Search PubMed⌕ Search

Biomedical subjects

C Upton

Publications and source records attributed to C Upton.

At least 73 records · Page 4Linked to original sources
PubMed1992

Analogues of triprolidine: structural influences upon antihistamine activity.

The synthesis of some geometrical isomers related to triprolidine is reported. Previous configurational assignments, by UV and proton NMR, are validated by high field nuclear Overhauser enhancement methods and the isomeric purity of tested E- and Z-isomers was greater than 99.5% as assessed by an HPLC method developed for these compounds. Affinity constants for triprolidine (E and Z) in guinea-pig ileum showed a potency ratio of approximately 600 whereas at cerebellar sites this ratio was only approximately 100, suggesting that the H1 receptor in these two tissues may not be identical. In-vivo tests using a lethal dose of compound 48/80 (a potent histamine-releasing agent) demonstrated that triprolidine itself was the most active compound to protect the animal among all the isomeric compounds tested: in all isomeric pairs the E-configuration possessed superior activity over Z. The disposition of the aryl groups in these geometrically constrained compounds mimics that seen in the structurally related chiral pheniramines which are sp3 hybridized and whose absolute stereochemistry is known.

Animals↗
PubMed1991

T2 open reading frame from the Shope fibroma virus encodes a soluble form of the TNF receptor.

A transcriptionally active open reading frame (T2) from Shope Fibroma Virus was recently shown to have striking sequence homology with members of a new superfamily of cell surface proteins, including a receptor for human tumor necrosis factor. Here we report that recombinant T2 protein expressed in COS cells is a soluble, secreted glycoprotein which specifically binds human TNF alpha and beta, and inhibits binding of these cytokines to native TNF receptors on cells. T2 binding of TNF is not inhibited by nerve growth factor, although the nerve growth factor receptor is also a member of the same family, nor by nine other recombinant cytokines. Further, the repeating domain structure of T2 most closely resembles that of the type I TNF receptor (p75) and is significantly different from other family members, including the type II TNF receptor (p55). Since T2 possesses a leader sequence but lacks a transmembrane domain, these results confirm the original suggestion (1) that T2 represents a soluble form of the type I TNF receptor which is secreted from virally infected cells, and whose function is to immunosuppress the host by abrogating the potentially destructive effects of TNF. This is the first such virally-encoded soluble cytokine receptor to be identified, and may represent a more general mechanism by which viruses subvert the host immune system.

Amino Acid Sequence↗
PubMed1991

Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence.

Poxviruses are known to contain a large number of open reading frames, particularly near the termini of the viral genome, that are not required for growth in tissue culture. However, many of these gene products are believed to play important roles in determining the virulence of the virus by modulating the host immune response to the infection. Recently it has been shown that Shope fibroma virus encodes, within the terminal inverted repeats, a protein (T2) related to the cellular tumor necrosis factor receptor (TNFR) and which specifically binds both TNF alpha and TNF beta. We have sequenced the terminal regions of two other Leporipoxviruses (myxoma virus and malignant rabbit fibroma virus) that are extremely invasive and capable of inducing extensive immunosuppression in rabbits and demonstrate that they also encode a closely related T2 homolog with all the structural motifs predicted for a secreted TNF binding protein. To investigate the biological role of the T2 protein, we have inactivated the myxoma virus T2 gene within each copy of the viral TIR by the insertion of a dominant selectable marker (Escherichia coli guanosine phosphoribosyltransferase) and selection of the recombinant virus in the presence of mycophenolic acid. The success of the inactivation of both copies of T2 was confirmed by the loss a broad protein band (52-56 kDa) of the predicted size for T2 from the profile of proteins secreted from mutant virus-infected BGMK cells at early times after infection. Although the T2-minus recombinant myxoma virus grew normally in tissue culture, upon infection of susceptible rabbits the viral disease was observed to be significantly attenuated. The majority of infected rabbits were able to mount an effective immune response to the infection and completely recovered. These survivor rabbits became immune to subsequent challenge with wild type myxoma virus. We conclude that the T2 viral protein is an important secreted virulence factor and that it in all likelihood functions by compromising the antiviral effects of TNF. We propose the term "viroceptor" to describe viral-encoded homologs of cellular lymphokine receptors whose function is to intercept the activity of the cognate lymphokine in order to short circuit the host immune response to the viral infection.

