only at early postnatal stages. In immunohistochemistry, PLCβ3-immunoreactivity was distinctly found in most basally located cells of immature ducts, while the immunoreactivity was weakly seen in terminal tubule cells without significant immunoreactivity in adjacent acinar cells. In contrast, the immunoreactivity was distinctly found in some basal cells of adult excretory ducts, and it was ultrastructurally localized densely in close association with bundles of tonofilaments in the cells. The present finding suggests the possibility that Ca2+ signaling governed by phospholipase Cβ3 is involved in the differentiation of ductal basal cells into apical cells through control of keratin molecule(s) in the cells.

Phosphorylcholine is located in Aggregatibacter actinomycetemcomitans fimbrial protein Flp 1

Phosphorylcholine (ChoP) is covalently incorporated into bacterial surface structures, contributing to host mimicry and promoting adhesion to surfaces. Our aims were to determine the frequency of ChoP display among Aggregatibacter actinomycetemcomitans strains, to clarify which surface structures bear ChoP, and whether ChoP-positivity relates to serum killing. The tested oral (N = 67) and blood isolates (N = 27) represented 6 serotypes. Mab TEPC-15 was used for immunoblotting of cell lysates and fractions and for immunofluorescence microscopy of cell surface-bound ChoP. The lysates were denatured with urea for hidden ChoP or treated with proteinase K to test whether it binds to a protein. Three ChoP-positive and two ChoP-negative strains were subjected to serum killing in the presence/absence of CRP and using Ig-depleted serum as complement source. Cell lysates and the first soluble cellular fraction revealed a < 10 kDa band in immunoblots. Among 94 strains, 27 were ChoP positive. No difference was found in the prevalence of ChoP-positive oral (21/67) and blood (6/27) strains. Immunofluorescence microscopy corresponded to the immunoblot results. Proteinase K abolished ChoP reactivity, whereas urea did not change the negative result. The TEPC-15-reactive protein was undetectable in Δflp1 mutant strain. The survival rate of serotype-b strains in serum was 100% irrespective of ChoP, but that of serotype-a was higher in ChoP-positive (85%) than ChoP-negative (71%) strains. The results suggest that a third of rough-colony strains harbor ChoP and that ChoP is attached to fimbrial subunit protein Flp1. It further seems that ChoP-positivity does not enhance but may reduce A. actinomycetemcomitans susceptibility to serum killing.

Expression of novel "LOCGEF" isoforms of ARHGEF18 in eosinophils.

Genomic, transcriptomic and proteomic databases indicate that the N-terminal 322 residues encoded by the presumptive LOC100996504 gene, which is adjacent to the ARHGEF18 guanine nucleotide exchange factor gene on chromosome 19, constitute the N-terminal portion of a 1361-residue isoform of ARHGEF18, dubbed LOCGEF-X3. LOCGEF-X3 arises from the use of a leukocyte-specific alternative transcriptional start site and splicing that bypasses the initial noncoding exon of the canonical 1015-residue ARHGEF18 isoform, p114. Eosinophil LOCGEF-X3 was amplified and cloned, recombinant LOCGEF-X3 was expressed, and anti-ARHGEF18 antibody was found to recognize a band in immunoblots of eosinophil lysates that co-migrates with recombinant LOCGEF-X3. PCR of eosinophils revealed minor amounts of transcripts for X4 and X5 isoforms of LOCGEF that arise from differential splicing and differ from the X3 isoform at their extreme N-termini. No p114 transcript or protein band was detected in eosinophils. Immunostaining with anti-ARHGEF18 antibody revealed relocalization of LOCGEF and RHOA from the periphery of round unstimulated eosinophils to the 2 poles of eosinophils polarized by treatment with IL5, CCL11, or IL33 in suspension. Canonical p114 ARHGEF18 has been implicated in maintenance of epithelial cell polarity. We suggest that the "LOC" portion of LOCGEF, which is unlike any other protein domain, has unique functions in control of polarity in activated eosinophils and other leukocytes.

CRISPR/Cas9-mediated knockout of Lim-domain only four retards organ of Corti cell growth.

