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E Appella

Publications and source records attributed to E Appella.

At least 289 records · Page 16Linked to original sources
PubMed1984

Amino acid sequence of a segment of the Acanthamoeba myosin II heavy chain containing all three regulatory phosphorylation sites.

The actin-activated Mg2+-ATPase activity of myosin II from the soil amoeba Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues on the myosin II heavy chain. Partial chymotryptic digestion of 32P-labeled myosin II cleaves from the tail end of the myosin II heavy chain a small peptide which contains all three phosphorylation sites. During purification the phosphorylated peptide is resolved into several different species as a result of heterogeneity both in phosphate content and in size (probably due to chymotryptic cleavage at the carboxyl terminus). However, all forms of the peptide have an identical amino terminus. The sequence of the first 58 residues of the peptide is: N-S-A-L-E-S-D-K-Q-I10-L-E-D-E-I-G-D-L-H- E20-K-N-K-Q-L-Q-A-K-I-A30-Q-L-Q-D-E-I-D-G-T- P40-S-S-R-G-G-S-T-R-G-A50-S-A-R-G-A-S-V-R. The phosphorylated serines are at positions 46, 51, and 56. The first 36 residues of the sequence display a repeating 3-4-3-4 pattern of hydrophobic residues suggesting that this section of the peptide forms an alpha-helical coiled-coil structure. A -Gly-Thr-Pro sequence at residues 38-40 disrupts the alpha-helix and, at the same point, the repeating pattern of non-polar residues is lost. It is likely that the residues extending from Gly-38 to the end of the myosin II tail, which include the 3 phosphorylatable serines, form a randomly coiled or small globular structure. This is the first report of the sequence around the regulatory phosphorylation sites on any myosin heavy chain.

Adenosine Triphosphatases↗
PubMed1984

Isolation of a tumor-associated transplantation antigen (TATA) from an SV40-induced sarcoma. Resemblance to the TATA of chemically induced neoplasms.

A tumor rejection antigen (TATA), obtained from the cytosol of a BALB/c mKSA sarcoma induced by SV40 virus, has been partially purified. This partially purified antigen is strikingly immunogenic against mKSA, providing more than 90% inhibition of growth at levels of 10-30 micrograms. This antigen preparation does not protect against challenge with another SV40-induced BALB/c sarcoma VLM which, however, shares a group-specific TATA with mKSA. The antigen also does not immunize against challenge with the methylcholanthrene-induced sarcomas of BALB/c mice, Meth A, CI-4, CII-7 and CII-10, each of which has its own unique TATA. Binding assays, using ELISA, failed to detect any SV40 antigen in the antigen preparation despite the fact that the large T antigen of SV40 (or fragments of it) constitutes the immunodominant TATA of mKSA.

Animals↗
PubMed1984

Role of a disulfide bridge in the immune function of major histocompatibility class I antigen as studied by in vitro mutagenesis.

Polymorphic major histocompatibility class I antigens have highly conserved disulfide bridges in the second and third external domains. To study the role of a disulfide bridge, we have introduced a mutation into the mouse H-2Ld gene by oligonucleotide-directed site-specific mutagenesis, disrupting the disulfide bridge in the second domain of the protein by changing cysteine at amino acid position 101 into serine. Upon introduction of the mutant gene into L cells, the mutant transplantation antigens were synthesized, inserted into the membrane, and displayed on the cell surface, indicating that the disulfide bridge is not essential for surface expression of the H-2 antigen. Binding studies carried out with 16 monoclonal antibodies specific for the H-2Ld antigen showed that most of the allodeterminants are lost or greatly altered in the mutant antigen. Further, almost complete loss of the recognition by H-2Ld-specific alloreactive cytotoxic T cells was observed. These results indicate that polymorphic determinants are dependent on a protein folding pattern dictated by the disulfide bridge. However, two antibodies previously found to react with antigenic sites present in the first and third domains were reactive with the mutant, implying an element of domain independence with respect to the determinants recognized by these antibodies.

Animals↗
PubMed1983

Identification of the c-myc oncogene product in normal and malignant B cells.

Antiserum to a synthetic peptide corresponding to the carboxyl-terminus of the human c-myc protein immunoprecipitated a 48,000-dalton protein from a number of normal and malignant human and mouse cells. The size of the protein is consistent with the potential coding region predicted from the c-myc nucleotide sequence, and is the same for malignant cells carrying either a rearranged or an unrearranged c-myc oncogene. Because c-myc transcripts are expressed at higher levels in malignant than in normal B cells, it appears that an increased level of the c-myc protein rather than a change in the gene product is the relevant factor in determining transformation.

