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E Appella

Publications and source records attributed to E Appella.

At least 235 records · Page 13Linked to original sources
PubMed1989

Prediction of major histocompatibility complex binding regions of protein antigens by sequence pattern analysis.

We have previously experimentally analyzed the structural requirements for interaction between peptide antigens and mouse major histocompatibility complex (MHC) molecules of the d haplotype. We describe here two procedures devised to predict specifically the capacity of peptide molecules to interact with these MHC class II molecules (IAd and IEd). The accuracy of these procedures has been tested on a large panel of synthetic peptides of eukaryotic, prokaryotic, and viral origin, and also on a set of overlapping peptides encompassing the entire staphylococcal nuclease molecule. For both sets of peptides, IAd and IEd binding was successfully predicted in approximately 75% of the cases. This suggests that definition of such sequence "motifs" could be of general use in predicting potentially immunogenic peptide regions within proteins.

Amino Acid Sequence↗
PubMed1989

Identification and characterization of a p53 gene mutation in a human osteosarcoma cell line.

The nuclear phosphoprotein p53 occurs at elevated levels in many transformed cells. Mutant forms of mouse p53 possess enhanced transforming activity compared with wild type p53. Mutant mouse p53 proteins form complexes with the 70 kDa family of heat shock proteins (HSPs). We previously demonstrated an association between p53 and the 70 kDa HSPs in the human osteosarcoma (HOS) derived cell line HOS-SL. We report here the molecular cloning and sequencing of the p53 gene from HOS-SL cells, and demonstrate that it is in fact mutant. Further, analysis of similar HOS-derived cell lines demonstrates that they also encode the same mutant form of p53, whereas the wild type form of p53 appears to be lost in these cells. Stability studies demonstrate an increased half life of the p53 protein in these cells, in keeping with its association with the HSP 70 proteins. A potential role for this p53 mutant in the transformation process is discussed.

Base Sequence↗
PubMed1988

Mechanisms influencing the immunodominance of T cell determinants.

The preferential recognition of certain amino acid sequences from foreign protein antigens by T cells is referred to as T cell epitope immunodominance. To determine the mechanisms underlying this phenomenon, we have studied the correlation between the interaction of a series of synthetic peptides encompassing the entire hen egg-white lysozyme (HEL) sequence with class II molecules of the H-2k haplotype, and T cell responsiveness to these peptides. After HEL priming, three immunodominant T cell epitopes were found: two, included in the HEL sequences 51-61 and 112-129, were recognized in association with I-Ak molecules, and one, included in sequence 1-18, in association with I-Ek molecules. Accordingly, these peptides bound to the appropriate class II molecule, as demonstrated by competition for antigen presentation. Several other HEL peptides, although capable of associating with class II molecules, were not immunodominant. The absence of immunodominance has been shown to arise by three different mechanisms: (a) competition by an immunodominant peptide for presentation in vivo, (b) failure to generate the peptide during antigen processing, and (c) an inherently poor capacity of the T cell repertoire to respond to a particular peptide-MHC complex.

Animals↗
PubMed1988

Synthesis and biological characterization of monocyte-derived neutrophil chemotactic factor.

MDNCF is a human monocyte-derived, 72-residue chemotactic peptide, which has sequence similarity with members of a family of pro-inflammatory cytokines. The peptide was synthesized by the solid-phase method, and is identical to the natural peptide in amino acid composition, sequence and chemotactic potency. MDNCF forms two loops via a neighboring pair of disulfide bridges, the probable locations of which are residues 7-34 and 9-50. Reduction and alkylation eliminated chemotactic activity. MDNCF fragments 7-37, 30-72 and 17-72 were all biologically inactive. The data suggest that the region of the clustered pair of disulfide bridges is important for biological activity.

Alkylation↗
PubMed1988

Enzymatic activity and filament assembly of Acanthamoeba myosin II are regulated by adjacent domains at the end of the tail.

Polyclonal antibodies raised against a synthetic peptide consisting of the last 19 amino acids at the end of the coiled-coil region of the heavy chains inhibited the actin-activated Mg2+-ATPase activity of myosin II and its ability to form filaments. Antibodies against a synthetic peptide corresponding to the 21 adjacent amino acids at the beginning of the non-helical tailpiece, which include the three regulatory phosphorylatable serines, had no effect on either activity.

Acanthamoeba↗
PubMed1988

The three-dimensional structure of a DNA duplex containing looped-out bases.