Lim-domain only 4 (LMO4) plays a critical role in mediating the ototoxic side-effects of cisplatin, a highly effective anti-cancer drug. However, the signaling mechanism by which cochlear LMO4 mediates otopathology is yet to be fully understood. Knockout cell culture models are useful tools for investigating the functional roles of novel genes and delineating associated signaling pathways. Therefore, LMO4 knockout organ of Corti cells were generated by using the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system. Successful knockout of LMO4 in UB/OC1 cells was verified by the absence of LMO4 protein bands in immunoblots. Though the Knockout of LMO4 retarded the growth rate and the migratory potential of the cells it did not inhibit their long-term viability as the LMO4 knockout UB/OC1 cells were able to survive, proliferate, and form colonies. In addition, the knockout of LMO4 did not alter the expression of myosin VIIa, a biomarker of hair cells, suggesting that the knockout cells retain important characteristic features of cochlear sensory receptor cells. Thus, the findings of this study indicate that CRISPR/Cas9 system is a simple and versatile method for knocking out genes of interest in organ of Corti cells and that LMO4 knockout UB/OC1 cells are viable experimental models for studying the functional role of LMO4 in ototoxicity.

Sumoylation of histone deacetylase 1 regulates MyoD signaling during myogenesis

Sumoylation, the conjugation of a small ubiquitin-like modifier (SUMO) protein to a target, has diverse cellular effects. However, the functional roles of the SUMO modification during myogenesis have not been fully elucidated. Here, we report that basal sumoylation of histone deacetylase 1 (HDAC1) enhances the deacetylation of MyoD in undifferentiated myoblasts, whereas further sumoylation of HDAC1 contributes to switching its binding partners from MyoD to Rb to induce myocyte differentiation. Differentiation in C2C12 skeletal myoblasts induced new immunoblot bands above HDAC1 that were gradually enhanced during differentiation. Using SUMO inhibitors and sumoylation assays, we showed that the upper band was caused by sumoylation of HDAC1 during differentiation. Basal deacetylase activity was not altered in the SUMO modification-resistant mutant HDAC1 K444/476R (HDAC1 2R). Either differentiation or transfection of SUMO1 increased HDAC1 activity that was attenuated in HDAC1 2R. Furthermore, HDAC1 2R failed to deacetylate MyoD. Binding of HDAC1 to MyoD was attenuated by K444/476R. Binding of HDAC1 to MyoD was gradually reduced after 2 days of differentiation. Transfection of SUMO1 induced dissociation of HDAC1 from MyoD but potentiated its binding to Rb. SUMO1 transfection further attenuated HDAC1-induced inhibition of muscle creatine kinase luciferase activity that was reversed in HDAC1 2R. HDAC1 2R failed to inhibit myogenesis and muscle gene expression. In conclusion, HDAC1 sumoylation plays a dual role in MyoD signaling: enhancement of HDAC1 deacetylation of MyoD in the basally sumoylated state of undifferentiated myoblasts and dissociation of HDAC1 from MyoD during myogenesis.

Anti-HCV immunoblot indeterminate results in blood donors: non-specific reactivity or past exposure to HCV?

The significance of anti-HCV immunoblot (IB) indeterminate results can be difficult to determine. We analysed results for blood donors tested on the MP Diagnostics HCV Blot 3.0 IB assay to determine whether indeterminate results representing past exposure to HCV could be distinguished from those due to non-specific reactivity. Results for all donors tested by IB during the study period (July 2010 to December 2013) were included in this study. Of 131 donors tested by IB, 34 (26.0%) were negative, 38 (29.0%) were indeterminate, and 59 (45.0%) were positive. There was no significant difference in IB band reactivity strength between indeterminate and positive donors. The PRISM HCV chemiluminescent immunoassay (ChLIA) sample to cut-off (s/co) ratio distribution for the indeterminate donors was significantly higher than for those with biological false reactivity (P=0·037), but significantly lower than for donors who were IB positive/HCV RNA negative (P<0·001) or IB not tested/HCV RNA positive (P<0·001). Of donors available for follow-up, 53.1% of the indeterminate group disclosed a putative risk factor for HCV infection compared to 39.4% (P<0·001) for the IB-negative group, 76.6% (P=0·065) for the IB-positive group and 83.4% (P<0·001) for the HCV RNA-positive group. The results of this study indicate that PRISM ChLIA s/co ratios >2·00 with IB indeterminate results predict exposure to HCV, particularly in the presence of putative risk factors for HCV infection. These findings may be applied to optimizing counselling of donors with indeterminate HCV results.

Retraction: 'An ~400kDa membrane-associated complex that contains one molecule of the resistance protein Cf-4' by Susana Rivas, Tatiana Mucyn, Harrold A. van den Burg, Jacques Vervoort and Jonathan D. G. Jones.

