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C E Seyfried

Publications and source records attributed to C E Seyfried.

13 recordsLinked to original sources
PubMed1994

Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5.

Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. We have purified human calnexin in association with the human interferon-gamma receptor and cloned calnexin cDNA from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind 45Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the "E-F hand" motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. We have also determined the cDNA sequences of mouse and rat calnexins. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35.

Amino Acid Sequence↗
PubMed1992

The human platelet-activating factor receptor gene (PTAFR) contains no introns and maps to chromosome 1.

Platelet-activating factor (PAF), a phospholipid, exhibits a variety of potent inflammatory bioactivities that are mediated by a specific cell surface receptor. The gene for the human PAF receptor (PTAFR) has been isolated by hybridization with a guinea pig probe. The coding sequence contains no intervening sequences. The encoded protein is highly homologous to the guinea pig PAF receptor (82% identity) and contains seven putative transmembrane domains. The PAF receptor therefore appears to be a member of the G protein coupled family of receptors and exhibits significant similarity to many members of the family. Analysis of somatic cell hybrids suggests that the PAF receptor is encoded by a single gene on human chromosome 1.

Amino Acid Sequence↗
PubMed1991

Allelic polymorphism in transcriptional regulatory regions of HLA-DQB genes.

Class II genes of the human major histocompatibility complex (MHC) are highly polymorphic. Allelic variation of structural genes provides diversity in immune cell interactions, contributing to the formation of the T cell repertoire and to susceptibility to certain autoimmune diseases. We now report that allelic polymorphism also exists in the promoter and upstream regulatory regions (URR) of human histocompatibility leukocyte antigen (HLA) class II genes. Nucleotide sequencing of these regulatory regions of seven alleles of the DQB locus reveals a number of allele-specific polymorphisms, some of which lie in functionally critical consensus regions thought to be highly conserved in class II promoters. These sequence differences also correspond to allelic differences in binding of nuclear proteins to the URR. Fragments of the URR of two DQB alleles were analyzed for binding to nuclear proteins extracted from human B lymphoblastoid cell lines (B-LCL). Gel retardation assays showed substantially different banding patterns to the two promoters, including prominent variation in nuclear protein binding to the partially conserved X box regions and a novel upstream polymorphic sequence element. Comparison of these two polymorphic alleles in a transient expression system demonstrated a marked difference in their promoter strengths determined by relative abilities to initiate transcription of the chloramphenicol acetyltransferase reporter gene in human B-LCL. Shuttling of URR sequences between alleles showed that functional variation corresponded to both the X box and upstream sequence polymorphic sites. These findings identify an important source of MHC class II diversity, and suggest the possibility that such regulatory region polymorphisms may confer allelic differences in expression, inducibility, and/or tissue specificity of class II molecules.

Alleles↗
PubMed1989

Sequence analysis of HLA class II domains: characterization of the DQw3 family of DQB genes.

HLA class II allelic variants within the DQw3-related family of genes carry distinct allo-specificities and have been implicated in specific HLA-disease associations, such as insulin-dependent diabetes mellitus. To investigate the nucleotide variations which characterize DQw3 genes, we applied a novel cDNA cloning strategy that uses a single-stranded vector/primer system to facilitate DNA sequencing of allelically variable gene families. Using a DQB-specific primer sequence and M13 bacteriophage as the cloning vector, direct cloning and sequencing of multiple DQB genes was performed without the need for second strand synthesis or for subcloning. Sequence analysis from eight lymphoblastoid cell lines selected to represent different ethnic backgrounds revealed three DQw3-related DQB genes, DQB3.1, 3.2, and 3.3, corresponding to the newly designated HLA-DQw7, w8, and w9 specificities, respectively. An unusual Pro-Pro couplet at codons 55-56 is characteristic of all DQw3-positive sequences and may be contributing to the broad DQw3 allospecificity. Comparisons among ethnically disparate DQw3-related sequences showed no additional expressed or silent nucleotide substitutions among these DQB alleles. Thus, polymorphism within the DQw3 family of genes appears to be extremely limited, with a paucity of nucleotide variations accumulated by evolutionary distance.

Alleles↗
PubMed1988

A specific nucleotide sequence defines a functional T-cell recognition epitope shared by diverse HLA-DR specificities.

An oligonucleotide probe that distinguishes the HLA-DR4, Dw14 allele from other HLA-DR4-associated class II genes was used to identify a common nucleotide sequence shared between the DR beta 1 locus encoding HLA-DR1 (Dw1), Dw16, and DRw10 haplotypes. The presence of this nucleotide sequence correlated with the ability of these haplotypes to stimulate an alloreactive proliferative T-cell clone (clone 14B) raised against a Dw14+ stimulator cell. Alleles of DR beta 1 that differ by one specific nucleotide variation at codon 71 neither hybridize to the Dw14-specific oligonucleotide nor stimulate clone 14B. These data demonstrate the presence of a shared epitope present on serologically distinct DR alleles that is recognized as a specific allodeterminant by a single T-cell clone.

Base Sequence↗
PubMed1987

Functional polymorphisms among HLA-DR4+ DR beta chains associated with limited peptide diversity.

