UVB radiation and 17-hydroxygeranyllinalool diterpene glycosides provide durable resistance against mirid (Tupiocoris notatus) attack in field-grown Nicotiana attenuata plants.

Depending on geographical location, plants are exposed to variable amounts of UVB radiation and herbivore attack. Because the role(s) of UVB in the priming and/or accumulation of plant defence metabolites against herbivores are not well understood, we used field-grown Nicotiana attenuata plants to explore the effects of UVB on herbivore performance. Consistent with previous reports, UVB-exposed plants accumulated higher levels of ultraviolet (UV)-absorbing compounds (rutin, chlorogenic acid, crypto-chlorogenic acid and dicaffeoylspermidine). Furthermore, UVB increased the accumulation of jasmonic acid, jasmonoyl-L-isoleucine and abscisic acid, all phytohormones which regulate plant defence against biotic and abiotic stress. In herbivore bioassays, N. attenuata plants experimentally protected from UVB were more infested by mirids in three consecutive field seasons. Among defence metabolites measured, 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) showed strongly altered accumulation patterns. While constitutive HGL-DTGs levels were higher under UVB, N. attenuata plants exposed to mirid bugs (Tupiocoris notatus) had still more HGL-DTGs under UVB, and mirids preferred to feed on HGL-DTGs-silenced plants when other UVB protecting factors were eliminated by UVB filters. We conclude that UVB exposure not only stimulates UV protective screens but also affects plant defence mechanisms, such as HGL-DTGs accumulation, and modulates ecological interactions of N. attenuata with its herbivores in nature.

The major phosphorylation site of the NADPH oxidase component p67phox is Thr233.

Phosphorylation of p67phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC-MS suggested that Thr233 was the phosphorylated residue. Mutagenesis of Thr233 to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr233 has been identified as the major phosphorylation site of p67phox, which is situated in a proline-rich domain.

Identification of regions of the Wiskott-Aldrich syndrome protein responsible for association with selected Src homology 3 domains.

Src homology 3 (SH3) domains have been shown to mediate selected interactions between signaling molecules and are essential for the activation of a number of receptor-driven pathways. The Wiskott-Aldrich syndrome protein was identified as a protein that associated selectively with the SH3 domains derived from c-Src, p85alpha, phospholipase Cgamma1, and c-Fgr. Significantly reduced association was detected to the N-terminal SH3 domain and the tandem SH3 domains of p47(phox), and no binding was detected to the SH3 domain of n-Src, the C-terminal SH3 domain of p47(phox), or either of the SH3 domains of p67(phox). Three peptides corresponding to potential Wiskott-Aldrich syndrome protein SH3 domain binding motifs were found to inhibit its association with c-Src, Fgr, and phospholipase Cgamma1 SH3 domains, but not the p85alpha SH3 domain. These peptides have the sequences MRRQEPLPPPPPPSRG, TGRSGPLPPPPPGA, and KGRSGPLPPVPLGI and show homology with other SH3 domain binding motifs. It is possible that the intracellular association of Wiskott-Aldrich syndrome protein with other signaling proteins is mediated by its SH3 domain-binding regions, and this may play a role in its putative function as a regulatory molecule in immune cells.

Hepatocyte nuclear factor 6, a transcription factor that contains a novel type of homeodomain and a single cut domain.

Tissue-specific transcription is regulated in part by cell type-restricted proteins that bind to defined sequences in target genes. The DNA-binding domain of these proteins is often evolutionarily conserved. On this basis, liver-enriched transcription factors were classified into five families. We describe here the mammalian prototype of a sixth family, which we therefore call hepatocyte nuclear factor 6 (HNF-6). It activates the promoter of a gene involved in the control of glucose metabolism. HNF-6 contains two different DNA-binding domains. One of these corresponds to a novel type of homeodomain. The other is homologous to the Drosophila cut domain. A similar bipartite sequence is coded by the genome of Caenorhabditis elegans.

Wiskott-Aldrich syndrome protein (WASp) is a binding partner for c-Src family protein-tyrosine kinases.