Rivas, S., Mucyn, T., van den Burg, H.A., Vervoort, J. and Jones, J.D.G. (2002a) An ∼400kDa membrane-associated complex that contains one molecule of the resistance protein Cf-4. Plant J. 29, 783-796. https://doi.org/10.1046/j.1365-313X.2002.01254.x. Rivas, S., Romeis, T. and Jones, J.D.G. (2002b) The Cf-9 disease resistance protein is present in an ∼420-Kilodalton heteromultimeric membrane-associated complex at one molecule per complex. Plant Cell, 14, 689-702. https://doi.org/10.1105/tpc.010357.

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion.

Prion diseases of cattle include the classical bovine spongiform encephalopathy (C-BSE) and the atypical H-type BSE (H-BSE) and L-type BSE (L-BSE) strains. Although the C- and L-BSE strains can be detected and discriminated by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays, no such test has yet been described for the detection of H-BSE or the discrimination of each of the major bovine prion strains. Here, we demonstrate an RT-QuIC assay for H-BSE that can detect as little as 10(-9) dilutions of brain tissue and neat cerebrospinal fluid samples from clinically affected cattle. Moreover, comparisons of the reactivities with different recombinant prion protein substrates and/or immunoblot band profiles of proteinase K-treated RT-QuIC reaction products indicated that H-, L-, and C-BSE have distinctive prion seeding activities and can be discriminated by RT-QuIC on this basis.

CD147 expression predicts biochemical recurrence after prostatectomy independent of histologic and pathologic features.

BackgroundCD147 is an MMP-inducing protein often implicated in cancer progression. The purpose of this study was to investigate the expression of CD147 in prostate cancer (PCa) progression and the prognostic ability of CD147 in predicting biochemical recurrence after prostatectomy.MethodsPlasma membrane-localized CD147 protein expression was quantified in patient samples using immunohistochemistry and multispectral imaging, and expression was compared to clinico-pathological features (pathologic stage, Gleason score, tumor volume, preoperative PSA, lymph node status, surgical margins, biochemical recurrence status). CD147 specificity and expression were confirmed with immunoblotting of prostate cell lines, and CD147 mRNA expression was evaluated in public expression microarray datasets of patient prostate tumors.ResultsExpression of CD147 protein was significantly decreased in localized tumors (pT2; p = 0.02) and aggressive PCa (≥pT3; p = 0.004), and metastases (p = 0.001) compared to benign prostatic tissue. Decreased CD147 was associated with advanced pathologic stage (p = 0.009) and high Gleason score (p = 0.02), and low CD147 expression predicted biochemical recurrence (HR 0.55; 95 % CI 0.31–0.97; p = 0.04) independent of clinico-pathologic features. Immunoblot bands were detected at 44 kDa and 66 kDa, representing non-glycosylated and glycosylated forms of CD147 protein, and CD147 expression was lower in tumorigenic T10 cells than non-tumorigenic BPH-1 cells (p = 0.02). Decreased CD147 mRNA expression was associated with increased Gleason score and pathologic stage in patient tumors but is not associated with recurrence status.ConclusionsMembrane-associated CD147 expression is significantly decreased in PCa compared to non-malignant prostate tissue and is associated with tumor progression, and low CD147 expression predicts biochemical recurrence after prostatectomy independent of pathologic stage, Gleason score, lymph node status, surgical margins, and tumor volume in multivariable analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1559-4) contains supplementary material, which is available to authorized users.

Abstract 1990: Identification and biochemical characterization of HMP19, a tumor/metastasis suppressor in pancreatic cancer

