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Biomedical subjects

J M Brewer

Publications and source records attributed to J M Brewer.

At least 19 recordsLinked to original sources
PubMed2004

Microscopic colitis with granulomatous inflammation.

AIMS: To present four cases in which the clinical and endoscopic findings were consistent with microscopic colitis, but the inflammatory infiltrate included a conspicuous granulomatous reaction. Microscopic colitis is defined as a syndrome of chronic watery diarrhoea with a chronic inflammatory cell infiltrate in the colonic mucosa and without significant abnormalities at colonoscopy. It encompasses both collagenous and lymphocytic colitis. METHODS AND RESULTS: In all cases the clinical course and endoscopic findings were unlike Crohn's disease and no infectious agents were identified. In all cases the main symptom was frequent watery diarrhoea, all were female and there were no endoscopic findings apart from mild mucosal erythema. Histologically, an active chronic inflammatory infiltrate was accompanied by scattered non-necrotizing granulomas, often closely associated with crypt epithelium (cryptolytic or pericryptal granulomas). In three of the patients the symptoms began after antibiotic use or had worsened with antibiotic use. Two of the patients were on allopurinol at the time of the onset of symptoms. In two of the patients symptoms have continued for more than 10 years. One patient died as a result of medical complications relating to severe diarrhoea and dehydration. CONCLUSIONS: Microscopic colitis rarely may be characterized by granulomatous inflammation. Such patients should not be regarded as having Crohn's disease.

Adult↗
PubMed2001

Neuropeptide Y rapidly reduces Period 1 and Period 2 mRNA levels in the hamster suprachiasmatic nucleus.

The mammalian suprachiasmatic nucleus (SCN) contains the main circadian clock. Neuropeptide Y (NPY) that is released from the intergeniculate leaflet of the lateral geniculate body to the SCN, acts in the SCN to advance circadian phase in the subjective day via the NPY Y2 receptor. We used semi-quantitative in situ hybridization to determine the effect of NPY on circadian clock genes, Period 1 (Per1) and Period 2 (Per2), expression in SCN slices. Addition of NPY to the brain slices in the subjective day resulted in reduction of Per1 and Per2 mRNA levels 0.5 and 2 h after treatment. NPY Y1/Y5 and Y2 agonists decreased Per1 within 0.5 h. These results suggest that NPY may induce phase shifts by mechanisms involving or resulting in reduction of Per1 and Per2 mRNA levels.

Animals↗
PubMed2001

On-line dialysis as a sample preparation technique for ion chromatography.

The use of on-line dialysis as a sample preparation technique for ion chromatography is described. A fully automated sample preparation device coupled to an ion chromatographic system for the determination of anions and cations in various matrices is presented. The method was based on stopped-flow dialysis, where the samples were continuously dialyzed for 10 min while the acceptor solution was stationary within the recipient channel. The matrices examined, without additional sample treatment, included milk, untreated wastewater, fruit juice, engine coolant, and a multivitamin tablet. The analyte recoveries for anions and cations in various matrices ranged from 87 to 106%. In addition, multiple sample injections were performed and repeatabilities were found in the range of 0.2 to 4%.

Anions↗
PubMed2001

Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes).

The ability of non-ionic surfactant vesicles to induce systemic immune responses in mice following oral immunisation was studied using a standard antigen (bovine serum albumin), a synthetic measles peptide and an influenza sub-unit vaccine. The effectiveness of this formulation was significantly increased by incorporating bile salts (in particular deoxycholate) into the formulation. We have named the resulting vesicles bilosomes. We found that the most effective immunisation protocol was to give two doses of vaccine three days apart and then repeat this protocol two weeks later. Following this method, preparation of measles peptide in bilosomes produced a specific cell mediated response, as measured by splenocyte proliferation and IL-2 production. Of particular significance, these studies demonstrate that oral administration of bilosomes incorporating the influenza sub-unit vaccine could induce as potent an antibody response as the parenterally administered vaccine containing the same quantity of antigen. In addition, the Th1/Th2 balance, as measured by antibody subclasses, was similar whether animals were immunised by the oral or the parenteral vaccine route. As bilosomes are prepared from naturally occurring lipids and have no apparent toxicity associated with their use, they represent a useful modification of conventional lipid vesicle based systems for the oral delivery of proteins and peptides.

