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Biomedical subjects

J A Thomson

Publications and source records attributed to J A Thomson.

At least 73 records · Page 4Linked to original sources
PubMed1995

The brn-2 gene regulates the melanocytic phenotype and tumorigenic potential of human melanoma cells.

The Oct transcription factors N-Oct-3 and N-Oct-5 are differentially expressed in normal melanocytes, melanoma tumors and cell lines. We have cloned the human brn-2 gene and have shown that it encodes both the N-Oct-3 and N-Oct-5 octamer binding activities detected in melanoma cells. The brn-2 genomic locus has been mapped to chromosome 6q16 and although chromosomal aberrations are common in this region in melanoma, no deletion or rearrangement of the brn-2 gene in melanoma cell lines was observed. Sequencing of the entire gene showed that there are no intervening sequences within the open reading frame. Antisense RNA-mediated inhibition of brn-2 gene expression in melanoma cells was associated with a change in morphology and loss of melanocytic and neural crest markers, including the melanocyte transcription factor microphthalmia and the TYRP pigmentation genes. In addition, loss of brn-2 in these cells resulted in the complete loss of ability to form tumors in SCID and nu/nu mice. These results suggest roles for brn-2 in the determination of the melanocytic lineage and in the tumorigenic phenotype of melanoma.

Animals↗
PubMed1995

Isolation of a primate embryonic stem cell line.

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81) that are characteristic of human embryonal carcinoma cells. R278.5 cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers but differentiate or die in the absence of fibroblasts, despite the presence of recombinant human leukemia inhibitory factor. R278.5 cells allowed to differentiate in vitro secrete bioactive chorionic gonadotropin into the medium, express chorionic gonadotropin alpha- and beta-subunit mRNAs, and express alpha-fetoprotein mRNA, indicating trophoblast and endoderm differentiation. When injected into severe combined immunodeficient mice, R278.5 cells consistently differentiate into derivatives of all three embryonic germ layers. These results define R278.5 cells as an embryonic stem cell line, to our knowledge, the first to be derived from any primate species.

Animals↗
PubMed1995

X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex.

The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12-FK506.

Amino Acid Sequence↗
PubMed1995

Design, synthesis and structure of non-macrocyclic inhibitors of FKBP12, the major binding protein for the immunosuppressant FK506.

We have synthesized a series of non-macrocyclic ligands to FKBP12 that are comparable in binding potency and peptidyl prolyl isomerase (PPIase) inhibition to FK506 itself. We have also solved the structure of one of these ligands in complex with FKBP12, and have compared that structure to the FK506-FKBP12 complex. Consistent with the observed inhibitory equipotency of these compounds, we observe a strong similarity in the conformation of the two ligands in the region of the protein that mediates PPIase activity. Our compounds, however, are not immunosuppressive. In the FKBP12-FK506 complex, a significant portion of the FK506 ligand, its 'effector domain', projects beyond the envelope of the binding protein in a manner that is suggestive of a potential interaction with a second protein, the calcium-dependent phosphatase, calcineurin, whose inhibition by the FKBP 12-FK506 complex interrupts the T-cell activation events leading to immunosuppression. In contrast, our compounds bind within the surface envelope of FKBP12, and induce significant changes in the structure of the FKBP12 protein which may also affect calcineurin binding indirectly.

Journal Article↗
PubMed1995

Comparative X-ray structures of the major binding protein for the immunosuppressant FK506 (tacrolimus) in unliganded form and in complex with FK506 and rapamycin.

FK506 (tacrolimus) is a natural product now approved in the US and Japan for organ transplantation. FK506, in complex with its 12 kDa cytosolic receptor (FKBP12), is a potent agonist of immunosuppression through the inhibition of the phosphatase activity of calcineurin. Rapamycin (sirolimus), which is itself an immunosuppressant by a different mechanism, completes with FK506 for binding to FKBP12 and thereby acts as an antagonist of calcineurin inhibition. We have solved the X-ray structure of unliganded FKBP12 and of FKBP12 in complex with FK506 and with rapamycin; these structures show localized differences in conformation and mobility in those regions of the protein that are known, by site-directed mutagenesis, to be involved in calcineurin inhibition. A comparison of 16 additional X-ray structures of FKBP12 in complex with FKBP12-binding ligands, where those structures were determined from different crystal forms with distinct packing arrangements, lends significance to the observed structural variability and suggests that it represents an intrinsic functional characteristic of the protein. Similar differences have been observed for FKBP12 before, but were considered artifacts of crystal-packing interactions. We suggest that immunosuppressive ligands express their differential effects in part by modulating the conformation of FKBP12, in agreement with mutagenesis experiments on the protein, and not simply through differences in the ligand structures themselves.

Journal Article↗
PubMed1995

Risk factors for the development of amoxycillin-clavulanic acid associated jaundice.

OBJECTIVES: To identify risk factors for the development of amoxycillin-clavulanic acid associated jaundice. DESIGN: Retrospective case-control study. Cases were selected from those reported to the Adverse Drug Reactions Advisory Committee from the time of introduction of amoxycillin-clavulanic acid to Australia in 1986 until December 1993. SUBJECTS: Thirty-four cases, defined as individuals who developed jaundice within eight weeks of starting amoxycillin-clavulanic acid, with a biochemical picture of cholestasis, normal calibre bile ducts and no other recognised causes of jaundice or recent use of other hepatotoxic drugs, were selected. For each case, four controls who had been prescribed amoxycillin-clavulanic acid without developing jaundice were randomly selected from the patient register of the prescribing doctor. RESULTS: Increasing age was a risk factor for amoxycillin-clavulanic acid associated jaundice; patients over 55 years had an odds ratio of 16.1 (95% confidence interval [CI], 2.9-88.9) compared with patients less than 30 years. Men had an odds ratio of 2.5 (95% CI, 1.1-5.4) compared with women, although the proportion of men in the study group was larger than in the reported cases overall. History of serious medical illness, drug dose, route and duration of therapy, other medications, smoking and previous drug allergies or use of amoxycillin-clavulanic acid were not significantly associated with jaundice. CONCLUSIONS: Because of the higher risk of jaundice with increasing age, the risk-benefit ratio of amoxycillin-clavulanic acid should be carefully considered in older patients. Further assessment is necessary to clarify the association between jaundice and male sex.

Adult↗
PubMed1995

Substitution of charged residues into the hydrophobic core of Escherichia coli thioredoxin results in a change in heat capacity of the native protein.

Two site-directed mutants of Escherichia coli thioredoxin (L78K and L78R) were designed to study the effect of placing a charged residue in the hydrophobic core of the protein. Both mutants retain catalytic activity in the assembly of phage M13. Thermal denaturation of both these mutant proteins at pH 7.0 shows a reduction of stability of approximately 4 kcal.mol-1 with respect to the oxidized wild-type form. The thermal denaturation of the protein fits a dimeric state model. A significant reduction in the change in heat capacity (delta Cp) on unfolding is observed compared to oxidized wild-type thioredoxin. We present data to indicate that this reduction in delta Cp is attributable to structural perturbations resulting in localized unfolding of the native protein and exposure to solvent of residues that are buried in the wild-type protein.

Calorimetry, Differential Scanning↗
PubMed1995

Transcriptional induction and expression of the endoglucanase celA gene from a ruminal Clostridium sp. ("C. longisporum").

Northern (RNA) blot analysis of RNA from Clostridium sp. revealed induction of transcription of the celA gene when barley beta-glucan was used as carbon source, while no celA mRNA was detected after growth on cellobiose. Western blots (immunoblots), prepared by using a rabbit antiserum raised against CelA protein purified from Escherichia coli, revealed the extracellular location of CelA in Clostridium sp. Despite the absence of detectable celA mRNA, significant quantities of CelA were detected in the culture supernatant during growth on cellobiose. This finding indicated a low constitutive expression of celA. A 6.7-fold increase in the total beta-glucanase specific activity in the extracellular fraction was observed during growth on beta-glucan. The transcriptional start site of celA was mapped by extension and was found to be the same in Clostridium sp. and in E. coli expressing the cloned celA gene. A consensus E. coli -10 promoter region (AATAAT), but not a -35 promoter region, could be identified. Two direct repeats (TATTGAATTTAT) separated by 15 nucleotides flank the region where the consensus -35 promoter regions would have been. The size of the celA mRNA transcript corresponded with the size of the open reading frame. A potential stem-loop structure was found 18 nucleotides downstream of the 3' stop codon, which could be responsible for termination of transcription.

Amino Acid Sequence↗
PubMed1995

A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vitro.

A virus-encoded serine proteinase mediates four site-specific cleavages in the hepatitis C virus polyprotein. In addition to the catalytic domain, which is located in the N-terminal one-third of nonstructural protein NS3, the 54-residue NS4A protein is required for cleavage at some but not all sites. Here, we provide evidence for a non-ionic detergent-stable interaction between NS4A and the NS3 serine proteinase domain and demonstrate that the central region of NS4A plays a key role in NS4A-dependent processing. Hydrophobic residues, in particular Ile-29, were shown to be important for NS4A activity, and a synthetic peptide, spanning NS4A residues 22 to 34, could substitute for intact NS4A in a cell-free trans cleavage assay. Furthermore, NS4A mutations, which abolished or inhibited processing, correlated with destabilization of the NS3-NS4A complex. These results suggest that a stable interaction exists between the central region of NS4A and the NS3 catalytic domain which is required for NS4A-dependent processing. Since NS4A is required for processing at certain serine proteinase-dependent cleavage sites, this interaction may represent a new target for development of antiviral compounds.

Amino Acid Sequence↗
PubMed1995

Transmission of thyrotoxicosis of autoimmune type by sibling allogeneic bone marrow transplant.

This case report is on a 25-year-old male with acute myeloblastic leukaemia who achieved a remission after cytoxic therapy and whole-body irradiation. He received a bone marrow transplant from his human leucocyte antigen (HLA)-identical sister who had unsuspected autoimmune thyroid disease. Nine months later the bone marrow recipient developed thyrotoxicosis of autoimmune type at a time when his circulating lymphocytes were of 46XX karyotype. It is suspected that the thyrotoxicosis resulted from the transplantation of a clone of lymphocytes predisposed to the production of thyroid-stimulating autoantibodies.

Adult↗
PubMed1994

Structure and mechanism of interleukin-1 beta converting enzyme.

Interleukin-1 beta converting enzyme (ICE) processes an inactive precursor to the proinflammatory cytokine, interleukin-1 beta, and may regulate programmed cell death in neuronal cells. The high-resolution structure of human ICE in complex with an inhibitor has been determined by X-ray diffraction. The structure confirms the relationship between human ICE and cell-death proteins in other organisms. The active site spans both the 10 and 20K subunits, which associate to form a tetramer, suggesting a mechanism for ICE autoactivation.

Amino Acid Sequence↗
PubMed1994

A nonconsensus octamer-recognition sequence (TAATGARAT-motif) identifies a novel DNA binding protein in human Merkel cell carcinoma cell lines.

We have established a number of cell lines from Merkel cell carcinoma (MCC) of the skin. In many respects these cell lines resemble those established from small cell lung cancer (SCLC) and it is difficult to differentiate between metastatic MCC and SCLC on morphological or histochemical criteria. Both are thought to be neuroendocrine tumours and may express a number of neuroendocrine markers. SCLC cell lines express the octamer-DNA binding transcription factor brn-2 gene products N-Oct3 and N-Oct5, which are restricted to the neuroectodermal cell lineage. In DNA binding studies using a consensus octamer recognition site we have found that 4 of 8 MCC cell lines examined expressed brn-2 in at least trace amounts compared with 3 SCLC cell lines which all expressed brn-2 proteins at high levels. Moreover, these DNA binding studies were extended by using a high-affinity brn-2 recognition site related to the degenerate octamer TAATGARAT-motif. This identified a novel DNA binding protein in a subset of MCC cell lines. The protein was absent from the three SCLC cell lines, melanoma cells and brain tissue. This binding activity, which we term Merkel cell DNA nuclear (MNF), was shown to be specific by competitive inhibition with oligonucleotide binding sites and was not inhibited by polyclonal antisera against the Oct-1, Oct-2 or Brn-2 proteins. This protein may serve as a unique marker for MCC compared with SCLC cells and may be involved in regulating the Merkel cell phenotype.

Base Sequence↗
PubMed1994

Investigation of the STR locus HUMTH01 using PCR and two electrophoresis formats: UK and Galician Caucasian population surveys and usefulness in paternity investigations.

Two hundred unrelated Caucasians from the UK and 210 from Galicia (NW Spain) have been genotyped for the HUMTH01 locus using polymerase chain reaction (PCR) amplification followed by high sieving agarose electrophoresis (UK samples) and polyacrylamide electrophoresis (Galician samples). Allele and genotype frequencies obtained from both populations were in close agreement to those seen by other workers and chi 2 tests of the two populations showed that both were in Hardy-Weinberg equilibrium. The potential usefulness of HUMTH01 in paternity investigations was analysed by constructing, within each population, false family trios, each with a non-father, in order to estimate the actual exclusion rate. The observed rate of exclusion was in close agreement with the expected rate for both populations. Examination of the mother-child pairs in the false families showed a common allele in every case and no evidence of mutation or non-Mendelian inheritance was observed. The HUMTH01 locus shows an informative polymorphism and the production and analysis of population databases is easily achieved. The use of PCR followed by electrophoresis in either gel format appears to provide a quick and straightforward method for investigating the HUMTH01 locus.

Adult↗
PubMed1994

Enthalpy of hydrogen bond formation in a protein-ligand binding reaction.

Parallel measurements of the thermodynamics (free-energy, enthalpy, entropy and heat-capacity changes) of ligand binding to FK506 binding protein (FKBP-12) in H2O and D2O have been performed in an effort to probe the energetic contributions of single protein-ligand hydrogen bonds formed in the binding reactions. Changing tyrosine-82 to phenylalanine in FKBP-12 abolishes protein-ligand hydrogen bond interactions in the FKBP-12 complexes with tacrolimus or rapamycin and leads to a large apparent enthalpic stabilization of binding in both H2O and D2O. High-resolution crystallographic analysis reveals that two water molecules bound to the tyrosine-82 hydroxyl group in unliganded FKBP-12 are displaced upon formation of the protein-ligand complexes. A thermodynamic analysis is presented that suggests that the removal of polar atoms from water contributes a highly unfavorable enthalpy change to the formation of C=O...HO hydrogen bonds as they occur in the processes of protein folding and ligand binding. Despite the less favorable enthalpy change, the entropic advantage of displacing two water molecules upon binding leads to a slightly more favorable free-energy change of binding in the reactions with wild-type FKBP-12.

Carrier Proteins↗
PubMed1994

Site selectivity of daunomycin.

We have reexamined the binding properties of the antitumor drug daunomycin using double-helical oligonucleotides 16 base pairs long that were designed to contain preferred binding sites for the drug. The preferred sites are contained in a six base pair core which is flanked on the 5' and 3' ends by tracts of adenines. The flanking sequences, which augment helix stability and reduce and effects, were chosen because daunomycin is known to bind poorly to poly(dA).poly(dT). Four major sequences were examined in the six base pair core: CGTACG, TAGCTG, TCATCC, and (TA)3 and compared with calf thymus DNA. A randomly generated 16 bp sequence containing no A tracts and a sequence containing only tracts of As and Ts were also used. Fluorometric, absorption, calorimetric, and stopped-flow techniques were used to examine the binding. The affinity of the drug for oligomers containing known binding sites was comparable to or enhanced relative to that for calf thymus bulk DNA. Association constants ranged from 1.0 x 10(8) to 3.0 x 10(7) M-1. The strongest core binding site found was CGTACG, but its affinity is only 2-fold larger than that of other core sequences. Appreciable binding to the flanking A tracts was observed. An oligonucleotide which incorporates the CGTACG sequence in a short hairpin helix binds an order of magnitude more weakly. Complex lifetimes measured by stopped flow generally increase with equilibrium stability; the kinetics confirm the existence of a set of weaker sites. The exothermic binding enthalpy for daunomycin with the CGTACG core sequence is more than twice as large as for the TATATA sequence. Binding to dA20.dT20 is endothermic, and a less exothermic component can be detected in the calorimetric binding curve of the oligomers containing flanking A tracts.

Animals↗
PubMed1994

Coat protein-mediated resistance in transgenic plants.

This review describes the proposed mechanism(s) of classical virus cross-protection in plants, followed by those suggested for coat protein-mediated resistance (CP-mediated resistance). Although both have common features, cross-protection is thought to be a complex response caused by the replication and expression of the entire viral genome, whereas the resistance conferred by the expression of a virus coat protein gene is more limited. The term genetically engineered cross-protection is frequently used because in many cases the phenotype of resistance mimics that of cross-protection. However, CP-mediated resistance, although a narrow term, more accurately describes the resistance that results from the expression of a virus CP gene in transgenic plants.

Capsid↗