Recifin A, Initial Example of the Tyr-Lock Peptide Structural Family, Is a Selective Allosteric Inhibitor of Tyrosyl-DNA Phosphodiesterase I.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDP1 activity resulted in the discovery of a new class of knotted cyclic peptides from the marine sponge sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be an unprecedented 42-residue cysteine-rich peptide named recifin A.

IL-7-induced phosphorylation of the adaptor Crk-like and other targets.

IL-7 is required for T cell differentiation and mature T cell homeostasis and promotes pro-B cell proliferation and survival. Tyrosine phosphorylation plays a central role in IL-7 signaling. We identified by two-dimensional electrophoresis followed by anti-phosphotyrosine immunoblotting and mass spectrometry sixteen tyrosine phosphorylated proteins from the IL-7-dependent cell line D1.

Discovery and Characterization of a Biologically Active Non-ATP-Competitive p38 MAP Kinase Inhibitor.

Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer.

RSK-mediated phosphorylation in the C/EBP{beta} leucine zipper regulates DNA binding, dimerization, and growth arrest activity.

The bZIP transcription factor C/EBPbeta is a target of Ras signaling that has been implicated in Ras-induced transformation and oncogene-induced senescence (OIS). To gain insights into Ras-C/EBPbeta signaling, we investigated C/EBPbeta activation by oncogenic Ras. We show that C/EBPbeta DNA binding is autorepressed and becomes activated by the Ras-Raf-MEK-ERK-p90(RSK) cascade.

Interleukin-7 regulates Bim proapoptotic activity in peripheral T-cell survival.

Interleukin-7 (IL-7) is critical for T-cell development and peripheral T-cell homeostasis. The survival of pro-T cells and mature T cells requires IL-7. The survival function of IL-7 is accomplished partly through induction of the antiapoptotic protein Bcl-2 and inhibition of proapoptotic proteins Bax and Bad.

Label-free global serum proteomic profiling reveals novel celecoxib-modulated proteins in familial adenomatous polyposis patients.

Celecoxib, a selective inhibitor of cyclooxygenase-2 (Cox-2), was efficacious in clinical prevention trials of patients with familial adenomatous polyposis (FAP) and sporadic colorectal cancer. To identify as yet poorly defined molecular determinants of celecoxib efficacy, a multidimensional serum fractionation approach was used coupled with nanospray tandem mass spectrometry to perform label-free global proteomic profiling of serum samples from the FAP/celecoxib prevention trial. Subsequently, the application of an algorithm for large-scale biomarker discovery on comparative serum proteomic profiles of pre- and post-celecoxib treatment samples identified 83 potentially celecoxib-responsive proteins from various cellular compartments, biological processes and molecular functions.

Cyclin-dependent kinase 5 phosphorylation of human septin SEPT5 (hCDCrel-1) modulates exocytosis.

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system, where it is involved in neuronal migration, synaptic transmission, and survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates.

IL-6 enhances the nuclear translocation of DNA cytosine-5-methyltransferase 1 (DNMT1) via phosphorylation of the nuclear localization sequence by the AKT kinase.

The epigenetic programming of genomic DNA is accomplished, in part, by several DNA cytosine-5-methyltransferases that act by covalently modifying cytosines with the addition of a methyl group. This covalent modification is maintained by the DNA cytosine-5-methyltransferase-1 enzyme (DNMT1), which is capable of acting in concert with other similar enzymes to silence important tumor suppressor genes. IL-6 is a multifunctional mediator of inflammation, acting through several major signaling cascades, including the phosphatidylinositol-3-kinase pathway (PI-3-K), which activates protein kinase B (AKT/PKB) downstream.

PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex.

PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX.

Structure and activity of the atypical serine kinase Rio1.

Rio1 is the founding member of the RIO family of atypical serine kinases that are universally present in all organisms from archaea to mammals. Activity of Rio1 was shown to be absolutely essential in Saccharomyces cerevisiae for the processing of 18S ribosomal RNA, as well as for proper cell cycle progression and chromosome maintenance. We determined high-resolution crystal structures of Archaeoglobus fulgidus Rio1 in the presence and absence of bound nucleotides.

Autophosphorylation of Archaeoglobus fulgidus Rio2 and crystal structures of its nucleotide-metal ion complexes.

The highly conserved, atypical RIO serine protein kinases are found in all organisms, from archaea to man. In yeast, the kinase activity of Rio2 is necessary for the final processing step of maturing the 18S ribosomal rRNA. We have previously shown that the Rio2 protein from Archaeoglobus fulgidus contains both a small kinase domain and an N-terminal winged helix domain.

Alternative p38 activation pathway mediated by T cell receptor-proximal tyrosine kinases.

Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal-regulated protein kinase MAPKs.

The parafibromin tumor suppressor protein is part of a human Paf1 complex.

Parafibromin, the product of the HRPT2 (hyperparathyroidism-jaw tumor syndrome 2) tumor suppressor gene, is the human homologue of yeast Cdc73, part of the yeast RNA polymerase II/Paf1 complex known to be important for histone modification and connections to posttranscriptional events. By purifying cellular parafibromin and characterizing its associated proteins, we have identified a human counterpart to the yeast Paf1 complex including homologs of Leo1, Paf1, and Ctr9. Like the yeast complex, the parafibromin complex associates with the nonphosphorylated and Ser2 and Ser5 phosphorylated forms of the RNA polymerase II large subunit.

