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Biomedical subjects

J A Thomson

Publications and source records attributed to J A Thomson.

At least 55 records · Page 3Linked to original sources
PubMed1998

Evidence for carbimazole as an antioxidant?

There is evidence in the literature to support the view that antioxidants are involved in the pathogenesis of Graves disease and that antioxidants may act as free radical scavengers. This study has compared the effects of a 12 month course of conventional Carbimazole therapy on peripheral blood antioxidant levels with those of a 12 month course of a higher dose treatment regime. Fifty seven patients were enrolled into the study. Those in Group 1 (n = 23) received a 12 month course of 60 mg/day Carbimazole. Those in Group 2 (n = 34) received 45 mg/day for the first month, 30 mg/day for the second and 20 mg/day for the remaining 10 months of treatment. T3 was added in both groups after 2-4 months to maintain patients euthyroid. Baseline samples were also obtained from 30 control subjects. Blood samples were taken for the measurement of plasma thiol (PSH), lysate thiol (LSH), superoxide dismutase (SOD) and caeruloplasmin (CP) and for routine thyroid function tests (TT4, TT3 and TSH). In untreated Graves' patients, serum levels of PSH and SOD were reduced and levels of LSH increased compared to controls. Following 2 months high dose Carbimazole therapy there was a significant increase in PSH levels and a significant reduction in CP levels compared to presentation levels. In the more conventional dose Group 2 patients PSH levels also rose significantly during the first 2 months of treatment. Levels for both groups were still significantly lower than the control group. After 12 months high dose Carbimazole therapy PSH levels had decreased so that they no longer differed from untreated levels. LSH and SOD levels still remained abnormal. CP levels continued to fall. Similar findings were obtained in those patients receiving the more conventional course of treatment. At no point was their any significant difference in antioxidant levels between the two treatment groups. The abnormal levels of antioxidants in the serum of untreated Graves' patients confirm their involvement in the pathogenesis of Graves' disease. Carbimazole therapy appeared to have only short term effects on the peripheral blood levels of the antioxidants measured. Carbimazole appeared to act only on the extra cellular markers of antioxidant activity (PSH, CP) although the disease itself had marked intracellular effects (LSH, SOD). These findings suggest that Carbimazole does not act as a free radical scavenger.

Adult↗
PubMed1997

Energetics of heme binding to native and denatured states of cytochrome b562.

Cytochrome b562 is a four-helix bundle protein containing a noncovalently bound b-type heme prosthetic group. For the first time, energetics of heme binding to an apocytochrome were measured by isothermal titration calorimetry. The heme is tightly bound to native apocytochrome b562, with a dissociation constant (Kd) of approximately 9 nM (DeltaG degrees = 11 kcal mol-1) at 25 degrees C. Unexpectedly, the thermally denatured apoprotein is also capable of specifically binding heme with modest affinity (Kd = 3 microM, DeltaG degrees = 7.6 kcal mol-1). This interaction results in the dependence of holocytochrome b562 stability on protein concentration in the submicromolar range.

Apoproteins↗
PubMed1997

Cloning and expression of a Bacillus thuringiensis (L1-2) gene encoding a crystal protein active against Glossina morsitans morsitans and Chilo partellus.

A local isolate of Bacillus thuringiensis,designated L1-2, that is toxic to Chilo partellus was found to be toxic to the adult tsetse fly, Glossina morsitans morsitans. The delta-endotoxin crystals derived from the isolate gave a major protein band with a molecular weight of Mr 130,000-140,000 on denaturing polyacrylamide gel electrophoresis. The sequence of the cloned gene was found to be similar to that of the B. thuringiensis subsp. kurstaki HD-73 cryIA(c) gene, having one amino acid difference at position 148 and four additional DNA differences.

