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  • Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen. 1848668

    Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.
    Document Type:
    Reference
    Product Catalog Number:
    14-111

  • ApoB-54.8, a truncated apolipoprotein found primarily in VLDL, is associated with a nonsense mutation in the apoB gene and hypobetalipoproteinemia. 1940616

    A new, large kindred with hypobetalipoproteinemia and a previously undescribed truncated form of apolipoprotein B (apoB) has been identified. The asymptomatic, Caucasian male proband (CK, aged 37 years) has total plasma cholesterol, triglyceride, low density lipoprotein-(LDL) cholesterol, high density lipoprotein- (HDL) cholesterol, and apoB concentrations of 108, 131, 32, 50, and 16 mg/dl, respectively. Plasma samples of 11 family members spanning three generations, which had less than 5th percentile concentrations of LDL-cholesterol, contained three apoB bands detected on immunoblots: the normal apoB-100 and apoB-48 and an unusual band of apparent molecular mass of 299,356 +/- 9580 daltons (approximately 54% the molecular weight of apoB-100). Additional immunoblotting experiments using several different anti-apoB monoclonal antibodies showed that the carboxyl terminal of apoB-100 had been deleted somewhere between amino acid residues 2148-2488. A segment of genomic DNA from the proband was amplified by polymerase chain reaction (PCR) between nucleotides 7491-7791 of Exon 26 of the apoB gene. The DNA segment was cloned into pGEM3Zf(-) and sequenced. A C----T transition was found at nucleotide 7665, resulting in a premature stop codon at amino acid residue 2486 corresponding to apoB-54.8. These results were confirmed by direct sequencing of PCR products from three apoB-54.8 positive and three apoB-54.8 negative kindred members. Allele-specific oligonucleotides were used to identify other affected family members. Cosegregation of apoB-54.8 with the C----T transition occurred in all cases. Based on haplotypes constructed from restriction fragment length polymorphism, variable number of tandem repeats, and 5' insertion/deletion analyses and from the presence or absence of apoB-54.8, it was possible to assign a single allele of apoB to the mutation throughout the family. In contrast with other shorter truncations such as apoB-31, apoB-40, and apoB-46, which are found with particles in the HDL density range, and apoB-89 that is found primarily with LDL, apoB-54.8 was found primarily in very low density lipoproteins, much less in LDL, and was virtually absent in HDL. This suggests that the length of the truncation may significantly affect the metabolism of the associated lipoprotein particles.
    Document Type:
    Reference
    Product Catalog Number:
    17-111

  • Regulation of RCK1 currents with a cAMP analog via enhanced protein synthesis and direct channel phosphorylation. 7782324

    We have recently shown that the rat brain Kv1.1 (RCK1) voltage-gated K+ channel is partially phosphorylated in its basal state in Xenopus oocytes and can be further phosphorylated upon treatment for a short time with a cAMP analog (Ivanina, T., Perts, T., Thornhill, W. B., Levin, G., Dascal, N., and Lotan, I. (1994) Biochemistry 33, 8786-8792). In this study, we show, by two-electrode voltage clamp analysis, that whereas treatments for a short time with various cAMP analogs do not affect the channel function, prolonged treatment with 8-bromoadenosine 3',5'-cyclic monophosphorothioate ((Sp)-8-Br-cAMPS), a membrane-permeant cAMP analog, enhances the current amplitude. It also enhances the current amplitude through a mutant channel that cannot be phosphorylated by protein kinase A activation. The enhancement is inhibited in the presence of (Rp)-8-Br-cAMPS, a membrane-permeant protein kinase A inhibitor. Concomitant SDS-polyacrylamide gel electrophoresis analysis reveals that this treatment not only brings about phosphorylation of the wild-type channel, but also increases the amounts of both wild-type and mutant channel proteins; the latter effect can be inhibited by cycloheximide, a protein synthesis inhibitor. In the presence of cycloheximide, the (Sp)-8-Br-cAMPS treatment enhances only the wild-type current amplitudes and induces accumulation of wild-type channels in the plasma membrane of the oocyte. In summary, prolonged treatment with (Sp)-8-Br-cAMPS regulates RCK1 function via two pathways, a pathway leading to enhanced channel synthesis and a pathway involving channel phosphorylation that directs channels to the plasma membrane.
    Document Type:
    Reference
    Product Catalog Number:
    14-111

  • Glucose production and substrate cycle activity in a fasting adapted animal, the northern elephant seal. 15755884