Amino Acid Sequence↗
PubMed1991

Identification and DNA sequence of the large subunit of the capping enzyme from Shope fibroma virus.

A 3.6-kb region of the Shope fibroma virus (SFV) BamHI D fragment located in the central region of the viral genome was sequenced. Three open reading frames (ORFs) were identified, D3R, D4L, and D5R. Each of these ORFs have a counterpart organized identically within the HindIII fragment D of the vaccinia virus genome (D1R, D2L, and D3R). Homology scores and assays of viral cores indicate that SFV D3R encodes the large subunit of the SFV mRNA capping enzyme.

Amino Acid Sequence↗
PubMed1990

Identification and DNA sequence of the Shope fibroma virus DNA topoisomerase gene.

The Shope fibroma virus (SFV) DNA topoisomerase gene has been identified and mapped to the BamHI D fragment near the midpoint of the genome. The DNA sequence of the SFV BamHI S fragment together with the contiguous BamHI-ClaI subfragment of BamHI D which encompasses the topoisomerase gene and two flanking genes has been determined and analyzed. Both the SFV DNA topoisomerase and the two flanking genes are closely related in terms of sequence and spatial organization to the homologous sequences from the midpoint of the vaccinia virus genome, indicating that these proteins are conserved not only in their sequence but also by position within the poxvirus genome. To confirm the assignment of the SFV gene, the putative SFV DNA topoisomerase has been expressed as an active fusion protein in Escherichia coli and this system should be useful in the analysis of topoisomerase function following the introduction of targeted mutations into the topoisomerase gene. The results of this work shed further light on the evolutionary relationship of the different poxvirus genera and indicate that central unique regions of the poxvirus genomes contain many of the essential viral genes and are thus highly conserved.

Amino Acid Sequence↗
PubMed1990

Myxoma virus and malignant rabbit fibroma virus encode a serpin-like protein important for virus virulence.

The leporipoxviruses Shope fibroma virus (SFV), the myxoma virus (MYX), and the SFV/MYX recombinant malignant rabbit fibroma virus (MRV) are closely related yet induce profoundly different diseases in the European rabbit. SFV, which produces a benign tumor at the site of inoculation, is cleared by the immune system after approximately 2 weeks whereas MYX and MRV induce a rapidly lethal systemic infection characterized by generalized suppression of host immune functions. DNA sequencing studies reveal that MRV and MYX possess homologous gene members of the T6/T8/T9 family originally described in the terminal inverted repeat (TIR) of SFV. We also describe a gene present in both MYX and MRV genomes, but which has apparently evolved in the SFV genome into a fragmented pseudogene that appears to contribute to the aggressive nature of MYX and MRV infections. Translation of this open reading frame, designated MYXOMA SERPIN 1 (SERP1), reveals a protein sequence with highly significant homology to the super-family of serine protease inhibitors (serpins) which also includes a number of other poxviral proteins. In the MYX genome the SERP1 gene lies entirely within the TIR sequences and is thus present as two copies, while in the MRV genome SERP1 is present in the unique sequences adjacent to the TIR boundary and hence is a single copy. The amino acid homology between the putative active site of SERP1 and those of other serpins predicts that the target enzyme will be different from the known catalog of serine antiprotease substrates. Deletion of this gene from MRV significantly attenuates the disease spectrum induced by the normally lethal virus. Although the MRV-S1 deletion construct (MRV with SERP1 gene deleted) grows in all tissue culture cells tested in a fashion identical to the MRV parent, the majority of rabbits infected with MRV-S1 are able to mount an effective immune response and totally recover from the virus infection to become resistant to subsequent challenge by MRV or MYX.

Amino Acid Sequence↗
PubMed1989

Transcriptional regulation of two cytotoxic T lymphocyte-specific serine protease genes.