Unpaired bases in DNA have been assigned a possible role in the mechanism of frameshift mutagenesis in sequences with repeated base pairs. They also occur in quasipalindromic DNA sequences, which have been implicated in mutagenesis where there are no repeated base pairs, through the formation of single-stranded hairpin loops. The conformation of unpaired bases in DNA has been the subject of numerous thermodynamic as well as high resolution NMR (nuclear magnetic resonance) studies (reviewed in ref. 4). The NMR studies in solution have shown that the duplex of the tridecamer DNA fragment d(CGCAGAATTCGCG) remains intact, and that the unpaired adenosines are stacked into the duplex. Having crystallized this oligonucleotide and determined its structure, we find its conformation in the crystal is close to that of a B-DNA duplex, with the two additional adenosines looped out from the double helix and causing little disruption of the rest of the structure.

Base Composition↗
PubMed1988

Crystal structure of 15-mer DNA duplex containing unpaired bases.

Errors during DNA replication or repair can lead to the presence of unpaired or inserted bases in the double helix, as well as to mismatched base pairs. So far only structures of the latter type have been characterized by X-ray crystallography. We report here a 3-A crystal structure of DNA 15-mer d(CGCGAAATTTACGCG), which forms a duplex with two unpaired adenine residues looped outside the B-type helix. This arrangement is in disagreement with the nuclear magnetic resonance spectroscopy results for the same 15-mer in solution, indicating polymorphic nature of the structure adopted by this sequence.

Adenosine↗
PubMed1988

Structures of amidohydrolases. Amino acid sequence of a glutaminase-asparaginase from Acinetobacter glutaminasificans and preliminary crystallographic data for an asparaginase from Erwinia chrysanthemi.

The complete amino acid sequence of a glutaminase-asparaginase from Acinetobacter glutaminasificans, for which a preliminary tertiary structure is available from crystallographic analysis, has been determined by automated Edman degradation of fragments produced by chemical and proteolytic cleavages. The protein consists of 331 amino acid residues and has a molecular weight of 35,500. The pattern of hydrophilic and hydrophobic regions is typical of a globular protein. A new crystal form of an Erwinia chrysanthemi 1125 asparaginase is reported. The space group is monoclinic C2, with unit cell parameters of: a = 107.8, b = 91.7, c = 129.2 A and beta = 91.7 degrees. A Vm of 2.25 A3/dalton was calculated for one tetramer of 35,100-dalton subunits per asymmetric unit. X-ray intensity data have been obtained to 2.2 A resolution. The point group symmetry of the Er. chrysanthemi tetramer is 222 from self-rotation function calculations. The relative orientations of an A. glutaminasificans glutaminase-asparaginase model and the Er. chrysanthemi asparaginase tetramer have been determined with the cross-rotation function, and translation function calculations have revealed a plausible location for the asparaginase tetramer in the crystal.

Acinetobacter↗
PubMed1988

Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor.

The cDNA coding for human monocyte-derived neutrophil-specific chemotactic factor (MDNCF) was cloned from LPS-stimulated human monocyte mRNA. The cDNA sequence codes for a polypeptide consisting of 99 amino acids, including a putative signal sequence. Comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of natural MDNCF shows that the mature functional protein comprises 72 amino acids, beginning with serine at residue 28. The deduced amino acid sequence shows striking similarity to several platelet-derived factors, a v-src-induced protein, a growth-regulated gene product (gro), and an IFN-gamma inducible protein. The availability of the MDNCF cDNA enabled us to use it as a probe to identify inducers of MDNCF mRNA expression in human PBMC. MDNCF mRNA was increased greater than 10-fold within 1 h after stimulation with LPS, IL-1, or TNF, but not by IFN-gamma, IFN-alpha, or IL-2. Furthermore, we also determined that LPS, IL-1, and TNF stimulated the mononuclear cells to produce biologically active MDNCF. This observation may account for the in vivo capacity of IL-1 and TNF to induce netrophil infiltrates.

Amino Acid Sequence↗
PubMed1988

Phosphorylation of intracellular precursors of human IL-1.

The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.

Amino Acid Sequence↗
PubMed1988

Modulation of metastatic potential by cell surface urokinase of murine melanoma cells.

