Pax5 (BSAP) regulates the murine immunoglobulin 3' alpha enhancer by suppressing binding of NF-alpha P, a protein that controls heavy chain transcription.

The Pax5 transcription factor BSAP (B-cell-specific activator protein) is known to bind to and repress the activity of the immunoglobulin heavy chain 3' alpha enhancer. We have detected an element--designated alpha P--that lies approximately 50 bp downstream of the BSAP binding site 1 and is required for maximal enhancer activity. In vitro binding experiments suggest that the 40-kDa protein that binds to this element (NF-alpha P) is a member of the Ets family present in both B-cell and plasma-cell nuclei. However, in vivo footprint analysis suggests that the alpha P site is occupied only in plasma cells, whereas the BSAP site is occupied in B cells but not in plasma cells. When Pax5 binding to the enhancer in B cells was blocked in vivo by transfection with a triple-helix-forming oligonucleotide an alpha P footprint appeared and endogenous immunoglobulin heavy chain transcripts increased. The triple-helix-forming oligonucleotide also increased enhancer activity of a transfected construct in B cells, but only when the alpha P site was intact. Pax5 thus regulates the 3' alpha enhancer and immunoglobulin gene transcription by blocking activation by NF-alpha P.

Chimeric plant calcium/calmodulin-dependent protein kinase gene with a neural visinin-like calcium-binding domain.

Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca2+ and Ca2+/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca(2+)-binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca(2+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca2+/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca(2+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approximately 56 kDa) binds calmodulin in a Ca(2+)-dependent manner. Furthermore, 45Ca-binding assays revealed that CCaMK directly binds Ca2+. The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca2+ signaling in plants.

Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Cellular protein kinase C isoform zeta regulates human parainfluenza virus type 3 replication.

Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.

Evidence for a role of endothelin 1 and protein kinase C in nitroglycerin tolerance.

We sought to examine mechanisms responsible for increased vasoconstriction that occurs during development of nitroglycerin tolerance. Rabbits were treated for 3 days with nitroglycerin patches (0.4 mg/hr), and their aortic segments were studied in organ chambers. This treatment resulted in attenuated in vitro relaxations to nitroglycerin and increased contractile sensitivity to angiotensin II, serotonin, phenylephrine, KCl, and a direct activator of protein kinase C, the phorbol ester phorbol 12,13-dibutyrate. The protein kinase C antagonists calphostin C (100 nM) and staurosporine (10 nM) corrected the hypersensitivity to constrictors in tolerant vessels, yet had minimal effects on constrictions in control vessels. Paradoxically, constrictions caused by endothelin 1 were decreased in nitrate-tolerant vessels. Immunocytochemical analysis revealed intense endothelin 1-like and big endothelin 1-like immunoreactivity in the media of nitroglycerin-tolerant but not of control aortas. The enhanced vasoconstriction to angiotensin II, serotonin, KCl, and phenylephrine could be mimicked in normal vessels by addition of subthreshold concentrations of endothelin 1, and this effect was prevented by calphostin C. We propose that increased autocrine production of endothelin 1 in nitrate tolerance sensitizes vascular smooth muscle to a variety of vasoconstrictors through a protein kinase C-mediated mechanism.

Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.

Early Alzheimer disease-like histopathology increases in frequency with age in mice transgenic for beta-APP751.

beta-Amyloid deposition and neurofibrillary tangle formation are two histopathological features of Alzheimer disease. We have previously reported that beta-amyloid immunoreactive deposits form in the brains of transgenic mice programmed for neuronal expression of the 751-amino acid isoform of human beta-amyloid precursor protein (beta-APP751) and now describe that these animals also display Alz50 intraneuronal immunoreactivity similar to that seen in early Alzheimer disease. This suggests that abnormal beta-APP expression and/or beta-amyloid deposition promotes pathogenic alterations in tau protein. The frequency of both beta-amyloid deposition and Alz50-positive neurons was twice as prevalent in brains from old (22 months) as compared to young (2-3 months) beta-APP751 transgenic mice. This increase in histopathology with age in beta-APP751 transgenic mice parallels the time-dependent progression seen in the human disease.

