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.

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.

Purification and properties of transgelin: a transformation and shape change sensitive actin-gelling protein.

We have purified the transformation and shape change sensitive isoform of an actin associated polypeptide doublet previously described by us (Shapland, C., P. Lowings, and D. Lawson. 1988. J. Cell Biol. 107:153-161) and have shown that it is evolutionarily conserved as far back as yeast. The purified protein: (a) binds directly to actin filaments at a ratio of 1:6 actin monomers, with a binding constant (Ka) of approximately 7.5 x 10(5) M-1; and (b) causes actin filament gelation within 2 min. Although these activities are controlled by ionic strength (and may be mediated by positively charged amino acid residues) the molecule remains as a monomer irrespective of ionic conditions. EM reveals that the addition of this protein to actin filaments converts them from a loose, random distribution into a tangled, cross-linked meshwork within 1 min, and discrete tightly aggregated foci after 10 min. By use of an "add-back" cell permeabilization system we can rebind this molecule specifically to actin filaments in cells from which it has previously been removed. Since the protein is transformation sensitive and gels actin, we have named it transgelin.

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.

MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2.

The process of post-replicative DNA-mismatch repair seems to be highly evolutionarily conserved. In Escherichia coli, DNA mismatches are recognized by the MutS protein. Homologues of the E. coli mutS and mutL mismatch-repair genes have been identified in other prokaryotes, as well as in yeast and mammals. Recombinant Saccharomyces cerevisiae MSH2 (MSH for MutS homologue) and human hMSH2 proteins have been shown to bind to mismatch-containing DNA in vitro. However, the physiological role of hMSH2 is unclear, as shown by the recent finding that the mismatch-binding factor hMutS alpha isolated from extracts of human cells is a heterodimer of hMSH2 and another member of the MSH family, GTBP. It has been reported that S. cerevisiae possesses a mismatch-binding activity, which most probably contains MSH2. We show here that, as in human cells, the S. cerevisiae binding factor is composed of MSH2 and a new functional MutS homologue, MSH6, identified by its homology to GTBP.

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 of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase.

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNA-specific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain.

A family of AP-2 proteins regulates c-erbB-2 expression in mammary carcinoma.

The proto-oncogene c-erbB-2 is overexpressed in 25-30% of breast cancers through increased transcription and amplification of the gene. We have previously described a factor, OB2-1 which upregulates c-erbB-2 transcription and which is closely related to the developmentally regulated transcription factor, AP-2. Further analysis of affinity purified OB2-1 has now shown that it is in fact a combination of proteins from three AP-2-related genes, the previously described AP-2alpha gene and two new human family members, AP-2beta and AP-2gamma whose cloning and characterisation are described here. All three AP-2 proteins show a high degree of homology and are capable of binding to the c-erbB-2 promoter as homo- or heterodimers. The three proteins can also activate a c-erbB-2 reporter construct, but AP-2alpha and AP-2gamma are 3-4 times more active in this regard than AP-2beta. In addition both AP-2alpha and AP-2gamma are expressed at elevated levels in the majority of c-erbB-2 overexpressing mammary tumour lines examined. Mechanisms which may have led to the increased AP-2 levels in these cells are discussed.

Purification and identification by amino acid sequence analysis of the subunits of the CD3 antigen of human tonsil T-lymphocytes.

The CD3 antigen was purified from human tonsils by immunoaffinity chromatography and preparative SDS-PAGE; the overall yield was 10%. Amino acid sequence analysis of the separated gamma, delta and epsilon polypeptides revealed that the gamma and epsilon polypeptides were blocked at the N-terminus, whereas the partial N-terminal amino acid sequence for the delta chain was identical to that described by Borst et al. [Nature 312, 455-485 (1984)]. The gamma and epsilon chains were cleaved with formic acid and cyanogen bromide respectively in order to obtain amino acid sequence data. The sequences obtained corresponded exactly to the amino acid sequences deduced from the nucleotide sequences of putative gamma and epsilon cDNA clones.

Accelerated high-sensitivity microsequencing of proteins and peptides using a miniature reaction cartridge.