Animals↗
PubMed1997

Purification and characterization of the NS3 serine protease domain of hepatitis C virus expressed in Saccharomyces cerevisiae.

cDNA encoding the putative core of the hepatitis C virus NS3 serine protease domain (residues 1-181 of NS3; NS3 (181)) was expressed as an N-terminally (His)6-tagged fusion protein in Saccharomyces cerevisiae. NS3 (181) protease activity was found in soluble cell lysates, and the N-terminal metal-chelating domain facilitated the efficient purification of active enzyme, using immobilized metal affinity chromatography. The purified NS3(181), protease activity was characterized by assaying the trans-cleavage of in vitro transcription-translation generated substrates, and subsequently a previously unobserved cleavage site within the NS5A region was identified. The inhibitory effect of known protease inhibitors was also examined. It is hoped that availability of this method for the expression and purification of the NS3(181) protease will facilitate the development of anti-hepatitis C therapies.

Amino Acid Sequence↗
PubMed1997

Nonsurgical embryo transfer in the common marmoset monkey.

A technique for nonsurgical embryo transfer in common marmosets was developed. Transfers were either synchronous (ST) or asynchronous (AT). Synchronous transfers (embryo donor and the embryo recipient ovulated on the same day) were performed 5 to 8 days post-ovulation. Asynchronous transfers (embryo donor had ovulated at least 2 days before the embryo recipient) were performed when the recipient was 2 to 4 days post-ovulation (donor was 6 to 8 days post-ovulation). Four pregnancies from nine transfers (44%) were established by AT, and three pregnancies were carried to term. Only 1 of 11 transfers (9%) from ST resulted in a pregnancy, which was lost by Day 40 of gestation. Significantly more infants were born from AT (6 infants from 17 embryos; 35%) than from ST (0 infants from 22 embryos; 0%; p < 0.005). This technique allows experimental analysis of primate postimplantation development and provides a tool for conservation of endangered Callithrichid species.

Animals↗
PubMed1997

Polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells.

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregated in two somewhat independent domains. The essential machinery of a serine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing in vivo. This may reflect economical packaging of essential viral replicative components, but it could also mean that there is functional interdependence between the two domains. In this study, a full-length NS3-NS4A complex was isolated after expression and autoprocessing in transiently transfected COS cells. The protein was used to examine the effects of polynucleotides on the NTPase, helicase, and protease activities. Unlike the previously reported behavior of a separately expressed NS3 helicase domain, the full NS3-NS4A complex demonstrated optimal NTPase activity between pH 7.5 and 8.5. All three NS3-NS4A activities were modulated by polynucleotides, with poly(U) having the most remarkable effect. These findings suggest that the domains within NS3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this complex HCV replicase component.

Acid Anhydride Hydrolases↗
PubMed1996

Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide.

An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design. We report here the 2.5 angstrom resolution X-ray crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator peptide. The protease has a chymotrypsin-like fold and features a tetrahedrally coordinated metal ion distal to the active site. The NS4A peptide intercalates within a beta sheet of the enzyme core.

Amino Acid Sequence↗
PubMed1996

Conformational changes and stabilization of inosine 5'-monophosphate dehydrogenase associated with ligand binding and inhibition by mycophenolic acid.

The effects of substrate, product, and inhibitor (mycophenolic acid) binding on the conformation and stability of hamster type II inosine 5'-monophosphate dehydrogenase (IMPDH) have been examined. The protein in various states of ligand occupancy was compared by analyzing susceptibility to in vitro proteolysis, the degree of binding of a hydrophobic fluorescent dye, secondary structure content as determined by far-UV circular dichroism spectra, and urea-induced denaturation curves. These analysis methods revealed consistent evidence that IMPDH undergoes a local reorganization when IMP or XMP bind. NAD+ produced no such effect. In fact, no evidence was found for NAD+ binding independently of IMP. It is proposed that IMPDH adopts an open conformation around its nucleotide binding sites in the absence of substrates and that binding of IMP stabilizes a closed conformation that has a higher affinity for NAD+. The data also suggest the enzyme remains in the closed configuration throughout the catalytic steps and then reverts to the open conformation with XMP release, thereby consummating the enzyme cycle. Mycophenolic acid inhibition appeared to impart even greater stability. We propose that localized conformational changes occur during the normal and mycophenolic acid-inhibited reaction sequences of IMPDH.