Tryptic peptide map analysis by high-pressure liquid chromatography of DR4- associated DR beta chains revealed limited structural variation within DR beta polypeptides. Comparison of 3H-leucine-labelled tryptic peptide maps of Dw4 and Dw14 homozygous cells identified distinct peaks corresponding to Dw4 and Dw14-associated DR beta polypeptides. HPLC analysis of cell line 256, heterozygous for two DR4-related specificities, Dw4 and Dw14, displayed both peptides, corresponding to the variable Dw4 and Dw14 chromatograms. This observation was confirmed using a deletion mutant cell line derived from 256 lacking Dw4-associated class II genes. The observed peptide variation correlated precisely with predicted nucleotide-derived amino acid sequences implicating amino acids 66-71 of the DR beta chain as contributing to HLA-D structural and functional polymorphisms.

Amino Acid Sequence↗
PubMed1986

Identification of HLA-Dw14 genes in DR4+ rheumatoid arthritis.

Genes of the major histocompatibility complex, HLA, are associated with susceptibility to rheumatoid arthritis (RA), but the aetiology of this chronic inflammatory disease is not known. Synthetic oligonucleotide DNA probes were constructed to distinguish between two closely related but distinct alleles encoding the HLA-DR4 specificity in patients with RA. With these allele-specific oligonucleotide probes an uncommon DR4 genetic variant, Dw14, was identified in 6 of 7 RA patients homozygous for HLA-DR4. This allele may play an important part in susceptibility to RA.

Alleles↗
PubMed1983

Regulation of protein synthesis in mitogen-activated bovine lymphocytes. Analysis of actin-specific and total mRNA accumulation and utilization.

In response to the mitogenic lectin concanavalin A, the rate of protein synthesis in bovine small lymphocytes increased about 3-fold in 10 h and about 6-fold in 24 h. The rate of synthesis of the cytoskeletal protein actin, which comprises about 10% of total protein synthesis, increased in parallel with total protein synthesis. The cellular levels of actin mRNA were determined at various times after mitogen addition using a cDNA probe derived from a recombinant plasmid carrying a fragment coding for bovine actin. Actin mRNA sequences were found to increase significantly within 3 h and to increase 3.6 +/- 1.2-fold within 10 h after mitogen addition. Therefore, the increase in actin synthesis after mitogenic activation seems to be accounted for by accumulation of actin mRNA sequences. Total translatable mRNA and poly(A) sequences also accumulated in parallel with total protein synthesis, suggesting that regulation of actin synthesis was not unique. Two direct studies of mRNA utilization indicated that there is little, if any, translational regulation of actin synthesis. A detailed examination of actin mRNA-containing polysomes and total polysomes revealed no significant change in the average size of polysomes after cell activation. Therefore, given that the rates of polypeptide elongation and termination are constant after mitogen addition (Kay, J.E., Ahern, T., Lindsay V.J., and Sampson, J. (1975) Biochim. Biophys. Acta 378, 241-250), the rate of translational initiation per active mRNA must be unchanged. In addition, no significant amount of unutilized, nonpolysomal actin mRNA was detected in total cell extracts from either unstimulated or activated cells. Together these data suggest that elevated protein synthesis in activated lymphocytes is regulated by mRNA level.

Actins↗
PubMed1982

Purification, properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis.

S-adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis(guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32,000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [3H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme synthesis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic activity. There was no further increase in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occurring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.

Adenosylmethionine Decarboxylase↗
PubMed1981

Regulation of the level of S-adenosylmethionine decarboxylase during lymphocyte mitogenesis.

Bovine lymphocytes were mitogenically activated by the lectin concanavalin A and the activity of the polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (SDC), was followed (R. H. Fillingame and D. R. Morris, Biochem. Biophys. Res. Commun. 52: 1020-1025, 1973). Enzyme activity began to increase at approximately 3 hours after cellular activation, reaching a peak at 9-12 hours. A second elevation of SDC activity coincided with the entry of the cells into S phase at 24 hours. The early and late increases in SDC activity were regulated by different mechanisms. The initial elevation of activity was due to approximately a 10-fold enhancement of the rate of enzyme synthesis; the enhanced rate of synthesis was maintained throughout the remainder of the culture period. The second increase in SDC activity was due to a lengthening of the intracellular half-life of the enzyme from 80 to 170 min. These changes in the rates of SDC synthesis and degradation fully account for the observed biphasic enzyme induction curve. In vitro translation of the SDC messenger RNA has been achieved and this will be used in further studies of the mechanism of enhanced enzyme synthesis.

Adenosylmethionine Decarboxylase↗
PubMed1977

Inhibition of the synthesis of polyamines and DNA in activated lymphocytes by a combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone).

The cancer chemotherapeutic drug, methylglyoxal bis(guanylhydrazone), inhibits the synthesis of spermidine and spermine, but allows continued putrescine production in small lymphocytes stimulated by concanavalin A. DNA replication in these cells is inhibited 50% while the synthesis of protein and RNA continues normally. When excess putrescine accumulation in the presence of methylglyoxal bis(guanylhydrazone) was inhibited with alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, the inhibition of DNA replication was accentuated, with still no effect on protein or RNA synthesis. No inhibition of DNA synthesis by the combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone) was observed when the inhibitors were added after accumulation of cellular polyamines. In addition, inhibition was reversed by exogenous putrescine, spermidine, or spermine. We conclude that putrescine can fulfill in part the role normally played by spermidine and spermine in DNA replication, and that blocking putrescine synthesis in the presence of methylglyoxal bis(guanylhydrazone) amplifies the polyamine requirement. The implications of this with regard to polyamine synthesis as a site of chemotherapy are discussed.

Animals↗