Greatly reduced automated protein sequencing degradation times of less than 25 min with concurrent on-line phenylthiohydantoin (PTH) derivative analysis times of less than 16 min have been achieved by using a miniaturized reaction cartridge with optimized chemical and analytical cycles. Using this method a wide range of standard and novel peptides and proteins have been sequenced with reproducibly high initial and repetitive cycle yields. In these accelerated analyses the recovery of the more labile PTH derivatives was markedly improved by using elevated pressure during cleavage steps and temperature programming throughout the Edman cycle.

Purification and internal amino acid sequence of the 80 kDa protein kinase C substrate from Swiss 3T3 fibroblasts. Homology with substrates from brain.

The acidic 80 kDa protein kinase C (PKC) substrate was purified from 2.3 x 10(10) Swiss 3T3 fibroblasts. Partial amino acid sequence data were obtained from five peptides generated by S. aureus V8 cleavage of the protein, enabling a total of 91 amino acid residues to be assigned. The sequences of these five peptides were compared to the deduced amino acid sequences of acidic 80-87 kDa PKC substrates from both actively proliferating A431 epidermal carcinoma cells, and fully differentiated neural tissue. Despite their similar physical properties, there was no homology between the peptides derived from the fibroblast 80 kDa protein and the PKC substrate from A431 cells. However, there was 66% homology with the 87 kDa bovine brain protein within the regions covered by the peptides about 30% of the total protein). Furthermore, comparison of the peptides from the fibroblast 80 kDa protein with proteolytic peptides derived from the acidic 80 kDa rat brain protein revealed an overall homology of 89%. These data provide the first direct evidence that the 80 kDa PKC substrate from Swiss 3T3 fibroblasts is closely related to the 80-87 kDa PKC substrates detected in fully differentiated neural tissue.

A new apolipoprotein Al variant, Trp50Arg, causes hereditary amyloidosis.

A man with hereditary non-neuropathic systemic amyloidosis had amyloid fibril protein subunits consisting of N-terminal fragments (residues 1-86, 1-92 and 1-93) of a previously unknown variant of apolipoprotein Al, Trp50Arg, encoded by a thymine-cytosine transition. This is the third reported amyloidogenic apoAl variant. All involve substitutions of single neutral amino acids by the cationic residue arginine, suggesting a common mechanism of amyloidogenesis. However, the phenotypic expression of these mutations varies both within and between the seven known families with hereditary apoAl amyloidosis. These findings should facilitate analysis of the molecular basis of fibrillogenesis and of factors that modulate amyloid deposition and its consequences 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.

Isolation, characterization and biological activity of a CRF-related diuretic peptide from Periplaneta americana L.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.

Isolation and characterization of grancalcin, a novel 28 kDa EF-hand calcium-binding protein from human neutrophils.

A novel 28 kDa protein, which we have named 'grancalcin', has been identified in human neutrophils. The protein was isolated from the cytosol and found to be a homodimer, with an apparent molecular mass of 55 kDa on gel filtration. Polyclonal antibodies were raised to the native protein. N-Terminal sequence analysis and tryptic-peptide sequence analysis was performed. The protein exhibits sequence similarity to sorcin, a 24 kDa calcium-binding protein over-expressed in certain multi-drug-resistant cell lines. It appears to be a member of the EF-hand family of calcium-binding proteins. The association of a high proportion of this protein with the membranes and granules in the presence of physiological concentrations of calcium may indicate a role in granule-membrane fusion and degranulation.

Identification and functional characterisation of the cellular activating transcription factor 43 (ATF-43) protein.