Anilino Naphthalenesulfonates↗
PubMed1996

Inhibition of IMPDH by mycophenolic acid: dissection of forward and reverse pathways using capillary electrophoresis.

The objective of this work was to contribute to the understanding of mechanisms for IMPDH inhibition. We over-expressed hamster type II IMPDH in Escherichia coli, purified the protein to apparent homogeneity, and used capillary electrophoresis to quantify enzyme turnover events accompanying inhibition by mycophenolic acid (MPA). We dissected two convergent pathways leading to MPA-inhibition; a rapid "forward" pathway beginning with substrates and linked to enzyme catalysis, and a slower "reverse" pathway apparently not involving catalysis. MPA-inhibition occurred rapidly in the forward direction by interrupting the enzyme turnover cycle, after IMP and NAD+ binding, after hydride transfer, and after NADH release. Slow inhibition, without substrate turnover, was achieved by incubating free enzyme with excess XMP and MPA. We propose that mycophenolic acid inhibits IMPDH by trapping a transient covalent product of the hydride transfer reaction (IMPDH approximately XMP*) before a final hydrolysis step that precedes XMP and enzyme release in the forward reaction pathway. Understanding the ligand occupancy of the protein has also proven important for producing homogeneous, chemically defined complexes for structural studies. IMPDH samples inhibited by MPA in the forward and reverse pathways yielded similar, high-quality crystals that are currently undergoing X-ray diffraction analyses.

Animals↗
PubMed1996

1-Aminocyclopropane-1-carboxylate deaminase genes from Pseudomonas strains.

Microbial ACC deaminase catalyses the conversion of 1-aminocyclopropane-1-carboxylate (ACC), the precursor to the phytohormone ethylene, to ammonia and alpha-ketobutyrate. We screened microorganisms for ACC degrading ability and cloned and sequenced the ACC deaminase genes from two Pseudomonas strains which displayed high enzyme activity. One of the genes was homologous with two previously sequenced ACC deaminase genes, but the other was different.

Amino Acid Sequence↗
PubMed1996

Dynamic NMR studies of ligand-receptor interactions: design and analysis of a rapidly exchanging complex of FKBP-12/FK506 with a 24 kDa calcineurin fragment.

Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP-12/ FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP-12/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA). Analysis of 1H-13C HSQC data acquired for the FKBP-12/ 13C-FK506 and FKBP-12/13C-FK506/CnB/BBD complexes indicates that FKBP-12/FK506 and CnB/BBD are in fast exchange in the quaternary complex. Comparison of proton line widths shows significant broadening of resonances along the macrocycle backbone at 13-CH, 13-OMe, 15-OMe, 18-CH2, 20-CH, 21-CH, and 25-Me, as well as moderate broadening on the macrocycle backbone at 17-Me, 24-CH, and the pyranose 12-CH2 protons. The tri-substituted olefin and cyclohexyl groups also show moderate broadening at the 27-Me, 28-CH, and 30-CH2 positions, respectively. Unexpectedly, little line broadening was observed for the allyl resonances of FK506 in the quaternary complex, although 13C longitudinal relaxation measurements suggest this group also makes contacts with calcineurin. In addition, intermolecular transfer NOE peaks were observed for the allyl 37-CH2, 21-CH, 30-CH2, 13-OMe, 15-OMe, 17-Me, 25-Me, and 27-Me groups, indicating that these are potential sites on the FK506 molecule that interact with calcineurin.

Amino Acid Sequence↗
PubMed1996

Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts.