Abstract The overwhelming majority of pancreatic cancer is not diagnosed until the cancer has metastasized, which leads to an abysmal average life expectancy (3 to 6 months post-diagnosis). Earlier detection and more effective treatments have been hampered by inadequate understanding of the underlying molecular mechanisms controlling metastasis. Metastasis suppressors inhibit metastasis without blocking orthotopic tumor growth and represent powerful new targets and biomarkers. We hypothesize that metastasis suppressors are involved in controlling metastasis in pancreatic cancer. Using an unbiased genome wide shRNA screen, we identified HMP19 as a putative metastasis suppressor in pancreatic ductal adenocarcinoma (PDAC). Functional validation was carried out by manipulating expression in PDAC cell lines. Knockdown of HMP19 in S2-028 cells increased orthotopic tumor growth and liver metastasis, while over-expression in S2-007 cells slowed tumor growth and suppressed metastasis in xenographs. HMP19 expression inversely correlated with in vitro migration, colony formation, proliferation, and cyclin E1 expression. In clinical samples, HMP19 expression inversely correlated with tumor size, plexus invasion, liver metastasis, and patient survival (p&amp;lt;0.05). HMP19 is essentially uncharacterized, except for reported sub-cellular localization in the Golgi apparatus in neuronal cells (Saberan-Djoneidi et al. 1994). HMP19 is localized throughout the secretory/endocytic pathways (fluorescence microscopy), including at the plasma membrane (surface biotinylation). HMP19 is predicted to be a type II, single-pass transmembrane protein with 3 domains (cytoplasmic; transmembrane; extracellular). However, the ability to biotinylate HMP19 in intact S2-028 cells expressing the predicted cytoplasmic+transmembrane domains suggest that HMP19 might be a type I transmembrane protein. The cytoplasmic tail and transmembrane domains are important for protein stability, while the presence of multiple bands in immunoblots (16, 19, 21, and 25 kDa) suggests that HMP19 is post-translationally modified by an as-yet defined mechanism. Interestingly, we found that HMP19 co-immunoprecipitates with and suppresses phospho-ERK nuclear localization. Here we describe the discovery and characterization of a novel PDAC metastasis suppressor and potential mechanism of action, which is in support of our hypothesis that metastasis suppressors control metastasis in pancreatic cancer. Support: National Foundation for Cancer Research, Hall Family Foundation, Kansas Bioscience Authority, and CA134981 Citation Format: Christopher R. Bohl, Hiroshi Kurahara, Shoji Natsugoe, Yuka Nishizono, Sitaram Harihar, Tomoo Iwakuma, Danny R. Welch. Identification and biochemical characterization of HMP19, a tumor/metastasis suppressor in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1990. doi:10.1158/1538-7445.AM2014-1990

Serological diagnosis of Besnoitia bennetti infection in donkeys (Equus asinus)

Besnoitiosis is an emerging infectious disease of donkeys (Equus asinus) in the United States for which there are currently no serologic methods of diagnosis. A study was performed to evaluate physical examination findings and 3 serologic assays for the detection of Besnoitia bennetti infection in donkeys. A prospective study of 416 donkeys from 6 privately owned herds across 5 U.S. states (New York, Pennsylvania, Vermont, Oregon, and Washington) was performed. Donkeys were examined for clinical lesions suggestive of besnoitiosis and evaluated for antibodies against B. bennetti using a fluorescent antibody test (FAT) and 2 immunoblot assays specific for bradyzoite and tachyzoite antigens, respectively. Donkeys were confirmed to be infected with B. bennetti by histology (cases; n = 32) and were compared to those with no clinical signs of besnoitiosis (controls; n = 384). Identifying clinical lesions in 2 or more locations correctly identified infected donkeys 83% of the time. Donkeys with besnoitiosis had significantly higher FAT titers (P < 0.001) and numbers of bradyzoite (P < 0.001) and tachyzoite (P < 0.001) immunoblot bands than control donkeys. The sensitivity and specificity of the serologic assays for detecting besnoitiosis was 88% and 96% for FAT, 81% and 91% for bradyzoite immunoblot, and 91% and 92% for tachyzoite immunoblot, respectively. Fluorescent antibody and immunoblot assays are effective at identifying donkeys with besnoitiosis and provide a more efficient and less invasive diagnostic alternative to histology.

Improvement of immunoprecipitation/immunoblotting of endogenous protein by TrueBlot antibody

To improve the immunoblotting of immunoprecipitated proteins and decrease the interference of immunoprecipitation antibody in the interaction of endogenous proteins. Transient transfect cells with fusion protein expression vector containing the targeted S5b gene and the FLAG tag, the transfected cells or untransfected cells were harvested to study the exogenous or endogenous protein interaction. The total cell lysate was immunoprecipitated by specific antibody. Then the eluted immunocomplex was separated by SDS-PAGE, and the TrueBlot antibody or conventional antibody was used as the secondary antibody for immunoblotting detection of S5b and its partner (Rpt1 and Rpt2). Clear immunoblotting bands for S5b, Rpt1 and Rpt2 were obtained. TrueBlot antibody prefers the immunoblot antibody to immunoprecipitation antibody, and decreases the interruption of immunoprecipitation antibody to display clear protein band.