Administration, Oral↗
PubMed2001

A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1.

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.

Anions↗
PubMed2001

Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases.

Liposomes prepared from naturally occurring biodegradable and nontoxic lipids are good candidates for local delivery of therapeutic agents. Treatment of arthritis by intra-articular administration of anti-inflammatory drugs encapsulated in liposomes prolongs the residence time of the drug in the joint. We have previously shown that intra-articular injection of human lactoferrin (hLf), a glycoprotein that possesses anti-inflammatory and antimicrobial activities, into mice with collagen-induced arthritis reduces inflammation. We have now investigated the possibility of using liposome-entrapped hLf as a delivery system to prolong hLf retention at sites of local inflammation such as the rheumatoid joint. Entrapment of hLf in negatively charged liposomes enhanced its accumulation in cultured human synovial fibroblasts from rheumatoid arthritis (RA) patients, compared with positively charged formulations or free protein. However, in the presence of synovial fluid, positively charged liposomes with entrapped hLf were more stable than the negatively charged formulations. In vivo experiments in mice with collagen-induced arthritis showed that the positive liposomes were more efficient in prolonging the residence time of hLf in the inflamed joint as compared with other liposomes. Thus, the amount of hLf retained in the joint after 2 hr was 60% of the injected dose in the case of positive liposomes and only 16% for negative pH-sensitive liposomes. The results suggest that entrapment of hLf in positively charged liposomes may modify its pharmacodynamic profile and be of therapeutic benefit in the treatment of RA and other local inflammatory conditions.

Animals↗
PubMed2000

Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica.

Formyltetrahydrofolate synthetase (FTHFS) from the thermophilic homoacetogen, Moorella thermoacetica, has an optimum temperature for activity of 55-60 degrees C and requires monovalent cations for both optimal activity and stabilization of tetrameric structure at higher temperatures. The crystal structures of complexes of FTHFS with cesium and potassium ions were examined and monovalent cation binding positions identified. Unexpectedly, NH(4)(+) and K(+), both of which are strongly activating ions, bind at a different site than a moderately activating ion, Cs(+), does. Neither binding site is located in the active site. The sites are 7 A apart, but in each of them, the side chain of Glu 98, which is conserved in all known bacterial FTHFS sequences, participates in metal ion binding. Other ligands in the Cs(+) binding site are four oxygen atoms of main chain carbonyls and water molecules. The K(+) and NH(4)(+) binding site includes the carboxylate of Asp132 in addition to Glu98. Mutant FTHFS's (E98Q, E98D, and E98S) were obtained and analyzed using differential scanning calorimetry to examine the effect of these mutations on the thermostability of the enzyme with and without added K(+) ions. The addition of 0.2 M K(+) ions to the wild-type enzyme resulted in a 10 degrees C increase in the thermal denaturation temperature. No significant increase was observed in E98D or E98S. The lack of a significant effect of monovalent cations on the stability of E98D and E98S indicates that this alteration of the binding site eliminates cation binding. The thermal denaturation temperature of E98Q was 3 degrees C higher than that of the wild-type enzyme in the absence of the cation, indicating that the removal of the unbalanced, buried charge of Glu98 stabilizes the enzyme. These results confirm that Glu98 is a crucial residue in the interaction of monovalent cations with FTHFS.

Aspartic Acid↗
PubMed2000

The H159A mutant of yeast enolase 1 has significant activity.

The function of His159 in the enolase mechanism is disputed. Recently, Vinarov and Nowak (Biochemistry (1999) 38, 12138-12149) prepared the H159A mutant of yeast enolase 1 and expressed this in Escherichia coli. They reported minimal (ca. 0.01% of the native value) activity, though the protein appeared to be correctly folded, according to its CD spectrum, tryptophan fluorescence, and binding of metal ion and substrate. We prepared H159A enolase using a multicopy plasmid and expressed the enzyme in yeast. Our preparations of H159A enolase have 0.2-0.4% of the native activity under standard assay conditions and are further activated by Mg(2+) concentrations above 1 mM to 1-1.5% of the native activity. Native enolase 1 (and enolase 2) are inhibited by such Mg(2+) concentrations. It is possible that His159 is necessary for correct folding of the enzyme and that expression in E. coli leads to largely misfolded protein.

