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Biomedical subjects

M Parsons

Publications and source records attributed to M Parsons.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and expression of Trypanosoma cruzi ribosomal protein P0 and epitope analysis of anti-P0 autoantibodies in Chagas' disease patients.

Chagas' disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a major cause of heart failure in endemic areas. Antigenic mimicry by T. cruzi antigens sharing epitopes with host macromolecules has been implicated in the pathogenesis which is thought to have a significant autoimmune component. We report herein on the cloning and characterization of a full-length cDNA from a T. cruzi expression library encoding a protein, TcP0, that is homologous to the human 38-kD ribosomal phosphoprotein HuP0. The T. cruzi P0 protein shows a clustering of residues that are evolutionarily conserved in higher eukaryotes. This includes an alanine- and glycine-rich region adjacent to a highly charged COOH terminus. This "hallmark" domain is the basis of the crossreactivity of the highly immunogenic eukaryotic P protein family. We found that T. cruzi-infected individuals have antibodies reacting with host (self) P proteins, as well as with recombinant TcP0. Deletion of the six carboxy-terminal amino acids abolished the reactivity of the T. cruzi infection sera with TcP0. This is similar to the specificity of anti-P autoantibodies described for a subset of patients with systemic lupus erythematosus (SLE) (Elkon, K., E. Bonfa, R. Llovet, W. Danho, H. Weissbach, and N. Brot. 1988. Proc. Natl. Acad. Sci. USA. 85:5186). These results suggest that T. cruzi P proteins may contribute to the development of autoreactive antibodies in Chagas' disease, and that the underlying mechanisms of anti-P autoantibody may be similar in Chagas' and SLE patients. This study represents the first definitive report of the cloning of a full-length T. cruzi antigen that mimics a characterized host homologue in structure, function, and shared antigenicity.

Amino Acid Sequence↗
PubMed1992

The refinding of theory in clinical practice.

The analyst's use of his preconscious is central to understanding how clinical practice in psychoanalysis is informed by theory. One view is that we have a definitive conception of our theory, from which we derive our clinical practice by largely preconscious deliberation. Another view sees psychoanalytic concepts as elastic, related to each other along dimensions of meaning in a finite but flexible theoretical space. We do not, on this view, determine once for all what our theory is, but continually have to discover it by exploring our preconscious as we watch ourselves at work. Some detailed clinical material shows how I observed the evolution of my use of theory in a particular case. I had to refind theory which I already knew; familiar concepts had to become surprising. This also allowed theoretical understanding to emerge which was new to me. This implies an act of personal engagement with our theoretical concepts, rather than the pseudo-objective application of what are taken for facts. A survey of various positions in the philosophy of science shows that this is entirely consistent with the nature of scientific activity.

Austria↗
PubMed1991

Distinct patterns of tyrosine phosphorylation during the life cycle of Trypanosoma brucei.

Regulation of tyrosine phosphorylation is a critical element in controlling growth and differentiation in higher eukaryotes. We have determined that the protozoan Trypanosoma brucei, which diverged early in the eukaryotic lineage, possesses multiple proteins which react with a specific anti-phosphotyrosine antiserum. Anti-phosphotyrosine immunoprecipitates of [32P]orthophosphate-labeled cells were shown to contain phosphotyrosine by two-dimensional electrophoresis. Western analysis of cells from different stages of the life cycle demonstrates the appearance of tyrosine-phosphorylated proteins at 40-42 kDa during the transition from slender to stumpy blood-forms. Growth of procyclic form cells in orthovanadate resulted in increased levels of specific tyrosine-phosphorylated proteins. The demonstration of phosphotyrosine-containing proteins in T. brucei and their differential regulation during the life cycle suggests that tyrosine kinases and phosphatases may play an important role in the biology of primitive protozoa.

Animals↗
PubMed1991

A phosphoglycerate kinase-like molecule localized to glycosomal microbodies: evidence that the topogenic signal is not at the C-terminus.

The phosphoglycerate kinase (PGK) gene complex of Trypanosoma brucei contains three tandemly linked related genes. One gene encodes a cytoplasmic PGK, while another encodes a PGK isozyme localized to glycosomal microbodies. In this communication, we report that the third gene in this complex encodes a 56-kDa molecule which is also localized to the glycosomal core. DNA sequence analysis indicates that this gene contains multiple substitutions and a large insertion in the amino domain, but that it is very similar to the other PGK isozymes in the carboxy domain. The C-terminal tripeptide is identical to that of the cytoplasmic isozyme, and only one conservative change occurs in the last 25 amino acids. The encoded protein, p56, thus contrasts with the many peroxisomal microbody proteins in which the C-terminal tripeptide contains sufficient information for targeting to peroxisomes. Multiple mechanisms may exist for targeting proteins to the protein cores of microbody organelles. Comparisons of the DNA sequences of several alleles suggest that homologous recombination plays a role in the generation of allelic diversity.