    During prolonged fasting physiological mechanisms defend lean tissue from catabolism. In the fasting state, glucose is derived solely from gluconeogenesis, requiring some catabolism of amino acids for gluconeogenic substrates. This creates a conflict in animals undergoing fasts concurrently with metabolically challenging activities. This study investigated glucose metabolism in fasting and developing neonatal elephant seals. Glucose production and glucose cycle activity were measured early (2 weeks) and late (6 weeks) in the postweaning fasting period. Additionally the role of regulatory hormones on glucose production and glucose cycle activity were investigated. Glucose cycle activity was highly variable throughout the study period, did not change over the fasting period, and was not correlated with insulin or glucagon level. Endogenous glucose production (EGP) was 2.80+/-0.65 mg kg(-1) min(-1) early and 2.21+/-0.12 during late fasting. Insulin to glucagon molar ratio decreased while cortisol levels increased over the fast (t=5.27, 2.84; P=0.003, 0.04; respectively). There was no relationship between EGP and hormone levels. The glucose production values measured in this study were high and exceeded the estimated gluconeogenic substrate available. These data suggest extensive glucose recycling via Cori cycle activity occurring in northern elephant seals, and we propose a possible justification for this recycling.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Characterization of HDJ-2, a human 40 kD heat shock protein. 9839446

    Heat shock proteins (HSP) are a large and complex family of proteins that play important roles in cellular function and survival. In previous studies, cDNA for a 45 kD human HSP (HDJ-2) was cloned and shown to be homologous to DNA-J, a bacterial HSP [F.M. Ausubel, R. Brent, R. E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1997; A. Chellaiah, A. Davis, T. Mohanakumar, Cloning of a unique human homologue of the Escherichia coli DNAJ heat shock protein, Biochim. Biophys. Acta 1174 (1993) 111-113]. We have also shown that the expression of HDJ-2 is highly elevated in kidney allograft biopsies of kidneys undergoing rejection [Y.G. Alevy, D. Brennan, S. Durriya, T. Howard, T. Mohanakumar, Increased expression of the HDJ-2 heat shock protein in biopsies of human rejected kidneys, Transplantation 61 (1996) 963-967]. Because of the potential importance of HDJ-2 to disease pathogenesis, we carried out studies to characterize the structure and regulation of HDJ-2. Polyclonal and monoclonal antibodies that recognize recombinant HDJ-2 were prepared and used to localize its cellular expression. HDJ-2 was found to be farnesylated but not glycosylated. This HSP was ubiquitously expressed in all of the cell types we analyzed and was localized throughout the cytoplasm and around the nuclear membrane. However, upon heat shock it migrated to the Golgi, nucleolus, and the nuclear membrane. Northern blot analysis revealed two mRNA transcripts whose synthesis was not affected by heat shock. In addition, Western blot analysis showed that expression of HDJ-2 was also not affected by heat shock. Thus, our study shows the characterization of a HSP which, because of its migration pattern upon heat shock, is an excellent candidate for a protein chaperon.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1274
    Product Catalog Name:
    Anti-Nuclear Membrane Antibody

  • cAMP-responsive element modulator (CREM)α protein induces interleukin 17A expression and mediates epigenetic alterations at the interleukin-17A gene locus in patients wit ... 22025620

    IL-17A is a proinflammatory cytokine that is produced by specialized T helper cells and contributes to the development of several autoimmune diseases such as systemic lupus erythematosus (SLE). Transcription factor cAMP-responsive element modulator (CREM)α displays increased expression levels in T cells from SLE patients and has been described to account for aberrant T cell function in SLE pathogenesis. In this report, we provide evidence that CREMα physically binds to a cAMP-responsive element, CRE (-111/-104), within the proximal human IL17A promoter and increases its activity. Chromatin immunoprecipitation assays reveal that activated naïve CD4(+) T cells as well as T cells from SLE patients display increased CREMα binding to this site compared with T cells from healthy controls. The histone H3 modification pattern at the CRE site (-111/-104) and neighboring conserved noncoding sequences within the human IL17A gene locus suggests an accessible chromatin structure (H3K27 hypomethylation/H3K18 hyperacetylation) in activated naïve CD4(+) T cells and SLE T cells. H3K27 hypomethylation is accompanied by decreased cytosine phosphate guanosine (CpG)-DNA methylation in these regions in SLE T cells. Decreased recruitment of histone deacetylase (HDAC)1 and DNA methyltransferase (DNMT)3a to the CRE site (-111/-104) probably accounts for the observed epigenetic alterations. Reporter studies confirmed that DNA methylation of the IL17A promoter indeed abrogates its inducibility. Our findings demonstrate an extended role for CREMα in the immunopathogenesis of SLE because it contributes to increased expression of IL-17A.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Mesenchymal expression of Tbx4 gene is not altered in Adriamycin mouse model. 20182749

    The Adriamycin mouse model (AMM) is a reproducible teratogenic model of esophageal atresia/tracheo-esophageal fistula (EA/TEF). Tbx4 is a member of the T-box family of transcription factor genes, which is reported to play a key role in separation of the respiratory tract and the esophagus. Up-regulation of Tbx4 is reported to cause TEF in the chick. Optical projection tomography (OPT) is a technique that allows three-dimensional (3D) imaging of gene expression in small tissue specimens in an anatomical context. The aim of this study was to investigate the temporo-spatial expression of Tbx4 during the critical period of separation of the trachea and esophagus in normal and Adriamycin treated embryos using OPT.
    Document Type:
    Reference
    Product Catalog Number:
    07-633
    Product Catalog Name:
    Anti-HNF3β/FOXA2 Antibody