Amino Acid Substitution↗
PubMed2000

Antibody responses, cytokine levels and protection of mice immunised with HSV-2 antigens formulated into NISV or ISCOM delivery systems.

The immunogenicity of a type 2 herpes simplex virus (HSV-2) antigen preparation following its formulation into immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) was investigated in a murine model. The immune responses induced by each formulation were characterised by antigen specific total and subclass serum responses, and by lymphocyte proliferation and cytokine (interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-gamma (IFN-gamma)) production by in vitro restimulated spleen cells. The degree of protection afforded to mice by these various HSV-2 vaccine preparations against homologous (HSV-2) and heterologous (HSV-1) challenge infection was also determined. The findings suggest that formulation of the HSV-2 glycoprotein antigens with ISCOM or NISV delivery vehicles, and the methods used to prepare these formulations, influenced the immunogenicity of the final preparation. Higher IgG2a and neutralising antibody levels, IL-2 and IFN-gamma levels and lymphoproliferative responses were noted in mice immunised with the HSV-2 ISCOM formulated vaccine preparation. Furthermore, although HSV-2 antigens formulated in dehydration-rehydration NISV, or entrapped in NISV by freeze-thawing at 30 degrees C (HSV-2 NISV 30), also elicited relatively high antibody, IL-2 and IFN-gamma levels and relatively high lymphoproliferative responses, formulation of HSV-2 antigens by freeze-thawing with NISV at 60 degrees C (HSV-2 NISV 60) did not. There were no differences between any of the HSV-2 vaccine formulations in terms of IL-4 induction in in vitro stimulated spleen cell cultures. Almost complete protection against HSV-2 challenge was afforded by the HSV-2 ISCOM preparation, while partial protection against challenge infection was afforded by the HSV-2 NISV 30 vaccine formulation. The findings are discussed in relation to the nature of the immune mechanisms, particularly Th1- or Th2-like responses, that may be elicited by HSV-2 antigen preparations formulated into various delivery systems and the relevance of these immune responses to protection against HSV infection in the murine model.

Administration, Oral↗
PubMed1999

Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling.

Previous studies demonstrate that aluminium hydroxide adjuvant (alum) produces increased Th1 responses in IL-4-deficient mice compared with wild-type animals, although the continued production of IL-5 by spleen cells from these mice also indicates that Th2 responses are induced. In the present study, we demonstrate that alum can induce Th2-associated IL-4 and IL-5 production in the absence of IL-4 signaling in mice deficient in either IL-4Ralpha or Stat6. The Th2 responses observed could not be due to IL-13 as IL-13 responses are also impaired in IL-4Ralpha- and Stat6-deficient mice. We also detected higher levels of IL-4 in IL-4Ralpha gene-deficient, though not Stat6-deficient, mice compared with their wild-type counterparts. The increased levels of IL-4 could be explained by the IL-4R being unavailable to neutralize this cytokine in IL-4Ralpha-deficient mice. While levels of IL-5 production in IL-4Ralpha- or Stat6-deficient mice were similar to IL-4-deficient and wild-type mice, other type 2-associated responses, which are largely or wholly IL-4 dependent, such as the production of IgG1 or IgE Abs, were either reduced or absent. We conclude that alum adjuvants can induce IL-4 production and Th2 responses independently of IL-4 or IL-13, negating the requirement for an early source of IL-4 in the Th2 response induced by this adjuvant.

Adjuvants, Immunologic↗
PubMed1998

Lipid vesicle size determines the Th1 or Th2 response to entrapped antigen.