B-Lymphocytes↗
PubMed1983

Major histocompatibility complex-controlled, antigen-presenting cell-expressed specificity of T cell antigen recognition. Identification of a site of interaction and its relationship to Ir genes.

In previous work (5,6), we have reported studies on a T lymphocyte hybridoma clone and the peritoneal exudate T cells (PETLES) from B10.A(5R) mice primed with the cytochrome c carboxyl terminal peptide (residues 81-103) of the tobacco horn worm moth (Manducca sextus). As expected, since B10.A(5R) is a low responder to pigeon fragment 81-104, it was found that the B10.A(5R) lymphocytes were unable to respond to the pigeon cytochrome c 81-104 fragment presented on syngeneic B10.A(5R) antigen-presenting cells (APC). However, these same T lymphocytes did respond to the pigeon fragment when presented on B10.A APC. Thus, some structural difference between the pigeon and moth peptides had prevented B10.A(5R) APC from effectively presenting the pigeon fragment to moth-primed B10.A(5R) lymphocytes. This structural difference was found to be the deletion of an alanine at position -103 (Ala103) from the pigeon sequence in the moth peptide. Two additional T cell specificities were created by changing residue-99. These T cell populations from the B10.A(5R) showed an identical dependence on the Ala103 deletion when B10.A and B10.A(5R) APC were compared. The relationship of APC-expressed antigen specificity and MHC-linked immune responsiveness differences was also examined. The B10.A(5R) was found to be a high responder to each of three peptides that lack Ala103 but not to the Ala103-containing analogues. B10.A mice, in contrast, respond to both types of peptides. Utilizing allogeneic antigen-presentation to B10.A PETLES by pulsed APC, it was shown that the poor response of the B10.A(5R) to the Ala103-containing peptides was, in two of three cases, not associated with any differences in T cell repertoires but due to two different APC capabilities of B10.A and B10.A(5R). The exception apparently represents a case of T cell repertoire polymorphism between B10.A and B10.A(5R) that can also affect immune responsiveness.

Animals↗
PubMed1983

Developmental activation of the H-2K gene is correlated with an increase in DNA methylation.

Embryonal carcinoma cell lines present strong developmental analogies with early embryonic cells. Previous studies have shown that treatment of F9 teratocarcinoma cells with retinoic acid induces the expression of the classical transplantation antigens which are indispensable for effective interactions between cells. In contrast to several genes that were analyzed, all of which were highly and heterogeneously methylated in F9 cells, the H-2K gene was poorly methylated if at all. Activation of the H-2K gene upon differentiation of F9 cells was accompanied by an increase in DNA methylation. While increased methylation of the H-2K gene in one of the two homologous chromosomes was correlated with a low level of expression, increased methylation in both chromosomes was associated with a high level of expression. Treatment of differentiated F9 cells with 5-azacytidine resulted in inhibition of DNA methylation and a concomitant repression of the H-2K gene expression. Thus, in contrast to the many examples of an inverse correlation between the level of gene methylation and its transcriptional activity, expression of the H-2K gene is directly correlated with the extent of methylation. This finding offers an explanation whereby the hypomethylation observed in tumor cells may be responsible for the establishment and maintenance of a malignant state of growth.

Animals↗
PubMed1983

Amidination.

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Amidines↗
PubMed1983

Control of H-2 antigen and beta 2-microglobulin gene expression in mouse trophoblast cell clones.

We have investigated the expression of H-2 antigen and beta 2-microglobulin in recently established mouse trophoblast cell clones. These clones, derived from C57BL/6 (H-2b) or BALB/c (H-2d) strains, synthesized extra-embryonic endoderm- and trophectoderm-specific cytoskeletal proteins, termed "Endo A" and "Endo B," and no detectable SSEA-1 embryonic antigen. Flow microfluorometry indicated that H-2 antigen expression on the cell surface of trophoblast cells was very low, corresponding to 2-5% of the amounts found on differentiated teratocarcinoma cells (12-1a) and BALB/c 3T3 cells, respectively. Expression of beta 2-microglobulin was reduced to approximately equal to 14% of the amounts found on 12-la cells. Immunoprecipitation and polyacrylamide gel electrophoretic analysis indicated that the synthesis of H-2 antigen and beta 2-microglobulin in the trophoblast cells was lower than that found in normal spleen cells. In addition low, but unequal, levels of mRNA specific for H-2 antigen and beta 2-microglobulin were found in trophoblast cells by blot hybridization with cDNA probes. The low mRNA levels may be due to transcriptional control of the genes encoding H-2 antigen and beta 2-microglobulin.

Animals↗
PubMed1983

Antibodies against a nonapeptide of polyomavirus middle T antigen: cross-reaction with a cellular protein(s).