The expression of two serine proteases is induced by antigenic stimulation in cytotoxic T lymphocytes. Using nuclear run-on analysis the increase in steady state mRNA level has been shown to correspond to transcriptional activation. However, the two genes appear to be sequentially rather than coordinately induced. Both genes were shown to be more sensitive to DNase I digestion than a beta-globin gene in cytotoxic T cells. In addition, for the cytotoxic cell protease 1 gene the 5' region of the gene was more sensitive than the 3' end. Two DNaseI hypersensitive sites were seen in the 5' flanking sequences of both genes. The DNA sequences of the upstream regions of both genes were determined and compared. Although the two flanking sequences are overall quite dissimilar, there are short regions which are shared between the two CTL-protease genes. A number of these have been implicated in regulating the expression of other T cell genes.

Animals↗
PubMed1988

Organization of two genes encoding cytotoxic T lymphocyte-specific serine proteases CCPI and CCPII.

The genes encoding two recently described cytotoxic T cell proteases, CCPI and CCPII, have been isolated and sequenced. The organizations of the coding and noncoding portions of the two genes are very similar to each other and also to the gene encoding rat mast cell protease type II. Similarly to other serine protease genes, each of the active-site residues is contained on a separate exon; however, two introns were found in particularly interesting positions. One occurs within the postulated activation dipeptide and the other in a position close to the active-site Asp residue. This latter intron interrupts the amino acid sequence in the invariant core region of the protein. We believe that these genes represent a new subfamily of serine protease genes.

Amino Acid Sequence↗
PubMed1988

Tumorigenic poxviruses: fine analysis of the recombination junctions in malignant rabbit fibroma virus, a recombinant between Shope fibroma virus and myxoma virus.

Malignant rabbit fibroma virus (MRV) has been shown to be a lethal tumorigenic poxvirus of rabbits derived from a recombination event between Shope fibroma virus (SFV), which induces benign fibromas in rabbits, and myxoma virus, the agent of myxomatosis. We have cloned and sequenced all of the MRV recombination junctions, which are located near the left and right terminal inverted repeat (TIR) regions, and present a composite map of the MRV genome with respect to the relevant gene products. The two junctions closet to the MRV termini, at identical positions at the left and right ends, are at nucleotide 5272 and result in an in-frame fusion protein (ORF T-5) in which the N-terminal 232 aa are derived from an SFV sequence linked to a C-terminus derived from myxoma. At the left MRV TIR the recombination junction distal from the terminus maps to nucleotide 9946 but leaves the adjacent gene virtually unchanged from its SFV homolog. At the right terminus, the relevant junction sequences from MRV and myxoma could not be cloned in wild-type Escherichia coli but were maintained stably in a recA recBC sbcB host. The SFV/myxoma junction at this location maps 5' to a growth factor gene (SFGF) which is related to those encoding epidermal growth factor and transforming growth factor-alpha. As a result, the myxoma growth factor gene has been deleted in MRV and replaced in toto by the SFV gene. The recombination junction upstream from the SFGF gene creates an in-frame fusion in ORF T11-R in which the N-terminal amino acids are derived from myxoma and the remainder from SFV. In summary, MRV has received the following ORFs from SFV: at the left terminus T5 (fusion), T6, T7, and T8; at the right terminus, T5 (fusion), T6, T7, T8, T9-R, SFGF, and T11-R (fusion).

Animals↗
PubMed1988

Cloning and sequencing of the cytoplasmic precursor to the alpha subunit of rat liver mitochondrial succinyl-CoA synthetase.

Succinyl-CoA synthetase [succinate-CoA ligase (GDP-forming); EC 6.2.1.4] of rat liver, an alpha beta dimer, is a component of the enzymology of the tricarboxylic acid cycle and functions within the mitochondrial matrix. We have isolated and determined the sequence of a cDNA clone containing the coding sequence of the cytoplasmic precursor to the alpha subunit of this enzyme together with stretches of nontranslated sequence at the 5' and 3' ends. The translated amino acid sequence indicates the presence of a 27-residue N-terminal signal sequence for mitochondrial targeting. The amino acid sequence of the mature alpha subunit shows an extraordinary degree of homology to the alpha subunit of Escherichia coli succinyl-CoA synthetase, with greater than 70% of the residues identical. This suggests that the fundamental differences in the quaternary structures and catalytic functions of the mammalian and bacterial enzymes must be attributable to differences in the beta subunits. mRNA that hybridizes to the cloned DNA is approximately equal to 1800 nucleotide residues in length, confirming that each of the two subunits is encoded separately and does not arise by proteolysis of a primary gene product containing both subunits of the mature protein.

Amino Acid Sequence↗
PubMed1988

The structure of phenindamine base and salts in the solute state.

High-field NMR (13C and 1H) studies of phenindamine are reported which establish structures of the free base and some of its salts in the solute condition. The base exists as a mixture of two isomers which differ in double bond position (9-9a or 4a-9a) while most salts are 9-9a isomers. The clinically employed tartrate (Thephorin) is exceptional in being a 4a-9a ene. Salts of both double bond type exist in solution as mixtures of protonated epimers of variable epimeric ratio, that of the tartrate in D2O being approximately 1:1.

Chemical Phenomena↗
PubMed1987

Tumorigenic poxviruses: genomic organization and DNA sequence of the telomeric region of the Shope fibroma virus genome.

Shope fibroma virus (SFV), a tumorigenic poxvirus, has a 160-kb linear double-stranded DNA genome and possesses terminal inverted repeats (TIRs) of 12.4 kb. The DNA sequence of the terminal 5.5 kb of the viral genome is presented and together with previously published sequences completes the entire sequence of the SFV TIR. The terminal 400-bp region contains no major open reading frames (ORFs) but does possess five related imperfect palindromes. The remaining 5.1 kb of the sequence contains seven tightly clustered and tandemly oriented ORFs, four larger than 100 amino acids in length (T1, T2, T4, and T5) and three smaller ORFs (T3A, T3B, and T3C). All are transcribed toward the viral hairpin and almost all possess the consensus sequence TTTTTNT near their 3' ends which has been implicated for the transcription termination of vaccinia virus early genes. Searches of the published DNA database revealed no sequences with significant homology with this region of the SFV genome but when the protein database was searched with the translation products of ORFs T1-T5 it was found that the N-terminus of the putative T4 polypeptide is closely related to the signal sequence of the hemagglutinin precursor from influenza A virus, suggesting that the T4 polypeptide may be secreted from SFV-infected cells. Examination of other SFV ORFs shows that T1 and T2 also possess signal-like hydrophobic amino acid stretches close to their N-termini. The protein database search also revealed that the putative T2 protein has significant homology to the insulin family of polypeptides. In terms of sequence repetitions, seven tandemly repeated copies of the hexanucleotide ATTGTT and three flanking regions of dyad symmetry were detected, all in ORF T3C. A search for palindromic sequences also revealed two clusters, one in ORF T3A/B and a second in ORF T2. ORF T2 harbors five short sequence domains, each of which consists of a 6-bp short palindrome and a 10- to 18-bp larger palindrome. The significance of these palindromic domains in this ORF is unclear but the coincidence of the end of one larger palindrome with the end of the translated protein sequence that has homology with the B chain of insulin suggests that the palindromes may divide the T2 protein into several functional units. The salient organizational features of the complete SFV TIR are also discussed in light of what is known about other poxviral TIRs.

Amino Acid Sequence↗
PubMed1987

Tumorigenic poxviruses: transcriptional mapping of the terminal inverted repeats of Shope fibroma virus.

A composite transcriptional map for the entire 12.4-kb terminal inverted repeat (TIR) region of the Shope fibroma virus (SFV) genome has been determined. Northern blotting and S1-nuclease mapping were used to determine the regions which are transcribed, their temporal relationships, as well as the transcriptional initiation sites. Sequences representing the entire TIR are transcribed into poly(A)+ mRNA at both early and late times in the infection. Fifteen transcriptional initiation sites were mapped, 12 within the TIRs and 3 within the unique sequences close to the junction between the right TIR and the unique internal sequences. Ten of the 12 transcriptional initiation sites within the TIR and 2 of the 3 sites outside the right TIR correspond to the 5'-ends of the major open reading frames (ORFs) T1 to T9 plus the SFV growth factor gene. The 3 other initiation sites map within ORFs but near potential start codons for shorter polypeptides. All the expressed ORFs are tandemly arranged and transcribed toward the hairpin terminus. At early times during SFV infection of cultured rabbit cells, transcription of each ORF gives rise to a transcript of distinct size, while at late times termination of transcription is imprecise and substantial read-through into downstream sequences occurs. These results are discussed in light of recent observations on the related recombinant leporipoxvirus, malignant rabbit fibroma virus, which suggest that one or more gene products from this region of the SFV genome are implicated in viral tumorigenicity.