Understanding the factors that control the differential induction of Th1 and Th2 responses is a key immunologic objective with profound implications for vaccination and immunotherapy of infectious and autoimmune diseases. Using Ag formulated in lipid vesicles prepared from nonionic surfactants, we describe a novel mechanism influencing the balance of the Th1 or Th2 response. Our results indicate that inoculation of BALB/c mice with vesicles with a mean diameter > or = 225 nm preferentially induces Th1 responses, as characterized by increased titers of IgG2a in plasma and elevated IFN-gamma production by lymph node cells. However, preparation of the same quantity of Ag in vesicles with mean diameter of < or = 155 nm induces a Th2 response, as identified by IgG1 in the absence of IgG2a production and increased lymph node IL-5 production. Although large (> or = 225 nm) vesicles could induce IL-12 production, smaller vesicles (< or = 155 nm) could not. However, small vesicles did induce higher levels of IL-1beta production by macrophages than larger vesicles. The role of IL-12 in this response was confirmed in IL-12-deficient mice, whose spleen cells failed to produce IFN-gamma following in vivo priming with Ag prepared in large vesicles. Our results therefore indicate that macrophages respond to endocytosis of large or small vesicles by producing different patterns of cytokines that can subsequently direct the immune response toward a Th1 or a Th2 phenotype.

Animals↗
PubMed1998

Purification and properties of the heme- and iron-sulfur-containing heterodisulfide reductase from Methanosarcina thermophila.

The heterodisulfide reductase (HDR) from Methanosarcina thermophila was purified to homogeneity from acetate-grown cells. In the absence of detergents, HDR consisted of an eight-protein complex with hydrogenase activity. However, when HDR was purified in the presence of 0.6% Triton X-100, a two-subunit (53 and 27 kDa) high specific activity ( approximately 200 units mg-1) enzyme was obtained that lacked hydrogenase activity. Sedimentation equilibrium experiments demonstrated that HDR has a molecular mass of 206 kDa and a high partial specific volume (0.9 cm3/g), indicating that the purified protein contains a significant amount of bound lipid. Like the HDR from Methanosarcina barkeri [Kunkel, A., Vaupel, M., Heim, S., Thauer, R. K., and Hedderich, R. (1997) Eur. J. Biochem. 244, 226-234], it was found to contain two discrete b-type hemes in the small subunit and two distinct [Fe4S4]2+/1+ clusters in the large subunit. One heme is high-spin and has a high midpoint potential (-23 mV), whereas the other heme apparently is low-spin and exhibits a relatively low midpoint potential (-180 mV). Only the high-spin heme binds CO. The midpoint potentials for the two clusters are -100 and -400 mV. In the fully reduced state, a complicated EPR spectrum with g values of 2.03, 1.97, 1.92, and 1.88 was observed. This spectrum resembles that of 8Fe ferredoxins in the fully reduced state, indicating that the two clusters in HDR are near enough to experience relatively strong dipolar interactions. Kinetic studies in which CO oxidation is coupled to heterodisulfide reduction strongly indicate that a membrane-associated compound is the direct electron donor to HDR. An electron-transfer pathway is presented that postulates a mechanism for coupling electron transport to proton translocation.

Amino Acid Sequence↗
PubMed1998

Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism.

The S39A mutant of yeast enolase (isozyme 1), prepared by site-directed mutagenesis, has a relative Vmax of 0.01% and an activation constant for Mg2+ ca. 10-fold higher, compared with native enzyme. It is correctly folded. There is little effect of solvent viscosity on activity. We think that the loop Ser36-His43 fails to move to the 'closed' position upon catalytic Mg2+ binding, weakening several electrostatic interactions involved in the mechanism.

Mutation↗
PubMed1998

Neither interleukin-6 nor signalling via tumour necrosis factor receptor-1 contribute to the adjuvant activity of Alum and Freund's adjuvant.