Alleles↗
PubMed1990

Microbody phosphoglycerate kinase of Trypanosoma brucei: expression and complementation in Escherichia coli.

In the primitive eukaryotic parasite, Trypanosoma brucei, most of the enzymes of glycolysis are located within microbody organelles called glycosomes. Proteins destined for the glycosome are synthesized on free ribosomes and post-translationally translocated into the organelle. The gene, gPGK, encoding the glycosomal isozyme of phosphoglycerate kinase (gPGK), was cloned adjacent to a T7 promoter and cotransformed with a plasmid encoding T7 RNA polymerase into Escherichia coli Pgk-cells. Functional complementation occurred, but only after the creation of a ribosome-binding site by mutagenesis. This represents the first example of complementation of an E. coli mutant with a gene encoding a microbody protein. Enzymatically active recombinant gPGK was purified to near homogeneity by ion exchange chromatography from highly expressing E. coli. The recombinant protein will aid in studies of glycosomal biogenesis.

Animals↗
PubMed1990

Trypanosoma brucei: two-dimensional gel analysis of the major glycosomal proteins during the life cycle.

Kinetoplastid organisms possess a unique organelle, the glycosome, which compartmentalizes the Embden-Meyerhof segment of glycolysis and several other metabolic pathways. In Trypanosoma brucei many of the enzyme activities localized to the glycosome are stage regulated. Two-dimensional gel analysis was used to examine the characteristics, expression, and biosynthesis of the major glycosomal proteins. Two-dimensional gel maps of glycosomes from slender bloodforms and late intermediate-stumpy bloodforms (the precursors of procyclic forms) were indistinguishable, while those of procyclic form glycosomes showed extensive differences. Glycosomal phosphoenolpyruvate carboxykinase and malate dehydrogenase were identified to have subunit molecular weights of 60 and 34 kDa, respectively. We detected two hitherto undescribed glycosomal proteins, one of which is found only in bloodforms. All of the major proteins, except glucose phosphate isomerase, were highly basic. Stage regulation of glycosomal enzyme activities correlated with stage regulation of specific protein biosynthesis.

Animals↗
PubMed1990

Leishmania gp63 molecule implicated in cellular adhesion lacks an Arg-Gly-Asp sequence.

The parasitic protozoa Leishmania are intracellular pathogens which enter host cells through largely undefined mechanisms. One molecule thought to play an important role in this process is gp63, the major glycoprotein on the surface of the infective promastigote form. We have cloned and analyzed the gp63 gene from Leishmania chagasi, an etiologic agent of acute visceral leishmaniasis. The predicted amino acid sequence is highly homologous to that reported for Leishmania major, with the exception of a 56-amino-acid region. This region in L. major was predicted to contain an arginine-glycine-aspartic acid (RGD) sequence that was subsequently hypothesized to be involved in binding to the host cell. The L. chagasi gene lacks this sequence or indeed any RGD sequence, and further studies failed to confirm the existence of an RGD sequence in the L. major gp63 gene. Binding to the host cell surface must therefore be mediated by other sequences in gp63 or by other components of the Leishmania promastigote.

Amino Acid Sequence↗
PubMed1990

Some issues affecting termination. The treatment of a high-risk adolescent.

This paper addresses some of the difficulties of termination in the psychoanalytic psychotherapy of a deprived, high-risk adolescent girl, who had two previous treatments. Some commonly referred to issues are explored: termination and the developmental process of adolescence; the relationship between termination, mourning, and internalization; the need for the resolution of the transference and careful monitoring of the countertransference; the importance of the transference resolution of the oedipus complex and related issues concerning preoedipal development and identification; and the consolidation of ego strength.

Adolescent↗
PubMed1990

Characterization of an in vitro assay for import of 3-phosphoglycerate kinase into the glycosomes of Trypanosoma brucei.