Antibodies were raised against the sequence Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met -Glu, which represents a part of the middle T antigen of polyomavirus that is considered to be important in inducing the phenotype of transformed cells. The antibodies reacted with native as well as denatured middle T antigens. In addition, the antibodies immunoprecipitated a cellular protein with an apparent molecular weight of 130,000 (130K) from mouse and rat cells. In some cases, a 33K protein was also immunoprecipitated. Immunoprecipitation of middle T antigen as well as 130K and 33K proteins was blocked by the peptide. The antibodies labeled microfilaments of untransformed mouse, rat, human, and chicken cells by immunofluorescence. This labeling was also blocked by the peptide. The labeling pattern and distribution under a variety of conditions were indistinguishable from those of anti-actin antibodies, although no evidence has been obtained to indicate that the anti-peptide antibodies react with actin. The 130K protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than chicken gizzard vinculin (130K) and slightly faster than myosin light-chain kinase of chicken smooth muscle (130K). Neither of these proteins absorbed the anti-peptide antibodies. The 33K protein does not seem to be tropomyosin (32K to 40K).

Animals↗
PubMed1983

The T lymphocyte response to cytochrome c. IV. Distinguishable sites on a peptide antigen which affect antigenic strength and memory.

The murine T cell proliferative response to the carboxyl terminal cyanogen bromide cleavage fragment 81-104 of pigeon cytochrome c (cyt) has been studied. Two interesting properties of this response have been previously described. First, T cells from B10.A mice primed with pigeon cyt 81-104 show more vigorous proliferation when restimulated with moth cyt 81-103 than when stimulated with pigeon cyt 81-104; that is, the B10.A T cell response to pigeon shows heteroclitic restimulation by moth. Second, T cells primed with the acetimidyl derivative (Am) of pigeon cyt 81-104 did not cross-react with the unmodified cyt fragments, but Am-moth cyt 81-103 still stimulated Am-pigeon cyt 81-104 primed T cells better than the Am-pigeon cyt 81-104 fragment. These results raised the issue of whether the antigenic sites on the fragments responsible for the specificity of T cell priming in vivo differed from the residues that contributed to the heteroclitic response of pigeon (or Am pigeon)-primed T cells to moth cyt c fragments. In this paper, synthetic peptide antigens were tested in order to identify which residues caused the heterocliticity of the moth fragment and which residues were involved in the antigenic differentiation of native and derivatized fragments. The heterocliticity of the T cell response to moth fragment 81-103 was found to be due to the deletion of the penultimate residue (Ala103) from the pigeon fragment. However, the ability to cause heterocliticity was not uniquely a property of this deletion. T cells from animals primed with peptides containing substitutions at positions 100 or 102 were also heteroclitically stimulated by the moth-like antigen. The observation that T cells could not be primed for recognition of the changes in peptide sequence that caused heteroclitic stimulation suggests that T cells do not directly recognize determinants in this region. The antigenically significant site of derivatization for T cell priming was found to be Lys99. Furthermore, substitution of a Gln at position 99 also resulted in elicitation of yet a third set of T cell clones specific for the presence of that residue. That is, the specificity of the primed T cell population was found to be altered by changes at residue-99, but no such alterations in specificity were demonstrable when T cells primed with peptides altered at residue-103, residue-102, or residue-100 were compared. Overall, the results demonstrate that the antigen can be divided into two functionally distinct sites that are in close physical proximity.

Alanine↗
PubMed1983

Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer.

Among the more than 20 H-2-like genes in the BALB/c mouse genome, there are two classical transplantation antigens (H-2Dd and H-2Ld) encoded at the D-end of the major histocompatibility complex. Here we report the identification of a bacteriophage clone that encodes H-2Dd. The H-2Dd gene was identified by nucleotide sequence analysis and by characterization of the new H-2 antigen expressed when the cloned gene was introduced into mouse L cells by DNA-mediated gene transfer. The previously identified H-2Ld gene was then compared with the H-2Dd gene. The two genes appear to have the same general structure, and for the 854 nucleotides that have been compared, the two genes are 89% homologous. The H-2Ld and H-2Dd antigens expressed on mouse L cells after DNA-mediated gene transfer were examined by immunologic criteria. The stably transformed cell lines express apparently normal levels of H-2Dd and H-2Ld on the cell surface as measured by quantitative immunofluorescence by using monoclonal anti-H-2 antibodies. They synthesize H-2Dd and H-2Ld at normal rates as determined by endogenous labeling and immunoprecipitation of cell extracts. They evoke a strong specific serologic response when used to immunize C3H mice. The newly expressed antigens are able to serve as targets for alloreactive T cells. These cloned genes provide good substrates for examining the evolution of two closely linked H-2 antigen genes. Comparison of the structures of these genes provides clues to the basis for the differential expression of these antigens and their different biologic functions.