We report the derivation of eight pluripotent cell lines from common marmoset (Callithrix jacchus) blastocysts. These cell lines are positive for a series of markers (alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that characterize undifferentiated human embryonal carcinoma cells and rhesus embryonic stem cells. All eight cell lines had a modal chromosome number of 46; seven cell lines were XX and one was XY. Two cell lines (Cj11 and Cj62) were cultured continuously for over a year and remained undifferentiated and euploid. In the absence of fibroblast feeder layers, these cell lines differentiated to multiple cell types, even in the presence of leukemia inhibiting factor. Differentiated cells secreted bioactive CG into the culture medium and expressed alpha-CG, beta-CG, and alpha-fetoprotein mRNA, indicating trophoblast and endoderm differentiation. Bioactive CG secretion in differentiating cells was increased substantially in the presence of GnRH agonist D-Trp6-Pro9-NHEt. When grown at high densities, these cells formed embryoid bodies with a close resemblance to early postimplantation embryos, including the formation of a yolk sac, amnion, and an embryonic disc with an early primitive streak. These results make these pluripotent cells strong candidates for marmoset embryonic stem cells.

Animals↗
PubMed1996

Ambulatory elderly patients of primary care physicians: functional, psychosocial and environmental predictors of need for social work care management.

With increasing numbers of elderly people, and the escalating costs of health care, screening becomes increasingly important for identifying those older people with social health care needs who appear in their primary care physicians' offices. Many people are not aware of available social services. Families with serious social problems are not finding the help they need. The aim of this study was to develop and refine a questionnaire as a screening tool to identify elderly outpatients in primary care settings who are at high risk for psychological, social or environmental needs. This study identified those ten factors at each site which were most indicative that further intervention was needed. There were consistencies among the coordinators across sites in terms of what factors triggered intervention. Activities of Daily Living (ADLs) and Instrumental Activities of Daily Living (IADLs) were more likely to be predictive of the coordinator's intervention than were other factors.

Activities of Daily Living↗
PubMed1996

An increased incidence of thyroid antibodies in patients with Turner's syndrome and their first degree relatives.

Previous studies have suggested that there may be a link between Turner's syndrome and autoimmunity. The numbers involved in these studies have tended to be small and few studies have included family members. This study has compared the incidence of thyroid antibodies in the serum of 60 patients with Turner' syndrome and 50 of their mothers with 127 controls. Total T4 and TSH levels were also measured. Of the 60 patients with Turner's syndrome 18 (30%) were positive for either thyroid peroxidase (TPO) and/or thyroglobulin antibodies. The peak incidence of thyroid antibodies occurred at 13 years of age. 11 (22%) of the mothers were also antibody positive. The incidence of thyroid antibodies was significantly higher in both the patients with Turner's Syndrome (30 vs 1.7% p < 0.001) and their mothers (22 vs 6.6% p < 0.05) than in the control groups. The increased incidence of thyroid antibodies found in these patients and their mothers confirms that there is an association between Turner's Syndrome and autoimmunity. However unlike previous studies we found more patients were positive for thyroglobulin than TPO antibodies.

Adolescent↗
PubMed1996

Hemorrhagic typhlocolitis associated with attaching and effacing Escherichia coli in common marmosets.

We describe the necropsy results from three common marmosets presented during an outbreak of hemorrhagic diarrhea in a colony of 230 common marmosets (Callithrix jacchus). Necropsy revealed consistent hemorrhagic typhlocolitis and variable ileitis associated with gram-negative bacilli closely adherent to enterocytes. Electron microscopy revealed bacilli attached intimately to shallow cup-like projections of enterocyte apical membranes with loss of microvilli (attaching and effacing). Escherichia coli isolates from affected marmosets were serogroup O26 and were positive for the E. coli attaching and effacing locus and negative for shiga-like toxin I and II, heat-stable enterotoxins a and b, and heat-labile enterotoxin by DNA probe hybridization. Results of a Vero cell cytotoxicity assay confirmed that the E. coli isolates did not produce shiga-like toxin. The increased availability of diagnostic probes for specific virulence factors such as attaching and effacing E. coli should lead to a greater understanding of the frequency of E. coli as a cause of diarrhea in nonhuman primates.

Animals↗