scholarly journals THE FUNCTIONAL DISSECTION OF AN ANTIGEN MOLECULE: SPECIFICITY OF HUMORAL AND CELLULAR IMMUNE RESPONSES TO GLUCAGON

1971 ◽  
Vol 133 (6) ◽  
pp. 1294-1308 ◽  
Author(s):  
George Senyk ◽  
E. Brady Williams ◽  
Danute E. Nitecki ◽  
Joel W. Goodman
Keyword(s):  
Lymphoid Cells ◽  
Delayed Hypersensitivity ◽  
Response Patterns ◽  
Amino Terminal ◽  
Transforming Activity ◽  
The Difference ◽  
Different Response ◽  
Carboxy Terminal ◽  
Cellular Immune ◽  
Immune Assays

Bovine glucagon, a polypeptide of 29 amino acids, was immunogenic in guinea pigs. The immunologic determinants of glucagon were investigated using isolated tryptic peptides of the hormone. Antibodies from virtually all of more than two dozen animals had specificity primarily for the amino-terminal heptadecapeptide (NM) and showed little or no binding with the carboxy-terminal undeca- and dodecapeptides (C). The smallest synthetic peptide of a series initiated at residue 16 which measurably bound antibody comprised residues 5–16 of glucagon. In cellular immune assays, both NM and C elicited delayed cutaneous reactions and inhibited the migration of peritoneal cells from immune animals. However, only intact glucagon and its C fragment stimulated lymphoid cells to synthesize DNA. While glucagon was somewhat more active than C, the addition of NM to C did not enhance its transforming activity. The smallest synthetic carboxy-terminal peptide with discernible transforming activity comprised residues 19–29 of glucagon. In both native and synthetic C peptide preparations, the undecapeptide was generally more active than the dodecapeptide, although cells from different animals gave different response patterns. The difference between the two is the presence of arginine at the amino-terminus of the peptide chain. Thus, the recognition specificity of populations of antigen-reactive cells from different animals displays a variation which is at least superficially analogous to that of populations of antibody molecules. In limited experiments using NM and C peptides as immunogens, neither gave rise to delayed hypersensitivity or to glucagon-binding circulating antibody, following a regimen which invariably provoked these responses when glucagon itself served as the immunogen. These results indicate that glucagon was cleaved by trypsin along functional lines into two parts, one of which housed the major antigenic determinant and the other of which carried the major immunogenic determinant, and they are highly compatible with a two-cell mechanism of immune induction. An apparent dissociation between the capacity to provoke delayed hypersensitivity reactions and to transform antigen-reactive cells in culture was observed.

scholarly journals Cell Cycle-Regulated Processing of HEF1 to Multiple Protein Forms Differentially Targeted to Multiple Subcellular Compartments

1998 ◽  
Vol 18 (6) ◽  
pp. 3540-3551 ◽  
Author(s):  
Susan F. Law ◽  
Yu-Zhu Zhang ◽  
Andres J. P. Klein-Szanto ◽  
Erica A. Golemis
Keyword(s):  
Cell Cycle ◽  
Regulatory Protein ◽  
Focal Adhesions ◽  
Lymphoid Cells ◽  
Full Length ◽  
Amino Terminal ◽  
Multiple Protein ◽  
Docking Proteins ◽  
Carboxy Terminal ◽  
Cycle Regulation

ABSTRACT HEF1, p130Cas, and Efs/Sin constitute a family of multidomain docking proteins that have been implicated in coordinating the regulation of cell adhesion. Each of these proteins contains an SH3 domain, conferring association with focal adhesion kinase; a domain rich in SH2-binding sites, phosphorylated by or associating with a number of oncoproteins, including Abl, Crk, Fyn, and others; and a highly conserved carboxy-terminal domain. In this report, we show that the HEF1 protein is processed in a complex manner, with transfection of a single cDNA resulting in the generation of at least four protein species, p115HEF1, p105HEF1, p65HEF1, and p55HEF1. We show that p115HEF1 and p105HEF1 are different phosphorylation states of the full-length HEF1. p55HEF1, however, encompasses only the amino-terminal end of the HEF1 coding sequence and arises via cleavage of full-length HEF1 at a caspase consensus site. We find that HEF1 proteins are abundantly expressed in epithelial cells derived from breast and lung tissue in addition to the lymphoid cells in which they have been predominantly studied to date. In MCF-7 cells, we find that expression of the endogenous HEF1 proteins is cell cycle regulated, with p105HEF1 and p115HEF1 being rapidly upregulated upon induction of cell growth, whereas p55HEF1 is produced specifically at mitosis. While p105HEF1 and p115HEF1 are predominantly cytoplasmic and localize to focal adhesions, p55HEF1 unexpectedly is shown to associate with the mitotic spindle. In support of a role at the spindle, two-hybrid library screening with HEF1 identifies the human homolog of the G2/M spindle-regulatory protein Dim1p as a specific interactor with a region of HEF1 encompassed in p55HEF1. In sum, these data suggest that HEF1 may directly connect morphological control-related signals with cell cycle regulation and thus play a role in pathways leading to the progression of cancer.