Amino Acid Sequence↗
PubMed1982

possible role of a retrovirus in the expression of tumor-specific antigens of the Meth A sarcoma.

The serologically defined tumor-specific surface antigen (TSSA) of the chemically-induced BALB/c Meth A sarcoma, highly restricted to one of 20 sarcomas of BALB/c origin, has been detected on a Moloney murine sarcoma virus (Mo-MuSV)-transformed BALB/c 3T3 cell lines, designated IIA(v). The immunogenicity of the IIA(v) cell in tumor-rejection assays was specific for the Meth A sarcoma, supporting the evidence for a close relationship between the TSSA and the tumor-associated transplantation antigen (TATA) of this tumor. Infection of SC-I cells with retroviruses present in cultured filtrates of IIA(v) cells resulted in Meth A antigen expression. The retroviruses associated with Meth A antigen expression have been tentatively identified as replication and/or transformation-defective XC- MuLV. The possible roles of Mo-MuSV and cellular genes of the BALB/c strain of mice in the expression of the Meth A antigen are discussed.

Animals↗
PubMed1982

Contribution of antigen-presenting cell major histocompatibility complex gene products to the specificity of antigen-induced T cell activation.

Previous studies from our laboratory showed that B 10.A mice are high responders to pigeon cytochrome c fragment 81-104, whereas'B 10.A(5R) mice are low responders. In the present studies, the C-terminal cyanogen bromide cleavage fragment and homologous synthetic peptides of tobacco horn worm moth cytochrome c were shown to be immunogenic in both B10.A and B10.A(5R) mice. These strains, however, showed different patterns of cross-reactivity when immune lymph node T cells were stimulated with cytochrome c fragments from other species. To examine the two patterns of responsiveness at a clonal level, cytochrome c fragment-specific T cell hybridomas were made and found to secrete interleukin 2 in response to antigen. The patterns of cross- reactivity of these B 10.A and B 10.A(5R) clones were similar to that seen in the whole lymph node population. Surprisingly, when these clones were tested for major histocompatibility complex (MHC)-restricted antigen recognition, they were all found to respond to antigen with both B10.A and B10.A(5R) antigen-presenting cells (APC). Furthermore, the cross-reactivity pattern appeared to be largely determined by the genotype of the APC, not the genotype of the T cell clone. That is, a given T cell clone displayed a different fine specificity when assayed with B10.A or B10.A(5R) APC. This observation indicates that the APC MHC gene product and antigen interact during the stimulation of the T cell response and that as a consequence the specificity of antigen-induced T cell activation is influenced by these MHC gene products. (During the preparation of this manuscript it has come to our attention that results similar to our own, concerning the fine specificity of cytotoxic T cell clones, have been obtained by Dr. T. R. Hunig and Dr. M. J. Bevan, Massachusetts Institute of Technology, Boston, MA. T. R. Hunig and M. J. Bevan. 1981. Specificity of T-cell clones illustrates altered self hypothesis. Nature. 294:460.)

Animals↗
PubMed1982

Purification and characterization of mouse beta-2 microglobulin: allelic variants from two different strains.

Beta-2 microglobulin (beta 2M) is a 12,000 dalton protein associated with membrane-bound cell surface antigens. Variants of beta 2M, beta 2MA and beta 2MB, were first detected by Michaelson et al. (Immunogenetics 11, 93-95, 1980). An improved method was used to purify beta 2MA and beta 2MB from BALB/c and C57BL/6 mouse livers, respectively. Reproducible yields of 10% were obtained. The purifications were accomplished by a 3 M sodium thiocyanate (NaSCN) extraction of a crude membrane fraction, an acid precipitation step, gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose and CM-cellulose in that order. The elution profile of beta 2MA and beta 2MB on the ion-exchange columns was found to be different, indicating the presence of structural changes. beta 2MA was found to be more acidic (pI = 7.35) than beta 2MB (pI = 7.68) by isoelectric focusing in gels. Complete sequence analysis of beta 2MA and partial sequence analysis of beta 2MB (61 of 99 residues) were performed by automated Edman degradation of the intact chain and of the overlapping peptides obtained by: (a) tryptic cleavage at arginines after acetimidation of lysine side chains, (b) BNPS-skatole cleavage at tryptophan residues and (c) hydroxylamine cleavage at asparagine-glycine linkages. A comparison of the primary structure of beta 2MA to the partial amino acid sequence obtained for beta 2MB revealed a single amino acid substitution (aspartic acid for alanine at position 85) that accounts for the differences in biochemical properties observed.

Alleles↗