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  • Biologic significance of fascin expression in clear cell renal cell carcinoma: systematic analysis of primary and metastatic tumor tissues using a tissue microarray techn ... 16979727

    OBJECTIVES: To investigate the biologic significance of fascin, a globular actin cross-linking protein, involved in cell adhesion and motility, in primary and metastatic renal cell carcinoma (RCC). METHODS: A total of 136 primary clear cell RCCs and 54 clear cell RCC metastases were stained immunohistochemically using a tissue microarray technique. Distinct cytoplasmic staining was considered positive, and the staining results were associated with the pT stage, Fuhrman grade, tumor size, sarcomatoid morphology, and metastasis-free survival. For multivariate testing, Cox's proportional hazards regression model was used. RESULTS: Fascin expression was noted in 13 (10%) of 136 primary and 25 (46%) of 54 metastatic RCC specimens (P 0.001). Fascin expression was associated with high tumor stage (2 [3%] of 70 pT1 versus 11 [17%] of pT2/pT3; P = 0.008), high tumor grade (3 [3%] of 88 grade 1-2 versus 10 [21%] of 48 grade 3-4; P = 0.002), and large tumor size (P 0.001). In addition, 8 (62%) of 13 RCCs with sarcomatoid morphology expressed fascin compared with 5 (4%) of 123 RCCs without sarcomatoid transformation (P 0.001). Metastatic disease was noted in 10 (77%) of 13 patients with fascin-positive RCC compared with 26 (21%) of 121 patients with fascin-negative RCC (P 0.001). Multivariate analysis revealed pT Stage 1 or greater (P 0.001, risk ratio [RR] 8.6, 95% confidence interval [CI] 2.8 to 26.5), Fuhrman grade greater than 2 (P 0.001, RR 12.7, 95% CI 4.6 to 35.4), fascin expression (P 0.001, RR 7.2, 95% CI 3.0 to 17.4), and female gender (P = 0.02, RR 2.5, 95% CI 1.1 to 5.5) as independent predictors of metastatic disease. CONCLUSIONS: Fascin immunoreactivity in RCC proved to be an independent predictor of metastatic disease and was demonstrated in almost one half of RCC metastases. Thus, fascin may be a promising molecular target for future cancer therapy.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3582
    Nombre del producto:
    Anti-Fascin Antibody, clone 55K2

  • Phosphorylation of S409/410 of TDP-43 is a consistent feature in all sporadic and familial forms of TDP-43 proteinopathies. 19125255

    Accumulation of hyperphosphorylated, ubiquitinated and N-terminally truncated TAR DNA-binding protein (TDP-43) is the pathological hallmark lesion in most familial and sporadic forms of FTLD-U and ALS, which can be subsumed as TDP-43 proteinopathies. In order to get more insight into the role of abnormal phosphorylation in the disease process, the identification of specific phosphorylation sites and the generation of phosphorylation-specific antibodies are mandatory. Here, we developed and characterized novel rat monoclonal antibodies (1D3 and 7A9) raised against phosphorylated S409/410 of TDP-43. These antibodies were used to study the presence of S409/410 phosphorylation by immunohistochemistry and biochemical analysis in a large series of 64 FTLD-U cases with or without motor neuron disease including familial cases with mutations in progranulin (n = 5), valosin-containing protein (n = 4) and linkage to chromosome 9p (n = 4), 18 ALS cases as well as other neurodegenerative diseases with concomitant TDP-43 pathology (n = 5). Our data demonstrate that phosphorylation of S409/410 of TDP-43 is a highly consistent feature in pathologic inclusions in the whole spectrum of sporadic and familial forms of TDP-43 proteinopathies. Physiological nuclear TDP-43 was not detectable with these mAbs by immunohistochemistry and by immunoblot analyses. While the accumulation of phosphorylated C-terminal fragments was a robust finding in the cortical brain regions of FTLD-U and ALS, usually being much more abundant than the phosphorylated full-length TDP-43 band, spinal cord samples revealed a predominance of full-length TDP-43 over C-terminal fragments. This argues for a distinct TDP-43 species composition in inclusions in cortical versus spinal cord cells. Overall, these mAbs are powerful tools for the highly specific detection of disease-associated abnormal TDP-43 species and will be extremely useful for the neuropathological routine diagnostics of TDP-43 proteinopathies and for the investigation of emerging cellular and animal models for TDP-43 proteinopathies.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MABN14

  • Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells. 23842753

    The IFN-inducible protein Irgm1 (LRG-47) belongs to the family of immunity-related GTPases that function in cell-autonomous resistance against intracellular pathogens in mice. Irgm1 deficiency is associated with a severe immunodeficiency syndrome. The protein has been variously interpreted as a direct effector molecule on bacterial phagosomes or on other organelles or as an inducer of autophagy. In this study, we re-examined one of these claims, namely that Irgm1 targets mycobacterial and listerial phagosomes. We found no colocalization of endogenous Irgm1, using two immunofluorescent staining techniques, either in fibroblasts or in macrophages. We demonstrated the predicted existence of two protein isoforms of Irgm1 derived from differential splicing and described immunological reagents for their detection. Both Irgm1 isoforms localize to the Golgi apparatus and weakly to mitochondria; however, only the long Irgm1 isoforms can be detected on endolysosomal membranes. Together with the previous observation that the general immunodeficiency phenotype of Irgm1(-/-) mice is reversed in Irgm1/Irgm3 double-deficient mice, our results argue against a direct effector function of Irgm1 at the bacterial phagosome. We discuss these findings in the context of evidence that Irgm1 functions as a negative regulator of other members of the immunity-related GTPase protein family.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts. 21051537

    We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p less than 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p less than 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.
    Tipo de documento:
    Referencia
    Referencia del producto:
    04-787

  • Monoclonal antibody detection of CD46 clustering beneath Neisseria gonorrhoeae microcolonies 16552073

    CD46 (membrane cofactor protein), a complement-regulatory protein that participates in innate and acquired immunity, also serves as a receptor for viral and bacterial pathogens. CD46 isoforms terminate in one of two cytoplasmic tails, Cyt1 or Cyt2, which differ in signaling and trafficking properties. Dissecting the functions of the two cytoplasmic tails in these cellular processes has been hampered by the absence of specific reagents. Here we report the construction of Cyt1- and Cyt2-specific monoclonal antibodies (MAbs). These MAbs recognize unique epitopes within the tails and can be used for immunofluorescence microscopy, immunoblotting, and immunoprecipitation. Studies of Neisseria gonorrhoeae-infected cells with the CD46 tail MAbs demonstrate the differential recruitment of Cyt1 and Cyt2 to the cortical plaque.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Prion protein oligomers in Creutzfeldt-Jakob disease detected by gel-filtration centrifuge columns. 19389076

    Prion diseases are diagnosed by the detection of accumulation of abnormal prion protein (PrP) using immunohistochemistry or the detection of protease-resistant abnormal PrP (PrP(res)). Although the abnormal PrP is neurotoxic by forming aggregates, recent studies suggest that the most infectious units are smaller than the amyloid fibrils. In the present study, we developed a simplified method by applying size-exclusion gel-filtration chromatography to examine PrP oligomers without proteinase K digestion in Creutzfeldt-Jakob disease (CJD) samples, and evaluated the correlation between disease severity and the polymerization degree of PrP. Brain homogenates of human CJD and non-CJD cases were applied to the gel-filtration spin columns, and fractionated PrP molecules in each fraction were detected by western blot. We observed that PrP oligomers could be detected by the simple gel-filtration method and distinctly separated from monomeric cellular PrP (PrP(c)). PrP oligomers were increased according to the disease severity, accompanied by the depletion of PrP(c). The separated PrP oligomers were already protease-resistant in the case with short disease duration. In the cases with quite severe pathology the oligomeric PrP reached a plateau, which may indicate that PrP molecules could mostly develop into amyloid fibrils in the advanced stages. The increase of PrP oligomers correlated with the degree of histopathological changes such as spongiosis and gliosis. The decrease of monomeric PrP(c) was unexpectedly obvious in the diseased cases. Dynamic changes of both oligomerization of the human PrP and depletion of normal PrP(c) require further elucidation to develop a greater understanding of the pathogenesis of human prion diseases.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AP192P
    Nombre del producto:
    Donkey Anti-Mouse IgG Antibody, HRP conjugate, Species Adsorbed

  • Development of a mouse monoclonal antibody for the detection of asymmetric dimethylarginine of Translocated in LipoSarcoma/FUsed in Sarcoma and its application in analyzi ... 25810899