BACKGROUND: Receptor-mediated signal transduction requires the assembly of multimeric complexes of signalling proteins, and a number of conserved protein domains, such as the SH2, SH3 and PH domains, are involved in mediating protein-protein interactions in such complexes. The identification of binding partners for these domains has added considerably to our understanding of signal-transduction pathways, and the purpose of this work was to identify SH3-binding proteins in haematopoietic cells. RESULTS: We performed affinity-chromatography experiments with a panel of GST-SH3 fusion proteins (composed of glutathione-S-transferase appended to various SH3 domains) to search for SH3-binding proteins in a human megakaryocytic cell line. Protein microsequencing identified one of the SH3-binding proteins as WASp, the protein that is defective in Wiskott-Aldrich syndrome (WAS) and isolated X-linked thrombocytopenia. WASp bound preferentially in vitro to SH3 domains from c-Src family kinases, and analysis of proteins expressed in insect cells using a baculovirus vector demonstrated a specific interaction between WASp and the Fyn protein-tyrosine kinase. Finally, in vivo experiments showed that WASp and Fyn physically associate in human haematopoietic cells. CONCLUSIONS: Haematopoietic cells from individuals with WAS exhibit defects in cell morphology and signal transduction, including reduced proliferation and tyrosine phosphorylation in response to stimulatory factors. Members of the c Src family of protein-tyrosine kinases, including Fyn, are involved in a range of signalling pathways - such as those regulating cytoskeletal structure - in both haematopoietic and non-haematopoietic cells. Our data suggest that binding of Fyn to WASp may be a critical event in such signalling pathways in haematopoietic cells.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

Cloning and expression of human G/T mismatch-specific thymine-DNA glycosylase.

Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3'-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of base-excision repair enzymes.

Identification of a specific Ins(1,3,4,5)P4-binding protein as a member of the GAP1 family.

Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) is produced rapidly from inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in stimulated cells. Despite extensive experimentation, no clearly defined cellular function has yet been described for this inositol phosphate. Binding sites specific for Ins(1,3,4,5)P4 have been identified in several tissues, and we have purified one such protein to homogeneity. Its high affinity for Ins(1,3,4,5)P4, and its exquisite specificity for this isomeric configuration, suggest it may be an Ins(1,3,4,5)P4 receptor. Here we report the cloning and characterization of this protein as a GTPase-activating protein, specifically a member of the GAP1 family. In vitro it shows GAP activity against both Rap and Ras, but only the Ras GAP activity is inhibited by phospholipids and is specifically stimulated by Ins(1,3,4,5)P4.

The cloning and sequence of the C isoform of PtdIns4P 5-kinase.

In this study we describe the purification and sequencing of the C isoform of platelet PtdIns4P 5-kinase. Subsequently a cDNA was isolated from a human circulating-leucocyte library, which when sequenced was shown to contain all of the peptides identified in the purified protein. In addition, expression of this cDNA in bacteria led to the production of a protein which was recognized by specific monoclonal antibodies raised to the bovine brain enzyme [Brooksbank, Hutchings, Butcher, Irvine and Divecha (1993) Biochem. J. 291, 77-82] and also led to the appearance of PtdIns4P 5-kinase activity in the bacterial lysates. Interestingly, the cDNA showed no similarity to any of the previously cloned inositide kinases. A search of the DNA databases showed that two proteins from Saccharomyces cerevisiae shared close similarity to this enzyme, one of which, the mss4 gene product, has been implicated in the yeast inositol lipid pathway. These data suggest that the PtdIns4P 5-kinases are a new family of inositide kinases unrelated to the previously cloned phosphoinositide 3/4-kinases.

GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells.

DNA mismatch recognition and binding in human cells has been thought to be mediated by the hMSH2 protein. Here it is shown that the mismatch-binding factor consists of two distinct proteins, the 100-kilodalton hMSH2 and a 160-kilodalton polypeptide, GTBP (for G/T binding protein). Sequence analysis identified GTBP as a new member of the MutS homolog family. Both proteins are required for mismatch-specific binding, a result consistent with the finding that tumor-derived cell lines devoid of either protein are also devoid of mismatch-binding activity.