Glycosomes are microbody organelles found in kinetoplastida, where they serve to compartmentalize the enzymes of the glycolytic pathway. In order to identify the mechanism by which these enzymes are targeted to the glycosome, we have modified the in vitro import assay developed by Dovey et al. (Proc. Natl. Acad. Sci. USA 85:2598-2602, 1988). This assay measures the uptake of in vitro-translated Trypanosoma brucei glycosomal 3-phosphoglycerate kinase (gPGK) by purified glycosomes. Up to 50% of the total 35S-gPGK in the glycosomal fraction was resistant to extraction by 3 M urea or treatment with proteinase K (500 micrograms/ml). The glycosome-associated 35S-gPGK could be chemically cross-linked to the endogenous glycosomal proteins to form a sodium dodecyl sulfate-resistant complex, suggesting that it is close to the intraglycosomal protein matrix. Deoxycholate solubilized the glycosome and thereby rendered the glycosome-associated 35S-gPGK fully susceptible to proteinase K. However, the glycosome-associated 35S-gPGK was not digested by proteinase K in the presence of Triton X-100, which cannot dissolve the glycosomal protein core. The 35S-gPGK synthesized in vitro was able to bind directly to protein cores, where it became resistant to urea extraction and proteinase K digestion. However, the 35S-gPGK-protein core complex exhibited a much higher density than the 35S-gPGK-glycosome complex and was readily separable in sucrose gradients. Thus, in our in vitro import assay, the 35S-gPGK appeared to associate with intact glycosomes, possibly reflecting import of protein into the organelle. Complete denaturation of the 35S-gPGK in 8 M urea prior to the assay enhanced the efficiency of its association with glycosomes. Native gPGK did not compete with the association of in vitro-translated gPGK unless it was denatured. The assay exhibited time and temperature dependence, but it did not require externally added ATP and was not inhibited by the nonhydrolyzable analogs adenosine-5'-(beta,gamma-imido)-triphosphate and gamma-S-ATP. However, the presence of 20 to 30 microM ATP inside the glycosome may fulfill the requirement for protein import.

Animals↗
PubMed1989

Elevated phosphoglycerate kinase mRNA but not protein in monomorphic Trypanosoma brucei: implications for stage-regulation and post-transcriptional control.

Phosphoglycerate kinase (PGK) is present in high levels in the glycosomes of bloodstream stage Trypanosoma brucei, but is virtually absent in procyclic stage glycosomes. Glycosomes isolated from slender and stumpy stage bloodforms show similar levels of PGK, although levels are slightly lower in stumpy forms. Lower levels of glycosomal PGK transcripts are observed in stumpy form RNA, paralleling the decrease in glycosomal PGK activity. Monomorphic strains and pleiomorphic strains show similar glycosomal PGK activity, but monomorphic strains have much higher levels of the glycosomal PGK transcript. In three separate cases, predominantly monomorphic strains derived from highly pleiomorphic strains showed increased levels of glycosomal PGK (gPGK) mRNA. gPGK synthesis rates in monomorphic and pleiomorphic strains were similar, and no significant differences in turnover were observed. These data suggest the possibility of translational control of gPGK protein levels in trypanosome bloodforms. The data also indicate that the metabolism of gPGK mRNA in highly passaged laboratory strains is altered, and counsel caution when attributing differences in transcript levels to stage-specific regulation.

Animals↗
PubMed1989

The pharmacological evaluation of LY 170680, a novel leukotriene D4 and E4 antagonist in the guinea-pig.

1. This paper describes the evaluation of LY170680 (5-less than 3-[2(R) (carboxyethylthio)-(S)-hydroxypentadeca 3(E)5(Z)-dienyl]phenyl greater than 1H tetrazole, as an antagonist of the cysteinyl leukotrienes C4, D4, E4, in a variety of in vitro and in vivo models. 2. In vitro, LY170680 was a potent, selective, and competitive antagonist of LTD4, and LTE4. It produced a concentration-dependent rightward displacement of the concentration-response curves elicited by either LTD4 or LTE4 on both the guinea-pig ileal and tracheal preparation. The pA2 values for LY170680 were estimated to be 8.1 +/- 0.2 (n = 8) and 8.1 +/- 0.1 (n = 6) on trachea, and 8.7 +/- 0.1 (n = 12) and 9.0 +/- 0.3 (n = 6) on ileum for both LTD4 and LTE4 respectively. The slopes of the Schild plots in these studies were all close to unity. 3. LY170680 was shown to be a modest antagonist of the LTC4-induced responses on guinea-pig ileum (pA2 7.0 +/- 0.2 n = 5), but had no discernable effects against contractions induced by histamine, prostaglandin E2 (PGE2), PGF2 alpha or acetylcholine. The compound also reduced the LTC4-induced responses on trachea, but in a non competitive manner. 4. Intravenous LY170680 reduced in a dose-dependent manner (ED50 3.8 mg kg-1, 60 min pretreatment) the fall in compliance in the anaesthetized guinea-pig, and the rise in total pulmonary resistance (TPR) (ED50 2.0 mg kg-1, 30 min pretreatment) in the artificially ventilated guinea-pig, produced by intraveous LTD4. 5. LY170680 (5mgkg-1, i.v.) was also effective in reducing the increase in TPR to intravenous antigen in sensitized animals pretreated with mepyramine, indomethacin and propranolol. 6. The compound was only moderately effective (ED5o 40mgkg-1 p.o.) in preventing the rise in TPR induced by intravenous LTD4, when given orally. 7. Inhaled LY170680 was particularly effective in preventing the increase in TPR produced by an exposure to aerosolised LTD4. An estimated 1-2 pg of LY170680 delivered to the airways by nebuliser 1 hour beforehand, produced a 6 fold lateral displacement to the right of the dose-response curve to LTD4. 8. Studies in conscious animals indicated that inhaled LY170680 produced a dose-dependent reduction in the increased volume of gas, trapped in the lung following exposure to aerosolised LTD4. Duration studies also indicated that an estimated inhaled dose of 10 g LY170680 significantly reduced the LTD4-induced' increase in trapped lung gas volume for at least 4 h. Inhaled LY170680 also reduced LTC4-induced increase in gas trapping in a manner similar to LTD4. However, histamine-induced gas trapping was unaffected.