We have carried out enzymatic, immunofluorescence, and surface iodination studies which show that B16 melanoma cells express the single chain form of the urokinase type plasminogen activator (uPA) on their cell surface, and that these cells are capable of plasminogen-dependent fibronectin degradation. The significance of the expression of surface single-chain uPA and uPA activity to the metastatic process was examined by preincubating melanoma cells with uPA modulating agents followed by i.v. injection of the cells into mice and enumeration of pulmonary nodules 17 days later. B16 cells that had been pretreated with anti-uPA immunoglobulins that were inhibitory to uPA activity invariably showed significantly decreased numbers of metastases compared to controls. On the contrary, pretreatment with plasmin, which is not only the product of the uPA catalyzed reaction but is also able to convert single-chain uPA to uPA, significantly increased the numbers of metastases. Control treatments, which included normal rabbit and mouse immunoglobulins, monovalent noninhibitory anti-uPA Fab fragments, and various monoclonal and polyclonal antibodies directed against other B16 cell surface antigens, did not affect the metastatic potential of the cells. Divalent inhibitory anti-uPA F(ab)2 fragments, on the contrary, inhibited metastasis as efficiently as intact IgG. The results support the hypothesis that proteolysis of extracellular matrix components by cell surface-localized uPA may be a critical step during the process of tumor cell invasion and metastasis.

Animals↗
PubMed1988

Growth-related expression of a 72,000 molecular weight poly(A)+ mRNA binding protein.

In this communication, we have studied a 72,000 mol w (p72) host protein which reacts with a mouse monoclonal antibody (PAb6) directed against antigenic determinants on the Simian virus 40 (SV40) large T antigen protein that map 5' of 0.42 map units on the viral genome. The p72 protein is an abundant basic (pI greater than 7) cytoplasmic protein found in both SV40-transformed and untransformed parental cells and in cell lines derived from normal human and tumor tissue. By two-dimensional gel analysis and Western blot analysis the p72 protein identified by PAb6 is indistinguishable from the 72,000 mol w protein PABP associated with the poly(A)+ tract of cytoplasmic messenger RNA molecules. In normal human peripheral blood mononuclear cells stimulated to proliferate with the T-cell-specific mitogenic lectin phytohemagglutinin the synthesis and cytoplasmic accumulation of p72 occurs very early during the G0----G1-phase transition. The p72 protein is also expressed in proliferating and differentiated human promyelocytic HL60 cells indicating that the expression of this protein is not strictly limited to cycling cells.

Animals↗
PubMed1988

Interaction of an immunodominant epitope with Ia molecules in T-cell activation.

The amino acid sequence corresponding to residues 107-116 of hen egg-white lysozyme (HEL) has been identified as containing an immunodominant T-cell epitope recognized in association with the I-Ed molecule. The immunodominance of this epitope in HEL-primed H-2d mice was demonstrated by analysis of the T-cell proliferative response induced by synthetic peptides covering almost the entire HEL sequence. All the T-cell hybridomas from H-2d mice analyzed recognize the HEL sequence 107-116 in association with the I-Ed molecule. Correlating with the restriction of T-cell recognition, HEL-(105-120)-peptide binds to I-Ed but not to I-Ad molecules. Conservative or semiconservative substitutions at positions 113 (Asn----Lys), 114 (Arg----His), or 115 (Cys----Ala) abrogate the ability of HEL-(105-120) to activate T cells. Substitutions at residues 113 and 115 affect T-cell recognition but not the binding to I-Ed molecules, whereas, as shown by binding data and competition experiments, an Arg----His substitution at position 114 profoundly impairs the capacity of the peptide to interact with I-Ed molecules. In agreement with these results, [Lys113]HEL-(105-120)-peptide but not [His114]HEL-(105-120)-peptide was found to be immunogenic in H-2d mice. Thus, a single semiconservative substitution drastically reduces binding capacity and abolishes immunogenicity, suggesting that a strict correlation exists between binding of a peptide to Ia molecules and its immunogenicity.

Amino Acid Sequence↗
PubMed1988

Interferon-induced transcription of a major histocompatibility class I gene accompanies binding of inducible nuclear factors to the interferon consensus sequence.

Interferon (IFN) induces transcription of major histocompatibility class I genes by way of the conserved cis-acting regulatory element, termed the IFN consensus sequence (ICS). Binding of nuclear factors to the ICS was studied in gel mobility shift assays with the 5' upstream region of the murine H-2Ld gene. We found that the ICS binds a constitutive nuclear factor present in lymphocytes and fibroblasts regardless of IFN treatment. Within 1 hr after IFN treatment, new ICS binding activity was induced, which consisted of at least two binding activities distinguished by their requirement for de novo protein synthesis. Methylation interference and competition experiments showed that both constitutive and induced factors bind to the same approximately equal to 10-base-pair binding site within the ICS. Site-directed mutagenesis of H-2Ld-chloramphenicol acetyltransferase fusion genes showed that mutations in the binding site, but not in other regions of the ICS, abolish transcriptional activation of class I genes by IFN, providing evidence that specific binding of nuclear factors to the ICS is an essential requirement for transcriptional induction. Finally, we show that IFN-inducible genes of various species share a sequence motif that is capable of competing for the nuclear factors identified here. We propose that specific protein binding to the conserved motif represents a basic mechanism of IFN-mediated transcriptional induction of a number of genes.