    RNA-binding protein Translocated in LipoSarcoma/FUsed Sarcoma (TLS/FUS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). We previously identified that TLS was associated with protein arginine methyltransferase 1 (PRMT1), and four arginine residues within TLS (R216, R218, R242 and R394) were consistently dimethylated. Protein arginine methylation is involved in various cellular events such as signal transduction, transcriptional regulation and protein-protein interactions.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Identification and characterization of a neuronal enriched novel transcript encoding the previously described p60Fe65 isoform. 21824145

    Fe65 is a multimodular adaptor protein that interacts with the cytosolic domain of the ?-amyloid precursor protein (APP), the major component of Alzheimer's disease (AD) senile plaques. In the work here presented, we describe the existence of a new Fe65 transcript variant (GenBank Accession EF103274). A unique 5' sequence of 69 nucleotides, spanning a region between exons 2 and 3 of the FE65 gene, was present in a yeast two-hybrid (YTH) clone from a human brain cDNA library. In silico analysis and RT-PCR revealed the presence of a novel exon of 133 bp, and we redefined the structure of the human FE65 gene. The novel exon 3a-inclusive transcript generates a shorter isoform, p60Fe65. The migration pattern of the p60Fe65 isoform was observed previously and attributed to an alternative translation initiation site within the p97Fe65 transcript. Here, we provide evidence for the origin of the previously unexplained p60Fe65 isoform. Moreover, Fe65E3a is expressed preferentially in the brain and the p60Fe65 protein levels increased during PC12 cell differentiation. This novel Fe65 isoform and the regulation of the splicing events leading to its production, may contribute to elucidating neuronal specific roles of Fe65 and its contribution to AD pathology.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-758

  • Digital microfluidic assay for protein detection. 24449893

    Global studies of the human proteome have revealed a plethora of putative protein biomarkers. However, their application for early disease detection remains at a standstill without suitable methods to realize their utility in the clinical setting. There thus continues to be tremendous interest in developing new technology for sensitive protein detection that is both low in cost and carries a small footprint to be able to be used at the point of care. The current gold standard method for protein biomarker detection is the ELISA, which measures protein abundance using bulky fluorescent scanners that lack portability. Here, we present a digital microfluidic platform for protein biomarker detection that is low in cost compared with standard optical detection methods, without any compromise in sensitivity. This platform furthermore makes use of simple electronics, enabling its translation into a portable handheld device, and has been developed in a manner that can easily be adapted to assay different types of proteomic biomarkers. We demonstrate its utility in quantifying not only protein abundance, but also activity. Interleukin-6 abundance could be assayed from concentrations as low as 50 pM (an order of magnitude lower than that detectable by a comparable laboratory designed ELISA) using less than 5 μL of sample, and Abelson tyrosine kinase activity was detectable in samples containing 100 pM of kinase.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-1050
    Nombre del producto:
    4G10® Platinum, Anti-Phosphotyrosine Antibody (mouse monoclonal cocktail IgG2b)

  • Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo. 25905636

    The level of drebrin, an evolutionarily conserved f-actin-binding protein that regulates synaptic structure and function, is reduced in the brains of patients with chronic neurodegenerative diseases such as Alzheimer's disease (AD) and Down's syndrome (DS). It was suggested that excitotoxic neuronal death caused by overactivation of NMDA-type glutamate receptors (NMDARs) occurs in AD and DS; however, the relationship between excitotoxicity and drebrin loss is unknown. Here, we show that drebrin is a novel target of calpain-mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. Notably, the NMDA-induced degradation of drebrin in mature neurons occurred concomitantly with a loss of f-actin. Furthermore, pharmacological inhibition of f-actin loss facilitated the drebrin degradation, suggesting a functional linkage between f-actin and drebrin degradation. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly in vitro. Furthermore, cerebral ischemia also induced the degradation of drebrin in vivo. These findings suggest that calpain-mediated degradation of drebrin is a fundamental pathology of neurodegenerative diseases mediated by excitotoxicity, regardless of whether they are acute or chronic. Drebrin regulates the synaptic clustering of NMDARs; therefore, degradation of drebrin under excitotoxic conditions may modulate NMDAR-mediated signal transductions, including pro-survival signaling. Overall, the results presented here provide novel insights into the molecular basis of cellular responses to excitotoxicity in vitro and in vivo.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB374
    Nombre del producto:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5