  • The neutrophil-activating proteins interleukin 8 and beta-thromboglobulin: in vitro and in vivo comparison of NH2-terminally processed forms. 2145175

    Isolation of the human neutrophil activating protein (NAP) interleukin 8 (IL8) from leukocytes has revealed that it is structurally related to beta-thromboglobulin (beta TG) from platelets. Both these proteins occur as natural mixtures of multiple forms, differing from each other by unequal truncation at the NH2 terminus. In this study we have compared IL8 and beta TG forms for in vitro and in vivo neutrophil activation. In contrast to IL8, none of the beta TG forms were found to exert granulocyte chemotactic activity in vitro, as measured in the agarose assay. However, fractions rich in the most extensively processed forms of beta TG (e.g. NAP-2) as well as pure NAP-2 did induce lactoferrin release from granulocytes, whereas fractions containing only the longer forms (e.g. connective tissue-activating peptide III) were inactive. In order to observe this in vitro effect, about 10-fold less IL8 (10 nM) than NAP-2 was required. In the presence of a vasodilator substance low doses (2-20 pmol) of IL8 and the shorter forms of beta TG caused granulocyte accumulation and plasma leakage in rabbit skin whereas the longer forms of beta TG again failed to do so. Finally, granulocytosis induction following i.v. injection was found to occur with NAP-2. At the maximal dose tested (250 pmol), this in vivo effect of NAP-2 was less pronounced than that of IL8. In the case of IL8, NH2-terminal processing did not seem to affect granulocyte stimulatory activity. It should be noted, however, that the extent of processing of IL8 is less than that occurring with beta TG. It can be concluded that the platelet factor beta TG, structurally related to the monokine IL8, can also play a role in neutrophil activation during inflammatory reactions.
    Document Type:
    Reference
    Product Catalog Number:
    17-191
    Product Catalog Name:
    MAP Kinase/Erk Assay Kit, non-radioactive

  • Expression profiles of a human pancreatic cancer cell line upon induction of apoptosis search for modulators in cancer therapy. 15557790

    We analyzed the differential gene expression in the pancreatic cancer cell line NP-18 upon induction of apoptosis caused by cyclin-dependent kinase inhibition triggered by either overexpression of the tumor suppressor gene p16(INK4A)using an adenoviral construction or incubation with the chemical inhibitors, roscovitine or olomoucine. Screening was performed using cDNA arrays from Clontech that allowed the determination of the expression of 1,176 genes specifically related with cancer. The analysis was carried out using the Atlas Image 2.01 (Clontech) and GeneSpring 4.2 (Silicon Genetics) softwares. Among the differentially expressed genes, we chose for further validation histone deacetylase 1 (HDAC1), von Hippel Lindau and decorin as upregulated genes, and Sp1, hypoxia-inducible factor-1 alpha and DNA primase as downregulated genes. The changes in the expression of these genes to mRNA were validated by quantitative RT-PCR and the final translation into protein by Western blot analysis. Inhibition of HDAC activity, Sp1 binding and DNA primase expression led to an increase in the level of apoptosis, both in parental cells and in doxorubicin-resistant cells. Therefore, these proteins could constitute possible targets to develop modulators in cancer chemotherapy that would increase or restore apoptosis.
    Document Type:
    Reference
    Product Catalog Number:
    06-720
    Product Catalog Name:
    Anti-HDAC1 Antibody

  • BML-111 alleviates acute lung injury through regulating the expression of lncRNA MALAT1. 29704485

    BML-111 is a lipoxin receptor agonist that plays a vital role on inflammation. MALAT1 is reported to mediate lung injury. ALI rat model was established using the method of venous cannula. Pulmonary microvascular endothelial cells (PMVEC) of rats were isolated using immunomagnetic separation method. Hematoxylin-eosin (HE) staining was performed to observe the lung injury degree. Real-time PCR and western blot were performed to detect the genes expression. ELIAS was used to determine the level of TNF-α and IL-6. RNA pull-down and RIP were carried out to affirm the relationship between MALAT1 and TLR4. The lung injury score and lung wet/dry weight ratio was significantly increased in ALI rats, while BML-111 treatment significantly decreased it, the HE staining directly revealed the lung injury. The expression of MALAT1 was decreased, while TLR4 was increased in ALI rats, BML-111 stimulation significantly reversed it. MALAT1 targets TLR4 to regulate its expression. TLR4 regulated the inflammation and cell apoptosis of PMVEC via NF-κB and p38 MAPK signaling pathway. The down-regulated MALAT1 mediates the mechanism of ALI by regulating of NF-κB and p38 MAPK signaling pathways via TLR4, while BML-111 stimulation significantly alleviated the ALI by regulating the expression of MALAT1.
    Document Type:
    Reference
    Product Catalog Number:
    17-700
    Product Catalog Name:
    Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit