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Biomedical subjects

J A Thomson

Publications and source records attributed to J A Thomson.

At least 91 records · Page 5Linked to original sources
PubMed1994

Nonsurgical uterine stage preimplantation embryo collection from the common marmoset.

A nonsurgical technique for the recovery of uterine stage preimplantation embryos was developed for the common marmoset (Callithrix jacchus). In 54 flush attempts, using 19 different animals, 54 morphologically normal embryos, seven unfertilized oocytes or degenerate embryos, and five empty zonae pellucidae were recovered, giving a recovery rate of 1.0 embryo per flush or 1.2 ovulation products per flush. Because the ovarian cycles of common marmosets can be synchronized with prostaglandin PGF2 alpha, multiple marmosets can be flushed in a short period, providing age-matched embryos for controlled experiments.

Animals↗
PubMed1994

Expression studies of pigmentation and POU-domain genes in human melanoma cells.

Human melanoma cell lines have been used to examine the regulation of the tyrosinase (TYR) and tyrosinase-related protein genes TRP-1 and TRP-2 in response to differentiating chemicals and UV irradiation. TRP-1 mRNA levels can be repressed by treatment with the differentiating chemicals DMSO and HMBA. There is little effect of UV irradiation on pigment synthesis by human melanoma cell lines or tyrosinase activity, with variable effects on the levels of the TYR, TRP-1, and TRP-2 gene transcripts. The human TRP-1 gene promoter has been isolated and its activity tested by transient cell transfection to begin an examination of signal transduction mechanisms operating in response to pigmenting and differentiating agents. To identify transcription factors that may be involved in melanocytic gene expression, we studied the N-Oct-3 and N-Oct-5 octamer-binding activities normally expressed in the neuroectodermal cell lineage and which are expressed at high levels in melanoma cells. POU-domain-containing cDNA have been isolated from the A2058 human melanoma cell line that are homologous to the brn-2 gene that encodes N-Oct-3 and N-Oct-5.

Acetamides↗
PubMed1994

Construction of a Bioinsecticidal Strain of Pseudomonas fluorescens Active against the Sugarcane Borer, Eldana saccharina.

A cryIA(c) gene was cloned from a native Bacillus thuringiensis strain showing activity against the sugarcane borer, Eldana saccharina. The sequence of the cloned gene was very similar to that of the B. thuringiensis subsp. kurstaki HD-73 cryIA(c) gene. The gene was introduced into an isolate of Pseudomonas fluorescens, capable of colonizing sugarcane, on two broad-host-range plasmids, pDER405 and pKT240, having copy numbers of 13 and 28, respectively. By using the Omegon-Km vector, the cry gene was introduced into the chromosome of P. fluorescens isolate 14. Bioassays on eldana larvae showed that the strain carrying the gene integrated into the chromosome was as toxic as one carrying it on pKT240. Glasshouse trials indicated that sugarcane treated with P. fluorescens 14::Omegon-Km-cry were more resistant to eldana damage than untreated sugarcane was.

Journal Article↗
PubMed1994

Hypothyroidism presenting with cardiac tamponade.

Two cases of cardiac tamponade initially suspected to be secondary to malignancy are presented. Primary hypothyroidism can cause pericardial effusions and thyroid function tests confirmed this diagnosis in these cases. Hypothyroidism should be considered as an underlying cause of pericardial effusion in cardiac tamponade.

Aged↗
PubMed1994

Concentration of ptaquiloside, a major carcinogen in bracken fern (Pteridium spp.), from eastern Australia and from a cultivated worldwide collection held in Sydney, Australia.

Two surveys of bracken fern for the concentration of the carcinogen ptaquiloside (PT) have been carried out, one of bracken fern from the eastern side of Australia and the other from a worldwide collection of bracken clones held in Sydney Australia. Bracken from eastern Australia contained concentrations of ptaquiloside ranging from 0 to 12,945 micrograms PT/g. From 91 samples 15% contained greater than 5,000 micrograms PT/g and 57% of samples contained more than 1,000 micrograms PT/g bracken on the dry weight basis. Ptaquiloside concentrations were highest in Pteridium revolutum and from P. esculentum from areas where bovine enzootic haematuria was known to occur. Bracken from the cultivated bracken clone collection from world-wide sources tended to have lower concentrations of ptaquiloside ranging from 0 to 9,776 micrograms PT/g. From 77 samples, 8% contained more than 5,000 micrograms PT/g and 35% contained more than 1,000 micrograms PT/g bracken. Samples from both the eastern Australia survey and the Australian representatives in the worldwide collection showed significantly higher concentrations of PT in the P. esculentum collected from the more southern states. In samples from the worldwide collection there were no statistical differences in the concentrations of PT in bracken between taxa.

Analysis of Variance↗
PubMed1993

15N NMR relaxation studies of the FK506 binding protein: backbone dynamics of the uncomplexed receptor.

Backbone dynamics of the major tacrolimus (FK506) binding protein (FKBP-12, 107 amino acids) have been studied using 15N relaxation data derived from proton-detected two-dimensional 1H-15N NMR spectroscopy. 15N spin-lattice relaxation rate constants (R1), spin-spin relaxation rate constants (R2), and heteronuclear NOEs were determined for over 85% of the backbone amide 15N nuclei. A model free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559; Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4559-4570] was used to derive values for the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and the chemical exchange line width (Rex) for each N-H bond vector. The final optimized overall correlation time (tau m) was 9.2 ns. The average order parameter (S2) describing the amplitude of motions on the picosecond time scale was found to be 0.88 +/- 0.06. Motions on the picosecond time scale are restricted at the N and C termini, consistent with previous NMR structural studies indicating well-defined beta-strands in these regions. With the exception of the flap region from resides 82 to 87, no regions appear to be significantly disordered on the picosecond time scale. Residues in several regions of the protein exhibit high Rex terms, indicating possible motions on the millisecond to microsecond time scale due to chemical exchange and/or conformational averaging effects. Possible effects of tacrolimus (FK506) binding on FKBP-12 dynamics are discussed in the context of previously determined solution structures for FKBP-12 in the uncomplexed [Michnick et al. (1991) Science 252, 836-839; Moore et al. (1991) Nature 351, 248-250] and complexed [Meadows et al. (1993) Biochemistry 32, 754-765] states.

Amides↗
PubMed1993

Probing hydration contributions to the thermodynamics of ligand binding by proteins. Enthalpy and heat capacity changes of tacrolimus and rapamycin binding to FK506 binding protein in D2O and H2O.

The stabilities of native proteins and protein-ligand complexes result from differential interactions among numerous polar and nonpolar atoms within the proteins and ligands and of these atoms with water. Delineation of the various energetic contributions of the stabilities of proteins or protein-ligand complexes in aqueous solution, and an evaluation of their structural basis, requires a direct account of the changes, in the interactions of the protein with the solvent, that accompany the folding or binding reactions. Two largely nonpolar, structurally related macrolide ligands, tacrolimus (also known as FK506) and rapamycin, each bind with high affinity to a common site on a small FK506 binding protein (FKBP-12) and inhibit its peptidylprolyl cis-trans-isomerase activity. In an effort to elucidate the influence of water on the thermodynamics of their binding reactions, we have measured the enthalpies of tacrolimus and rapamycin binding to FKBP-12, in buffered solutions of H2O (at pH 7.0) or D2O (at pD 7.0), by high-precision titration calorimetry in the temperature range 5-30 degrees C. For both tacrolimus and rapamycin binding, a large enthalpic destabilization of binding is observed in D2O relative to H2O, in the temperature range examined. Additionally, large negative constant pressure heat capacity changes are observed for the binding of the ligands in both H2O and D2O. A thermodynamic analysis is presented to identify the structural determinants of the differences in the energetics of binding in light and heavy water. The analysis suggests that a chief contributor to the observed enthalpic destabilization is the differential hydration, of protein and ligand atoms, by light and heavy water.

Carrier Proteins↗
PubMed1993

Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.

Amino Acid Sequence↗
PubMed1993

Cloning of an endo-(1-->4)-beta-glucanase gene, celA, from the rumen bacterium Clostridium sp. ('C. longisporum') and characterization of its product, CelA, in Escherichia coli.

A genomic library of Clostridium sp. ('C. longisporum') ATCC 49440 in the host Escherichia coli was screened for endo-beta-glucanases, and plasmids pCM64 and pCM4 were isolated. The nucleotide sequence of a 3620 bp fragment was found to contain a 1548 bp open reading frame (ORF), termed celA, which encodes an endo-(1-->4)-beta-glucanase, CelA, assigned to family A4. N-terminal amino acid sequence determination revealed that pCM64 encoded the full-length celA gene, including a signal sequence, while pCM4 carried a 5'-truncated celA gene expressed as an N-terminal fusion protein, CelA delta N', without a signal sequence. CelA was secreted into the periplasm in E. coli. In this organism, proteolytic cleavage of CelA at or near a putative linker region resulted in the appearance of two active polypeptides of molecular masses 57 and 47 kDa. The former was the full-length enzyme while the latter consisted of the catalytic domain from which the cellulose-binding domain (CBD) had been removed (CelA delta CBD). The intracellularly-located CelA delta N' was not subject to proteolytic degradation. The pH and temperature optima of CelA were pH 4.8 and 43 degrees C, respectively. CelA hydrolysed barley beta-glucan, lichenan, carboxymethylcellulose and xylan. It showed preferential activity against the larger cellooligosaccharides (cellohexaose and cellopentaose); cellotetraose was the smallest substrate degraded completely.

Amino Acid Sequence↗
PubMed1993

Transcriptional regulation of an endoglucanase and a cellodextrinase gene in Ruminococcus flavefaciens FD-1.

A gene which encodes a 35 kDa protein with both carboxymethylcellulase and xylanase activity was cloned from Ruminococcus flavefaciens FD-1 and the nucleotide sequence determined. The FD-1 gene, celE, and the celA gene, which encodes a cellodextrinase, were used as probes to analyse transcription in R. flavefaciens grown under different conditions. Transcription of both genes was induced when cellulose was added to cells growing in cellobiose. This induction continued after cellulose depletion and after cell division had ceased. Transcription of both genes was also induced by cellotriose, although the effect was not as pronounced as induction by cellulose and was greater for the celA gene than for the celE gene.

Amino Acid Sequence↗
PubMed1993

In vivo and in vitro expression of octamer binding proteins in human melanoma metastases, brain tissue, and fibroblasts.

The pattern of octamer sequence-specific DNA binding proteins expressed in human melanoma was examined in nuclear extracts of seven surgically-isolated tumors, short-term cultures of these tumors, and 25 human melanoma cell lines to determine the in vivo and in vitro distribution of the melanocytic-associated Oct-M1 and Oct-M2 octamer binding activities. In the biopsy tissue and cultured melanoma cells of a metastasis from the cerebellum, two other binding activities (N-Oct-2 and N-Oct-6) in addition to the Oct-M1, Oct-M2 and the generally expressed Oct-1 protein were detected; this profile was consistent with that seen in normal human and mouse brain tissue. Melanoma tissue removed from lymph nodes and cell lines established from them also showed Oct-1, Oct-M1, Oct-M2, and N-Oct-2. N-Oct-2 was distinguished from the comigrating Oct-2A activity by failure to react with Oct-2A-specific antibody. All but one of the 25 melanoma cell lines exhibited Oct-1, Oct-M1, and Oct-M2 and/or N-Oct-2 activity, whereas cultured normal melanocytes expressed only Oct-1 and Oct-M1. In contrast to murine fibroblasts, which express only Oct-1, human fibroblast strains also expressed Oct-2A binding activity, which was confirmed by reactivity with Oct-2A antibody and the presence of Oct-2A mRNA and indicated that Oct-2A has a more general role than that of a lymphoid-specific transcription factor. Overall, the results indicate that expression of neural-specific Oct factors in human melanoma is (1) aberrant compared with normal melanocytes, (2) can be modulated by the surrounding tissue in a brain metastasis, and (3) may be part of the altered program of differentiation accompanying transformation.

Animals↗
PubMed1993

Contact sensitization to oxazolone: involvement of both interferon-gamma and interleukin-4 in oxazolone-specific Ig and T-cell responses.

The synthesis and role of several lymphokines were examined during contact sensitization to oxazolone (OX). Application of OX to the skin of mice increased the delayed-type hypersensitivity (DTH) response to challenge, serum titres of OX-specific IgG1 and IgG2a, and draining lymph node cell (LNC) numbers. At day 3, LN contained detectable interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-2 or IL-3 mRNAs; IL-3 and higher levels of IL-4, IFN-gamma and GM-CSF mRNAs were measured after 24 hr culture with anti-CD3 antibody in OX-primed but not unprimed LNC. As a result of sensitization, LNC secreted IL-3 constitutively and produced elevated levels of IL-2, IL-3, IL-4 and IFN-gamma in response to anti-CD3 antibody; a similar but weaker lymphokine response was recalled by OX-protein conjugate. CD4+ cells were the major source of the anti-CD3-induced lymphokines except IFN-gamma, which was derived mainly from CD8+ cells. Since both IL-4 and IFN-gamma were synthesized by OX-primed LNC in vivo and in vitro, their role was investigated by administering anti-lymphokine antibodies at the time of sensitization. Anti-IL-4 treatment reduced OX-specific serum IgG1 titres without affecting IgG2a titres, whereas anti-IFN-gamma treatment reduced IgG2a but not IgG1 titres. Although neither antibody altered DTH responsiveness, anti-IFN-gamma treatment markedly increased IL-4 production by CD4+ LNC and reduced IFN-gamma production in vitro, particularly by CD4+ cells. We conclude that endogenous IL-4 and IFN-gamma reciprocally influence the isotype of the Ig response to OX and that IFN-gamma also affects the relative levels of IL-4 and IFN-gamma synthesis by CD4+ LNC.

Animals↗
PubMed1992

Heat capacity changes and hydrophobic interactions in the binding of FK506 and rapamycin to the FK506 binding protein.

Differential interactions among nonpolar moieties at protein/ligand interfaces, and of these nonpolar groups with water, collectively termed hydrophobic interactions, are widely believed to make important energetic contributions to the stability of protein/ligand complexes. Quantitative estimates of hydrophobic interactions, and an evaluation of their structural basis, are essential for obtaining structure-based predictions of the free energies of binding for the purpose of drug design. Two largely nonpolar, immunosuppressive agents, FK506 and rapamycin, each bind with high affinity to a common hydrophobic pocket on a small peptidylproline cis-trans isomerase known as FK506 binding protein (FKBP-12) and inhibit its activity. In an effort to elucidate the structural features of these ligands responsible for the observed energetics, we have undertaken an investigation of the thermodynamics of binding of FK506 and rapamycin to FKBP-12. Enthalpies of binding have been determined by high-precision titration calorimetry over a range of temperature, allowing estimates of heat capacity changes. By analyzing the distribution of changes in solvent-accessible surface area upon binding of FK506 to FKBP-12 from crystallographic data, it is found that 99% of the net surface buried upon binding involves nonpolar groups. This leads to a heat capacity change of FK506 binding, normalized to the amount of nonpolar surface, of -0.40 +/- 0.02 cal.K-1.mol-1.A-2 (1 cal = 4.18 J), a value similar to that obtained for the aqueous dissolution of hydrophobic substances. Our observations are discussed in view of the general nature of hydrophobic interaction processes.

Calorimetry↗