Amino Acid Sequence↗
PubMed1988

Induction of mitochondrial manganese superoxide dismutase by interleukin 1.

Interleukin 1 (IL 1) inhibits the growth of human melanoma A375 cells. To identify the subcellular events preceding inhibition of growth by IL 1, we have examined the effect of IL 1 on protein synthesis caused by A375 cells. IL 1 selectively and predominantly induced a 25-kDa polypeptide (p25) in A375 cells after 12 h. On subcellular fractionation, p25 was exclusively located in the 10,000 x g-pelleted (mitochondria-enriched) fraction. To identify the p25 moiety, it was purified to homogeneity by sequential chromatography on DEAE-Sephacel and reverse-phase, high-pressure liquid chromatography and its amino-terminal amino acid sequence was determined. The sequence of the 35 amino-terminal amino acids of the p25 moiety was identical to that of human manganese superoxide dismutase (Mn SOD). The enzymatic activities of SOD were induced only in the mitochondria-enriched fraction of IL 1-treated A375 cells. However, IL 1 also induced Mn SOD in normal human skin fibroblasts and peripheral blood mononuclear cells, whose growth was stimulated by IL 1. The results show that induction of Mn SOD by IL 1 is a common biochemical event in IL 1-responsive cells.

Cytosol↗
PubMed1988

Lack of influence of cyclosporine on antigen presentation to lysozyme-specific T cell hybridomas.

Cyclosporine (CsA) was tested for its ability to inhibit antigen presentation by spleen cells or the B lymphoma line A20 to T cell hybridomas specific for hen egg-white lysozyme (HEL). Antigen-presenting cells (APC) were treated with CsA or nonimmunosuppressive derivatives thereof during or prior to encounter with antigen. The APC were then washed extensively and incubated in CsA-free medium for 6 hr before the T hybridoma cells were added. Under these conditions, CsA had no effect on antigen presentation up to the cytostatic regimen (1 microgram/ml). Omission of the 6-hr interval between CsA treatment of APC and the addition of T hybridoma resulted in inhibition of interleukin 2 production, although the CsA concentrations required were 10-75-fold higher than the ones inhibiting T cells directly (IC50: 100-150 ng/ml vs. 2-10 ng/ml). The responses to both HEL and a synthetic peptide of HEL sequence 105-120 were inhibited, indicating that the step influenced by the drug was not antigen-processing. The nonimmunosuppressive derivatives remained ineffective under these conditions. The results illustrate that the carryover of CsA from APC to T cells can mimic a drug effect on antigen presentation. Therefore, the demonstration of a CsA effect on antigen presentation can only be considered as conclusive when the readout of APC function is not a T cell response.

Animals↗
PubMed1988

Specific interaction between a subset of the p53 protein family and heat shock proteins hsp72/hsc73 in a human osteosarcoma cell line.

We report here immunological evidence for the specific association between p53 and hsp72/hsc73 heat shock proteins in a human cell line derived from an osteosarcoma. The same association between p53 and hsp72/hsc73 was observed in HOS-TE85 clone 5 from which the HOS-SL cell line was derived. This association was indicated by the co-immunoprecipitation from HOS-SL of both p53 and hsp72/hsc73 proteins observed with either an anti-p53 monoclonal antibody (PAb421) or an antiserum specifically reacting with hsp72/hsc73 heat shock proteins. Furthermore, Western blot analysis allowed us to show that hsp72/hsc73 proteins did not share an epitope with p53, confirming that the co-immunoprecipitation of p53 and hsp72/hsc73 was attributable to the physical association of the proteins. Data obtained from SDS-PAGE show that the HOS-SL cells expressed two forms of p53 with distinct molecular weights. Both forms contained several species with different isoelectric points ranging between pH 6.0 and 6.5. The data obtained from both 1D and 2D gel analyses consistently show that the p53 proteins involved in the association with hsp72/hsc73 were mainly the species that migrated with the slower mobility in the SDS dimension. The possibility is discussed that the HOS-SL p53 variant forming a complex with hsp72/hsc73 contains an activating mutation for transformation.

Antibodies, Monoclonal↗