scholarly journals The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-alpha catenin fusion molecules.

1994 ◽  
Vol 127 (1) ◽  
pp. 235-245 ◽  
Author(s):  
A Nagafuchi ◽  
S Ishihara ◽  
S Tsukita
Keyword(s):  
Cell Adhesion ◽  
Cell Movement ◽  
Negative Regulator ◽  
Beta Catenin ◽  
Amino Terminal ◽  
Cytoplasmic Proteins ◽  
The Difference ◽  
Adhesion Activity ◽  
Carboxy Terminal ◽  
E Cadherin

The carboxyl terminus-truncated cadherin (nonfunctional cadherin) has no cell adhesion activity probably because of its failure to associate with cytoplasmic proteins called alpha and beta catenin. To rescue this nonfunctional cadherin as adhesion molecules, we constructed three cDNAs for fusion proteins between nonfunctional E-cadherin and alpha catenin, nE alpha, nE alpha N, and nE alpha C, where the intact, amino-terminal and carboxy-terminal half of alpha catenin, respectively, were directly linked to the nonfunctional E-cadherin, and introduced them into mouse L cells. The subcellular distribution and cell adhesion activity of nE alpha and nE alpha C molecules was similar to those of intact E-cadherin transfectants: they bound to cytoskeletons, were concentrated at cell-cell adhesion sites and showed strong cell adhesion activity. nE alpha N molecules, which also bound to cytoskeletons, showed very poor cell adhesion activity. Taken together, we conclude that in the formation of the cadherin-catenin complex, the mechanical association of alpha catenin, especially its carboxy-terminal half, with E-cadherin is a key step for the cadherin-mediated cell adhesion. Close comparison revealed that the behavior of nE alpha molecules during cytokinesis was quite different from that of intact E-cadherin, and that the intercellular motility, i.e., the cell movement in a confluent sheet, was significantly suppressed in nE alpha transfectants although it was facilitated in E-cadherin transfectants. Considering that nE alpha was not associated with endogenous beta catenin in transfectants, the difference in the nature of cell adhesion between nE alpha and intact E-cadherin transfectants may be explained by the function of beta catenin. The possible functions of beta catenin are discussed with a special reference to its role as a negative regulator for the cadherin-mediated cell adhesion system.

scholarly journals HETEROGENEITY OF THE CELLULAR IMMUNE RESPONSE

1971 ◽  
Vol 133 (2) ◽  
pp. 187-201 ◽  
Author(s):  
Robert C. Bast ◽  
Eleanor J. Manseau ◽  
Harold F. Dvorak
Keyword(s):  
Immune Response ◽  
Cellular Immune Response ◽  
Large Population ◽  
Specific Antigen ◽  
Lymphoid Cells ◽  
Lymphocyte Stimulation ◽  
Delayed Hypersensitivity ◽  
Skin Reactions ◽  
Cellular Immune

Lymph node cells from guinea pigs immunized with HSA in complete Freund's adjuvant were grown in cultures containing different concentrations of specific antigen. Stimulation of thymidine incorporation was induced with progressively lower concentrations of HSA at successive intervals after sensitization. Moreover, the intensity of delayed skin reactions and the magnitude of stimulation in vitro increased over the same interval. These events are considered compatible with an evolution of the cellular immune response resulting from the selection of lymphoid cells by decreasing concentrations of antigen in vivo. Cells from animals rendered tolerant to HSA failed to respond to specific antigen in culture. As tolerance waned, stimulation was achieved at high but not low antigen concentrations. Tolerance, measured by cutaneous reactivity or by lymphocyte stimulation, was less readily induced in animals sensitized with adjuvant containing a reduced concentration of mycobacteria. Lymph nodes from these animals contained a large population of cells reactive at high antigen concentration, presumably less susceptible to the toleragenic effect of intravenous antigen. The dissociation of delayed hypersensitivity and antibody formation observed early in the immune response and upon recovery from tolerance has permitted correlation of lymphocyte stimulation with delayed hypersensitivity and cutaneous basophil hypersensitivity respectively.