Administration, Inhalation↗
PubMed1988

A comparison of epinephrine and methoxamine for resuscitation from electromechanical dissociation in human beings.

Electromechanical dissociation (EMD) is an organized electrical depolarization of the heart without synchronous myocardial fiber shortening and, therefore, without cardiac output. Patients in EMD have a poor prognosis for resuscitation and long-term survival. The beneficial effect in resuscitation of epinephrine, the adrenergic agent currently recommended, has been shown to depend on stimulation of alpha-adrenergic vasoconstriction. The beta-adrenergic inotropic and chronotropic effects of epinephrine are theoretically detrimental by increasing myocardial oxygen consumption and subendocardial ischemia. The purpose of our study was to determine whether the pure alpha agonist methoxamine was superior to epinephrine in human beings in EMD as determined by survival at one hour. These two agents were compared in a prospective, randomized, and double-blinded study involving 80 patients with EMD of various causes seen in the emergency department and internal medicine inpatient service. The advanced cardiac life support (ACLS) algorithm (current at the time of our study) for resuscitation from EMD was used, with the blinded study drug (epinephrine 1 mg or methoxamine 10 mg) administered where the algorithm calls for epinephrine. Calcium and isoproterenol also were used in the majority of cases according to ACLS standards but never prior to the use of methoxamine or epinephrine. Survival data are summarized as: survival less than one hour, 22 patients receiving methoxamine, 22 receiving epinephrine; one to six hours, 15 patients receiving methoxamine, 13 patients receiving epinephrine; six to 12 hours, one patient receiving epinephrine; more than 24 hours but not surviving to discharge, three patients receiving methoxamine, two patients receiving epinephrine; and survival to discharge, one patient receiving epinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Biogenesis of glycosomes of Trypanosoma brucei: an in vitro model of 3-phosphoglycerate kinase import.

Glycosomes are intracellular, membrane-bound microbody organelles of trypanosomes and leishmania. Nine glycolytic enzymes are the major protein components of the glycosomes of Trypanosoma brucei long-slender bloodstream forms. Glycosomal proteins are believed to be synthesized in the cytoplasm and inserted across the glycosomal membrane posttranslationally. We have developed an in vitro protein import assay for the study of glycosomal biogenesis in T. brucei. All nine glycosomal glycolytic enzymes were detectable by immunoprecipitation and gel analysis of radiolabeled products derived from in vitro translation of total mRNA. Radiolabeled translational products were incubated with purified glycosomes isolated from bloodstream forms and digested with protease to remove proteins not imported into glycosomes. Gel analysis of reisolated glycosomes revealed that glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and 3-phosphoglycerate kinase (PGK) (EC 2.7.2.3) were apparently imported intact into the glycosome. Specificity of the protein import assay was verified by using translational products derived from cloned genes encoding T. brucei glycosomal PGK and its 95% homologous cytosolic isozyme. Glycosomal PGK was inserted into the glycosome in vitro with a 27.6% efficiency, but no imported cytosolic PGK was detectable. Preliminary data suggest that certain sequences between the N terminus and residue 123 may be important for import of glycosomal PGK. Our assay, combined with the potential use of genetically altered substrate proteins, may provide the opportunity to explore the recognition systems involved in glycosome biogenesis.

Adenosine Triphosphate↗