Interleukin 1 alpha activates two forms of p54 alpha mitogen-activated protein kinase in rabbit liver.

We have identified in rabbits two hepatic forms of T669 peptide kinases that are very strongly activated after systemic injection with the inflammatory cytokine interleukin 1 (IL-1). The T669 peptide contains a major phosphorylation site of the epidermal growth factor receptor, threonine 699 and is a substrate for mitogen-activated protein (MAP) kinases. The kinases were purified to homogeneity and corresponded to 50- and 55-kD proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequencing of 12 tryptic peptides of both kinases identified them as p54 MAP kinase alpha. This kinase belongs to the novel family of stress-activated protein kinases. This is the first evidence of IL-1 activating a specific protein kinase in vivo.

Primary localized orbital amyloidosis composed of the immunoglobulin gamma heavy chain CH3 domain.

1. Primary orbital amyloidosis is a rare form of localized amyloidosis in which the precursor protein has not previously been identified. We report here the first extraction of amyloid fibrils from a tissue biopsy containing orbital amyloid, and characterization of the fibril protein. 2. The N-terminal nine residues were identical with residues 278-286 and 275-283 of the third constant (CH3) domains of IgG1 and IgG4 gamma heavy chains, respectively. The mass of the fibril subunit protein was 6125Da by time-of-flight mass spectrometry, compared with the expected masses of 6169.9Da and 6214.9Da for the CH3 domains of gamma 1 from residue 278 and gamma 4 from residue 275, respectively. The fibril protein thus appeared to consist exclusively of an immunoglobulin heavy chain constant domain. 3. Only two examples of immunoglobulin heavy chain derived amyloid have been reported previously and both of these, as well as all published cases of the usual immunoglobulin light chain derived amyloid, contained variable domain sequence. The present case therefore represents a form of local, presumably clonal, B-cell/plasma-cell disorder characterized uniquely by deposition of an amyloidogenic immunoglobulin heavy chain constant domain fragment.

An SH3 domain and proline-rich sequence mediate an interaction between two components of the phagocyte NADPH oxidase complex.

Neutrophils possess a multicomponent NADPH oxidase system capable of producing large quantities of superoxide in a process known as the respiratory burst (1). Upon stimulation of a phagocytic cell, two cytosolic components of the oxidase, p67phox and p47phox, associate with a membrane-bound flavocytochrome b and a small GTP-binding protein to form a functional enzyme complex. Each of the Phox proteins contains two src homology 3 (SH3) domains, which are of unknown function but are potential mediators of protein-protein interactions between components of the activated oxidase. We have isolated a 47-kDa protein from lysates of differentiated HL60 cells that specifically bound to the carboxyl-terminal SH3 domain of p67phox and not to any other SH3 domain tested. This protein was identified as p47phox, and the putative SH3 domain binding site was located to a carboxyl-terminal proline-rich region. Proline-rich synthetic peptides based on this carboxyl-terminal region specifically inhibited the binding of p47phox to the carboxyl-terminal SH3 domain of p67phox, and sequential truncation defined a unique minimal sequence, which, although similar, does not match the consensus sequence defined for other SH3-binding proteins.

Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation.

Eosinophil accumulation is a prominent feature of allergic inflammatory reactions, such as those occurring in the lung of the allergic asthmatic, but the endogenous chemoattractants involved have not been identified. We have investigated this in an established model of allergic inflammation, using in vivo systems both to generate and assay relevant activity. Bronchoalveolar lavage (BAL) fluid was taken from sensitized guinea pigs at intervals after aerosol challenge with ovalbumin. BAL fluid was injected intradermally in unsensitized assay guinea pigs and the accumulation of intravenously injected 111In-eosinophils was measured. Activity was detected at 30 min after allergen challenge, peaking from 3 to 6 h and declining to low levels by 24 h. 3-h BAL fluid was purified using high performance liquid chromatography techniques in conjunction with the skin assay. Microsequencing revealed a novel protein from the C-C branch of the platelet factor 4 superfamily of chemotactic cytokines. The protein, "eotaxin," exhibits homology of 53% with human MCP-1, 44% with guinea pig MCP-1, 31% with human MIP-1 alpha, and 26% with human RANTES. Laser desorption time of flight mass analysis gave four different signals (8.15, 8.38, 8.81, and 9.03 kD), probably reflecting differential O-glycosylation. Eotaxin was highly potent, inducing substantial 111In-eosinophil accumulation at a 1-2 pmol dose in the skin, but did not induce significant 111In-neutrophil accumulation. Eotaxin was a potent stimulator of both guinea pig and human eosinophils in vitro. Human recombinant RANTES, MIP-1 alpha, and MCP-1 were all inactive in inducing 111In-eosinophil accumulation in guinea pig skin; however, evidence was obtained that eotaxin shares a binding site with RANTES on guinea pig eosinophils. This is the first description of a potent eosinophil chemoattractant cytokine generated in vivo and suggests the possibility that similar molecules may be important in the human asthmatic lung.

PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.

Phosphatidylinositol 3-kinase (PI 3-kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein-serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N-terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3-kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter-subunit serine phosphorylation in the regulation of the PI 3-kinase in vivo.

Isolation and characterization of a diuretic peptide common to the house fly and stable fly.

An identical CRF-related diuretic peptide (Musca-DP) was isolated and characterized from whole-body extracts of the house fly, Musca domestica, and stable fly, Stomoxys calcitrans. The peptide stimulates cyclic AMP production in Manduca sexta Malpighian tubules and increases the rate of fluid secretion by isolated Musca domestica tubules. The 44-residue peptide, with a mol.wt. of 5180, is amidated, and has the primary structure: NKPSLSIVNPLDVLRQRLLLEIARRQMKENTRQVELNRAILKNV-NH2. Musca-DP has a high percentage of sequence identity with other characterized CRF-related insect diuretic peptides.

Phospholipase D: a downstream effector of ARF in granulocytes.

Activation of the phospholipase D (PLD) pathway is a widespread response when cells are activated by agonists that bind receptors on the cell surface. A 16-kD cytosolic component can reconstitute guanosine triphosphate (GTP)-mediated activation of phospholipase D in HL60 cells depleted of their cytosol by permeabilization. This factor was purified and identified as two small GTP-binding proteins, ARF1 and ARF3. Recombinant ARF1 substituted for purified ARF proteins in the reconstitution assay. These results indicate that phospholipase D is a downstream effector of ARF1 and ARF3. The well-established role of ARF in vesicular traffic would suggest that alterations in lipid content by PLD are an important determinant in vesicular dynamics.

The GTPase dynamin binds to and is activated by a subset of SH3 domains.

Src homology 3 (SH3) domains have been implicated in mediating protein-protein interactions in receptor signaling processes; however, the precise role of this domain remains unclear. In this report, affinity purification techniques were used to identify the GTPase dynamin as an SH3 domain-binding protein. Selective binding to a subset of 15 different recombinant SH3 domains occurs through proline-rich sequence motifs similar to those that mediate the interaction of the SH3 domains of Grb2 and Abl proteins to the guanine nucleotide exchange protein, Sos, and to the 3BP1 protein, respectively. Dynamin GTPase activity is stimulated by several of the bound SH3 domains, suggesting that the function of the SH3 module is not restricted to protein-protein interactions but may also include the interactive regulation of GTP-binding proteins.

An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling.

Transmembrane signaling by the phospholipase C-beta (PLC-beta) pathway is known to require at least three components: the receptor, the G protein, and the PLC. Recent studies have indicated that if the cytosol is allowed to leak out of HL60 cells, then G protein-stimulated PLC activity is greatly diminished, indicating an essential role for a cytosolic component(s). We now report the complete purification of one component based on its ability to reconstitute GTP gamma S-mediated PLC activity and identify it as the phosphatidylinositol transfer protein (PI-TP). Based on the in vitro effects of PI-TP, we surmise that it is involved in transporting PI from intracellular compartments for conversion to PI bisphosphate (PIP2) prior to hydrolysis by PLC-beta 2/PLC-beta 3, the endogenous PLC isoforms present in these cells.