D-test: A New Test for Analyzing Scale Invariance Using Symbolic Dynamics and Symbolic Entropy

Methodology ◽  
2011 ◽  
Vol 7 (3) ◽  
pp. 88-95 ◽  
Author(s):  
Jose A. Martínez ◽  
Manuel Ruiz Marín
Keyword(s):  
Symbolic Dynamics ◽  
Scale Invariance ◽  
Rating Scales ◽  
Rating Scale ◽  
Marketing Research ◽  
Response Patterns ◽  
Measurement Scales ◽  
Improve Measurement ◽  
Entropy Measures ◽  
The Difference

The aim of this study is to improve measurement in marketing research by constructing a new, simple, nonparametric, consistent, and powerful test to study scale invariance. The test is called D-test. D-test is constructed using symbolic dynamics and symbolic entropy as a measure of the difference between the response patterns which comes from two measurement scales. We also give a standard asymptotic distribution of our statistic. Given that the test is based on entropy measures, it avoids smoothed nonparametric estimation. We applied D-test to a real marketing research to study if scale invariance holds when measuring service quality in a sports service. We considered a free-scale as a reference scale and then we compared it with three widely used rating scales: Likert-type scale from 1 to 5 and from 1 to 7, and semantic-differential scale from −3 to +3. Scale invariance holds for the two latter scales. This test overcomes the shortcomings of other procedures for analyzing scale invariance; and it provides researchers a tool to decide the appropriate rating scale to study specific marketing problems, and how the results of prior studies can be questioned.

Interaction Between Mutations in the suppressor of Hairy wing and modifier of mdg4 Genes of Drosophila melunogaster Affecting the Phenotype of gypsy-Induced Mutations

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 425-436 ◽  
Author(s):  
Pavel Georgiev ◽  
Marina Kozycina
Keyword(s):  
Mutagenic Effect ◽  
Induced Mutations ◽  
Binding Region ◽  
Direct Inhibition ◽  
Amino Terminal ◽  
Acidic Domain ◽  
Complete Inactivation ◽  
Insulation Effect ◽  
Gypsy Retrotransposon ◽  
Carboxy Terminal

Abstract The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers located distally from the promoter with respect to the position of the su(Hw)-binding region. Mutations in a second gene, modifier of mdg4, also affect the gypsy-induced phenotype. Two major effects of the mod(mdg4)lul mutation can be distinguished: the interference with insulation by the su(Hw)-binding region and direct inhibition of gene expression that is not dependent on the su(Hw)-binding region position. The mod(mdg4)lul mutation partially suppresses ct6, scD1 and Hw1 mutations, possibly by interfering with the insulation effect of the su(Hw)-binding region. An example of the second effect of mod(mdg4)lul is a complete inactivation of yellow expression in combination with the y  2 allele. Phenotypic analyses of flies with combinations of mod(mdg4)lul and different su(Hw) mutations, or with constructions carrying deletions of the acidic domains of the su(Hw) protein, suggest that the carboxy-terminal acidic domain is important for direct inhibition of yellow transcription in bristles, while the amino-terminal acidic domain is more essential for insulation.

scholarly journals p190 RhoGAP, the major RasGAP-associated protein, binds GTP directly.

1994 ◽  
Vol 14 (11) ◽  
pp. 7173-7181 ◽  
Author(s):  
R Foster ◽  
K Q Hu ◽  
D A Shaywitz ◽  
J Settleman
Keyword(s):  
Structural Features ◽  
Cellular Protein ◽  
Small Gtpases ◽  
Sequence Motifs ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Gtp Binding ◽  
Guanine Nucleotide Binding ◽  
Complex Forms ◽  
Carboxy Terminal

In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.