The potential contribution made by the inflammatory cytokines, interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) to the adjuvant activity of aluminium hydroxide gels (Alum) or Freund's complete adjuvant (FCA) was studied by comparing the immunological responses of IL-6- or TNF receptor 1- (p55; TNFR-1) deficient mice with immunocompetent control mice. While both TNFR-1- and IL-6-deficient mice primed with ovalbumin (OVA) prepared in either Alum or FCA produced similar IgG.1 responses in comparison to control mice, the pattern of T-helper type 1- (Th1) dependent IgG2a production was significantly altered. In TNFR-1-deficient mice, IgG2a responses were greater than in control mice when FCA, but not when Alum, was used as an adjuvant. Correspondingly, spleen cells from FCA-inoculated TNFR-1-deficient mice restimulated with antigen in vitro produced higher Th1 cytokine (interferon-gamma; IFN-gamma) levels with no alteration in Th2 cytokine (IL-4; IL-5, IL-6 and IL-10) production in comparison with wild-type mice. Higher levels of IgG2a were also detected in IL-6-deficient mice compared with wild-type mice following inoculation with OVA prepared in either FCA or in Alum. Furthermore, analysis of cytokine production by spleen cells revealed that both Th1 and Th2 cytokine production was higher in IL-6-deficient mice compared with control mice. As the majority of the effects of TNF-alpha are mediated via TNFR-1, we conclude that this cytokine inhibits the adjuvant activities of FCA. Furthermore, the results also imply that immunopotentiating effects of FCA or Alum adjuvant are both inhibited by IL-6.

Adjuvants, Immunologic↗
PubMed1997

Mechanism of enolase: the crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/enolase-phosphoenolpyruvate at 2.0 A resolution.

Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phospho-d-glycerate (PGA) to form phosphoenolpyruvate (PEP), is a homodimer in all eukaryotes and many prokaryotes. Here, we report the crystal structure of a complex between yeast enolase and an equilibrium mixture of PGA and PEP. The structure has been refined using 29 854 reflections with an F/sigma(F) of >/=3 to an R of 0.137 with average deviations of bond lengths and bond angles from ideal values of 0.013 A and 3.1 degrees , respectively. In this structure, the dimer constitutes the crystallographic asymmetric unit. The two subunits are similar, and their superposition gives a rms distance between Calpha atoms of 0.91 A. The exceptions to this are the catalytic loop Val153-Phe169 where the atomic positions in the two subunits differ by up to 4 A and the loop Ser250-Gln277, which follows the catalytic loop Val153-Phe169. In the first subunit, the imidazole side chain of His159 is in contact with the phosphate group of the substrate/product molecule; in the other it is separated by water molecules. A series of hydrogen bonds leading to a neighboring enolase dimer can be identified as being responsible for ordering and stabilization of the conformationally different subunits in the crystal lattice. The electron density present in the active site suggests that in the active site with the direct ligand-His159 hydrogen bond PGA is predominantly bound while in the active site where water molecules separate His159 from the ligand the binding of PEP dominates. The structure indicates that the water molecule hydrating carbon-3 of PEP in the PEP --> PGA reaction is activated by the carboxylates of Glu168 and Glu211. The crystals are unique because they have resolved two intermediates on the opposite sides of the transition state.

Crystallization↗
PubMed1997

Effect of site-directed mutagenesis of His373 of yeast enolase on some of its physical and enzymatic properties.

The X-ray structure of yeast enolase shows His373 interacting with a water molecule also held by residues Glu168 and Glu211. The water molecule is suggested to participate in the catalytic mechanism (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822). Replacement of His373 with asparagine (H373N enolase) or phenylalanine (H373F enolase) reduces enzymatic activity to ca. 10% and 0.0003% of the native enzyme activity, respectively. H373N enolase exhibits a reduced Km for the substrate, 2-phosphoglycerate, and produces the same absorbance changes in the chromophoric substrate analogues TSP1 and AEP1, relative to native enolase. H373F enolase binds AEP less strongly, producing a smaller absorbance change than native enolase, and reacts very little with TSP. H373F enolase dissociates to monomers in the absence of substrate; H373N enolase subunit dissociation is less than H373F enolase but more than native enolase. Substrate and Mg2+ increase subunit association in both mutants. Differential scanning calorimetric experiments indicate that the interaction with substrate that stabilizes enolase to thermal denaturation involves His373. We suggest that the function of His373 in the enolase reaction may involve hydrogen bonding rather than acid/base catalysis, through interaction with the Glu168/Glu211/H2O system, which produces removal or addition of hydroxyl at carbon-3 of the substrate.

Asparagine↗
PubMed1997

GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits. Purification, cDNA cloning, and bacterial expression.

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated Mr of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinity-purified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average Mr of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes.

Amino Acid Sequence↗