Functional characterization of the acyl carrier protein (PfACP) and beta-ketoacyl ACP synthase III (PfKASIII) from Plasmodium falciparum.

The genome of the malaria parasite, Plasmodium falciparum, appears to contain the proteins necessary for a Type II dissociated fatty acid biosynthetic system. Here we report the functional characterization of two proteins from this system. Purified recombinant acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase III (KASIII) from P.

A binding shift assay for the zinc-bound and zinc-free HIV-1 nucleocapsid protein by capillary electrophoresis.

Affinity capillary electrophoresis was used to detect a shift in mobility when a zinc ion binds to the highly basic nucleocapsid protein (NCp7) of HIV-1. NCp7 contains two Cys-X2- Cys-X4-His-X4-Cys zinc fingers. With constant concentrations of NCp7 as a receptor and various concentrations of zinc as a ligand in the sample buffer and the electrophoresis buffer, we observed changes in electrophoretic mobilities of NCp7 protein when complexes were formed with zinc.

Capillary zone electrophoresis of large DNA.

Capillary zone electrophoresis (CZE) of DNA 23.1 to 48.5 kb in length in polyacrylamide solutions of several concentrations provides evidence for polymer concentration and DNA length-dependent stretching and orientation of these species and suggests an effective separation at a polymer concentration of about 0.

Quantitation of human haptoglobin by ELISA system based on streptococcal haptoglobin receptors.

Streptococcus pyogenes cells with binding properties for human haptoglobin were used for quantitative determination of the acute phase protein, haptoglobin in various biological fluids. The S. pyogenes cells with protein surface antigen T4 served as solid phase in a microtitre ELISA system.

Advances in DNA electrophoresis in polymer solutions.

DNA electrophoresis in gels and solutions of agarose and polyacrylamide was objectively evaluated with regard to separation efficiency at optimal polymer concentrations. In application to DNA fragments, polyacrylamide gels were superior for separating fragments of less than 7800 bp, and agarose gels are the best choice for larger fragments. Agarose solutions are nearly as good as polyacrylamide gels for small DNA (< 300 bp).

[Activity of alanine aminopeptidase, beta-glucuronidase and N-acetyl-beta-d-glucosaminidase in urine of children with nephrotic syndrome].

Activity of alanine aminopeptidase (AAP), beta-glucuronidase, and N-acetyl-beta-D-glucosaminidase (NAG) in daily urine has been determined in 27 children with nephrotic syndrome, 14 children in remission, and 11 healthy children. It was found, that these enzymes activity is significantly increased in sick children in comparison with healthy ones. Similarly, the activity of AAP and NAG in daily urine is statistically significantly higher in children with remission, than that in healthy children.

Electrophoretic separation of S. pombe chromosomes in polyacrylamide solutions using a constant field.

Previous electrophoretic separations of megabase (Mb) sized DNA have been achieved in pulsed electric fields, using agarose gel as a matrix. The present study demonstrates separations of Mb sized DNA due to a retardation of migration in proportion to the concentration of uncrosslinked polyacrylamide of 5 x 10(6) molecular weight, using a constant electric field. Potentially, the method should be applicable to large DNA in general, greatly reducing the instrumental complexity of such separations and rendering them compatible with capillary electrophoresis apparatus.

Electrophoretic size separation of particles with diameters in the micron range, using polymer solutions.

Polystyrene-sulfate particles ranging in size from 536 to 2,170 nm diameter were subjected to electrophoresis (10 V cm-1, 25 degrees C) in K-MES, 0.03 M ionic strength, pH 6.12, 50 mM CHAPS in liquid polymer media contained in horizontal glass tubes of 1 mm ID.

Characterization of haptoglobin-binding properties of streptococci of serological group G.

Two group G streptococcal cultures (G 10187, G 11122) with surface antigen T4 possess surface receptors for human haptoglobin (Hp). G 10187 additionally interacted with immunoglobulin G and albumin, G 11122 with fibrinogen and fibronectin. Binding of 125I-Hp 2-1 was time-dependent, saturable, reversible in the presence of unlabelled Hp and could be inhibited by unlabelled human-Hp 2-1, -Hp 2-2, -Hp 1-1, Hp-hemoglobin complexes and by Hp preparations from pigs, horses and rabbits.

Further characterization of haptoglobin binding to streptococci of serological group A.

Certain group A streptococci with surface antigen T 4 possess surface receptors for human haptoglobin (Hp). Binding of 125I Hp 2-1 to two representative group A streptococcal cultures could be inhibited by unlabelled Hp 2-1, Hp 2-2 and Hp 1-1 but not by the alpha 1, alpha 2 or beta chains of Hp. Hp complexes formed with equine hemoglobin and asialo-Hp also reduced 125I-Hp 2-1 binding to group A streptococci.

Clearance of certain modified haptoglobins from the rabbit circulation.

1. Human haptoglobin (Hp) type 2-1 was subjected to the sulfanilazo-modification of tyrosine and histidine residues, the removal of sialic acid, and the reduction of disulfide bonds (isolation of alpha 2, alpha 1, beta subunits), respectively. Radioactively labeled preparations were administered intravenously to rabbits.