A conserved family of elav-like genes in vertebrates.

A large family of genes encodes proteins with RNA recognition motifs that are presumed to bind RNA and to function in posttranscriptional regulation. Neural-specific members of this family include elav, a gene required for correct differentiation and maintenance of neurons in Drosophila melanogaster, and a related gene, HuD, which is expressed in human neuronal cells. I have identified genes related to elav and HuD in Xenopus laevis, zebrafish, and mouse that define a family of four closely related vertebrate elav-like genes (elrA, elrB, elrC, and elrD) in fish, frogs, and mammals. In addition to protein sequence conservation, a segment of the 3'-untranslated sequence of elrD is also conserved, implying a functional role in elrD expression. In adult frogs, elrC and elrD are exclusively expressed in the brain, whereas elrB is expressed in brain, testis, and ovary. During Xenopus development, elrC and elrD RNAs are detected by late gastrula and late neurula stages, respectively, whereas a nervous system-specific elrB RNA species is expressed by early tadpole stage. Additional elrB transcripts are detected in the ovary and early embryo, demonstrating a maternal supply of mRNA and possibly of protein. These expression patterns suggest a role for different elav-like genes in early development and neuronal differentiation. Surprisingly, elrA is expressed in all adult tissues tested and at all times during development. Thus, the widely expressed elrA is expected to have a related function in all cells.

Tyrosine-phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming interferon-stimulated-gene factor 3.

Interferon alpha induction of transcription operates through interferon-stimulated-gene factor 3 (ISGF), a transcription factor two components of which are members of the newly characterized Stat family of transcription factors. Interferon alpha induces tyrosine phosphorylation of Stat1 and Stat2 proteins that associate and, together with a 48-kDa protein, form ISGF3. Evidence is presented that a heterodimer of Stat1 and Stat2 is present in ISGF3 and that Stat1 and the 48-kDa protein make precise contact, while Stat2 makes general contact, with the interferon-stimulated response element, the binding site of the ISGF3.

Identification of two flavivirus-like genomes in the GB hepatitis agent.

A subtractive PCR methodology known as representational difference analysis was used to clone specific nucleotide sequences present in the infectious plasma from a tamarin infected with the GB hepatitis agent. Eleven unique clones were identified, seven of which were examined extensively. All seven clones appeared to be derived from sequences exogenous to the genomes of humans, tamarins, Saccharomyces cerevisiae, and Escherichia coli. In addition, sequences from these clones were not detected in plasma or liver tissue of tamarins prior to their inoculation with the GB agent. These sequences were detected by reverse transcription-PCR in acute-phase plasma of tamarins inoculated with the GB agent. Probes derived from two of the seven clones detected an RNA species of > or = 8.3 kb in the liver of a GB-agent-infected tamarin by Northern blot hybridization. Sequence analysis indicated that five of the seven clones encode polypeptides that possess limited amino acid identity with the nonstructural proteins of hepatitis C virus. Extension of the sequences found in the seven clones revealed that plasma from an infected tamarin contained two RNA molecules > 9 kb long. Limited sequence identity with various isolates of hepatitis C virus and the relative positions of putative RNA helicases and RNA-dependent RNA polymerases in the predicted protein products of these molecules suggested that the GB agent contains two unique flavivirus-like genomes.

Synthesis of "Nod"-like chitin oligosaccharides by the Xenopus developmental protein DG42.

The Xenopus DG42 gene is expressed only between the late midblastula and neurulation stages of embryonic development. Recent database searches show that DG42 has striking sequence similarity to the Rhizobium NodC protein. NodC catalyzes the synthesis of chitin oligosaccharides which subsequently are transformed into bacterium-plant root signaling molecules. We find that the DG42 protein made in an in vitro coupled transcription-translation system catalyzes the synthesis of an array of chitin oligosaccharides. The result suggests the intriguing possibility that a bacterium-plant type of "Nod" signaling system may operate during early stages of vertebrate embryonic development and raises issues about the use of chitin synthase inhibitors as fungal-specific drugs.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.