Base Sequence↗
PubMed1987

Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha.

Myxoma virus, a Leporipoxvirus and agent of myxomatosis, was shown to possess a gene with the potential to encode an epidermal growth factorlike factor. Its relationship to other members of this family, including the poxvirus growth factors from Shope fibroma virus and vaccinia virus, was analyzed. Alignment of DNA sequences and related open reading frames of myxoma virus and Shope fibroma virus indicated colinearity of genes between these poxviruses.

Amino Acid Sequence↗
PubMed1987

The genome of Shope fibroma virus, a tumorigenic poxvirus, contains a growth factor gene with sequence similarity to those encoding epidermal growth factor and transforming growth factor alpha.

Degenerate oligonucleotide probes corresponding to a highly conserved region common to epidermal growth factor, transforming growth factor alpha, and vaccinia growth factor were used to identify a novel growth factor gene in the Shope fibroma virus genome. Sequence analysis indicates that the Shope fibroma growth factor is a distinct new member of this family of growth factors.

Amino Acid Sequence↗
PubMed1987

The subunits of succinyl-coenzyme A synthetase--function and assembly.

Succinyl-CoA synthetase is made up of two kinds of subunits, designated alpha and beta. The enzyme from Escherichia coli is an alpha 2 beta 2 tetramer (mol. mass. 142 kDa), whereas the mammalian mitochondrial species is an alpha beta dimer. By means of active enzyme centrifugation, we have shown that the active form of the bacterial enzyme is the tetramer even at very low assay concentrations, while the pig heart enzyme is a non-associating dimer over a wide concentration range. The E. coli enzyme shows distinct half-of-the-sites reactivity with respect to the phosphorylation of a histidine residue in the alpha-subunit that represents a step in catalysis. Many lines of evidence (hybrid enzyme formation, oxygen exchange kinetics, 31P-n.m.r. studies) suggest that co-operative interactions between alternatingly functional active sites on the two halves of the E. coli enzyme contribute to its catalytic efficacy. In further refining this model for catalysis, we have shown that the monothiophosphorylated E. coli enzyme does not catalyse exchange of 18O from the beta, gamma-bridge to the beta-non-bridge position of ATP, indicating that the enzyme does not undergo even transient bis-phosphorylation. As a first step in studying the in vivo synthesis and assembly of the enzyme in the mammalian mitochondrial matrix, we have cloned and sequenced a 900 bp cDNA fragment that encodes most of the alpha subunit of rat liver succinyl-CoA synthetase. The derived amino acid sequence shows an impressive degree of homology to that of the alpha subunit of the enzyme from E. coli. We have shown that the alpha subunit in rat liver is a discrete nuclear gene product, complete with cleavable signal sequence to specify mitochondrial targetting.

Animals↗
PubMed1986

A novel member of the serpin superfamily is encoded on a circular plasmid-like DNA species isolated from rabbit cells.

A novel member of the serpin family of serine protease inhibitors is presented. A plasmid-like DNA was isolated from rabbit cells by its homology to the genome of Shope fibroma virus (SFV), a tumorigenic poxvirus of rabbits, and was shown elsewhere to encode a serpin-like protein [(1986) Mol. Cell. Biol. 6, 265-276]. Although significant DNA homology exists between the rabbit plasmid serpin open reading frame and the SFV terminal inverted repeat DNA there is no intact serpin counterpart encoded by this region of the SFV genome. The alignment of the novel plasmid-borne polypeptide with the serpin family of proteins confirms its status within this group.

Animals↗