N-Terminal amino acid sequence of rat tonin: homology with serine proteases

1978 ◽  
Vol 56 (9) ◽  
pp. 920-925 ◽  
Author(s):  
N. G. Seidah ◽  
R. Routhier ◽  
M. Caron ◽  
M. Chrétien ◽  
S. Demassieux ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Terminal Sequence ◽  
Renin Substrate ◽  
Amino Terminal ◽  
Single Polypeptide Chain ◽  
Serine Protease Family ◽  
Terminal Amino ◽  
Single Polypeptide ◽  
Carboxy Terminal ◽  
Amino Terminal Sequence

In this paper, we present the amino-terminal sequence of rat tonin, an endopeptidase responsible for the conversion of angiotensinogen, the tetradecapeptide renin substrate, or angiotensin I to angiotensin II. It is shown that isoleucine and proline occupy the amino- and carboxy-terminal residues respectively. The N-terminal sequence analysis permitted the identification of 34 out of the first 40 residue s of the single polypeptide chain composed of 272 amino acids. The se results showed an extensive homology with the sequence of many serine proteases of the trypsin–chymotrypsin family. This information, coupled with the slow inhibition of tonin by diisopropylfluorophosphate, classified this enzyme as a selective endopeptidase of the active serine protease family.

scholarly journals The Catenin p120ctnInteracts with Kaiso, a Novel BTB/POZ Domain Zinc Finger Transcription Factor

1999 ◽  
Vol 19 (5) ◽  
pp. 3614-3623 ◽  
Author(s):  
Juliet M. Daniel ◽  
Albert B. Reynolds
Keyword(s):  
Transcription Factor ◽  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Mammalian Cells ◽  
Yeast Two Hybrid ◽  
Amino Terminal ◽  
Juxtamembrane Region ◽  
Carboxy Terminal ◽  
Two Hybrid ◽  
E Cadherin

ABSTRACT p120 ctn is an Armadillo repeat domain protein with structural similarity to the cell adhesion cofactors β-catenin and plakoglobin. All three proteins interact directly with the cytoplasmic domain of the transmembrane cell adhesion molecule E-cadherin; β-catenin and plakoglobin bind a carboxy-terminal region in a mutually exclusive manner, while p120 binds the juxtamembrane region. Unlike β-catenin and plakoglobin, p120 does not interact with α-catenin, the tumor suppressor adenomatous polyposis coli (APC), or the transcription factor Lef-1, suggesting that it has unique binding partners and plays a distinct role in the cadherin-catenin complex. Using p120 as bait, we conducted a yeast two-hybrid screen and identified a novel transcription factor which we named Kaiso. Kaiso’s deduced amino acid sequence revealed an amino-terminal BTB/POZ protein-protein interaction domain and three carboxy-terminal zinc fingers of the C2H2 DNA-binding type. Kaiso thus belongs to a rapidly growing family of POZ-ZF transcription factors that include the Drosophila developmental regulators Tramtrak and Bric à brac, and the human oncoproteins BCL-6 and PLZF, which are causally linked to non-Hodgkins’ lymphoma and acute promyelocytic leukemia, respectively. Monoclonal antibodies to Kaiso were generated and used to immunolocalize the protein and confirm the specificity of the p120-Kaiso interaction in mammalian cells. Kaiso specifically coprecipitated with a variety of p120-specific monoclonal antibodies but not with antibodies to α- or β-catenin, E-cadherin, or APC. Like other POZ-ZF proteins, Kaiso localized to the nucleus and was associated with specific nuclear dots. Yeast two-hybrid interaction assays mapped the binding domains to Arm repeats 1 to 7 of p120 and the carboxy-terminal 200 amino acids of Kaiso. In addition, Kaiso homodimerized via its POZ domain but it did not heterodimerize with BCL-6, which heterodimerizes with PLZF. The involvement of POZ-ZF proteins in development and cancer makes Kaiso an interesting candidate for a downstream effector of cadherin and/or p120 signaling.

scholarly journals Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

2007 ◽  
Vol 88 (12) ◽  
pp. 3270-3274 ◽  
Author(s):  
Marianne Bonvin ◽  
Jobst Greeve
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Point Mutations ◽  
Alternative Mechanism ◽  
Amino Terminal ◽  
Hbv Replication ◽  
B Virus ◽  
Carboxy Terminal ◽  
Deaminase Domain

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

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