The promoter motif CGTCA binds multiple cellular factors that mediate a variety of inducible events, including positive responses to raised cellular levels of cAMP and to the Adenovirus E1a protein. To date, at least ten mammalian cDNA clones have been isolated that encode distinct proteins capable of binding to this motif. However, in most cases the precise stimuli that may regulate these different factors have yet to be determined. We have previously shown that the abundant Hela protein ATF-43 forms a complex in vivo with the cyclic AMP response element binding protein (CREB). In this report we definitively show that ATF-43 is the product of the two published cDNA clones, ATF1 and TREB 36. We confirm that ATF1 efficiently heterodimerises with CREB and demonstrate that even though ATF1 and CREB homodimers, as well as the ATF1/CREB heterodimer efficiently bind to the CGTCA motif, the resulting DNA-protein complexes have significantly different stabilities. A region outside the DNA binding domain of ATF1 contributes to the instability of its interaction with DNA. We further show that despite ATF1's homology to CREB, it responds poorly to activation by protein kinase A. In light of our finding that in Hela cells the majority of CREB protein is heterodimerised with ATF1, we speculate on the functional significance of such heterodimers.

Alpha-enolase: a novel cytosolic autoantigen in ANCA positive vasculitis.

Anti-neutrophil cytoplasmic antibodies (ANCA) in sera from patients with clinically proven vasculitis have been described as reacting with proteins present in the granules of human neutrophils. We have studied sera from 59 ANCA positive patients to further characterize the antibody response. In addition to the antigens previously identified in the vasculitic syndromes (myeloperoxidase and serine proteinase 3) the majority of these sera contained antibodies that reacted with a cytosolic extract of neutrophils on Western blots. Nearly 40% of these sera had antibodies directed against a cytosolic protein(s) of molecular mass 48 kD. This protein was purified from neutrophil cytosol by ammonium sulphate fractionation, anion exchange and reverse phase chromatography. Amino acid sequence analysis of a proteolytic fragment of this protein identified it as alpha enolase. The anti-enolase antibodies only recognized the alpha isoform and were present in sera giving either a pANCA or cANCA staining pattern by indirect immunofluorescence. Antibodies to alpha enolase were also found in sera from patients with systemic lupus erythematosus, particularly those with renal disease. We conclude that the antibody response in ANCA positive vasculitis is not restricted to neutrophil granule proteins.

Three distinct DNA ligases in mammalian cells.

The major DNA ligase of proliferating mammalian cells, DNA ligase I, catalyzes the joining of single strand breaks in double stranded DNA and is active on a synthetic substrate of oligo(dT) hybridized to poly(dA). DNA ligase I does not catalyze the joining of an oligo(dT).poly(rA) substrate. Two additional DNA ligases, II and III, which can act on the latter substrate have been purified from calf thymus. DNA ligase II, which has been described previously, is a 72-kDa protein. DNA ligase III migrates as a 100-kDa protein in denaturing gel electrophoresis. Structural, immunochemical, and catalytic studies on the three DNA ligase activities strongly indicate that they are the products of three different genes.

Location of the active site for enzyme-adenylate formation in DNA ligases.

The enzyme-AMP reaction intermediate of the 102-kDa bovine DNA ligase I was digested with trypsin, and the adenylylated peptide was isolated by chromatography under conditions that maintain the acid-labile phosphoramidate bond. Microsequencing of the peptide showed that it contains an internal trypsin-resistant lysine residue, as expected for the site of adenylylation. Inhibition of DNA ligase I activity by pyridoxal 5'-phosphate also indicated the presence of a reactive lysine residue in the catalytic domain of the enzyme. Comparison of the known primary structures of several other DNA ligases with the adenylylated region of mammalian DNA ligase I allows their active sites to be tentatively assigned by sequence homology. The ATP-dependent DNA ligases of mammalian cells, fission yeast, budding yeast, vaccinia virus, and bacteriophages T3, T4, and T7 contain the active site motif Lys-Tyr/Ala-Asp-Gly-(Xaa)-Arg, with the reactive lysine residue flanked by hydrophobic amino acids. The distance between the postulated adenylylation site and the carboxyl terminus of the polypeptide is very similar in these ATP-dependent DNA ligases, whereas the size of the amino-terminal region is highly variable.

Homology between the subunits of S100 and a 10kDa polypeptide associated with p36 of pig lymphocytes.

The polypeptide of about 10,000-Mr which is associated with the p36 polypeptide of pig mesenteric lymph node lymphocytes has a N-terminal 51 amino acid sequence that is 56 and 42% homologous with the N-terminii of the alpha and beta subunits respectively of S100.