Biomedical subjects

J M Brewer

Publications and source records attributed to J M Brewer.

At least 37 records · Page 2Linked to original sources

PubMed1997

A conserved region in the sigma54-dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation.

Rhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between sigma54-RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with sigma54-holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for sigma54-holoenzyme. Mutagenesis was confined to the well-conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in sigma54-dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C-terminal half of the C3 region were efficiently cross-linked to sigma54 and the beta-subunit of RNA polymerase, suggesting that they bound normally to sigma54-holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N-terminal half of the C3 region had reduced affinities for sigma54 and the beta-subunit in the cross-linking assay. These data suggest that the N-terminal half of the C3 region of DctD contains a site that may contact sigma54-holoenzyme during open complex formation.

Amino Acid Sequence↗

PubMed1997

Homodimer formation by the individual subunits of bovine lutropin as determined by sedimentation equilibrium.

Although differing in their amino acid sequences, the folding patterns of the alpha and beta subunits of human choriogonadotropin are similar in the crystal structure of the HF-treated glycoprotein hormone. Each subunit forms a cystine-knot motif like that found in several growth factors that form homodimers and heterodimers. In order to ascertain if the alpha and beta subunits can self-associate, e.g. to form homodimers, sedimentation equilibrium at various glycoprotein concentrations and temperatures was used to study the subunits of bovine lutropin, which are expected to exhibit conformations like those of the choriogonadotropin subunits. Each subunit was found to form homodimers with Kd values of 0.3 and 0.1 mM for alpha and beta respectively at 37 degrees C. Self-association was weakly exothermic for alpha and endothermic for beta; entropic factors made a major contribution for each. It is unlikely that homodimer formation of either subunit would be physiologically important, although homodimers may form to some extent intracellularly because of the relatively high concentrations during biosynthesis.

Animals↗

PubMed1997

Cytokines and the mechanisms of action of vaccine adjuvants.

There is an increasing trend away from classical attenuated or killed whole pathogen vaccines towards developing chemically defined preparations. Paradoxically, in order to be effective, these defined vaccines require incorporation into adjuvants, of which very little is actually understood about how or why they work. It is, therefore, of little surprise that, in this context, adjuvants have been referred to as 'the immunologists' dirty little secret'. [Janeway CA, Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor Symp Quantit Biol (1989) 54: 1-13]. However, modern techniques in immunology have made available a number of powerful tools that will allow a more complete dissection of how traditional adjuvants work, knowledge of which will not only facilitate the rational design of new adjuvants but also further clarify our understanding of the immune system. The initial studies described in this review indicate that cytokines play an important, if not the central, role in the ability of adjuvants to influence both the quantity and quality of immune responses.

Adjuvants, Immunologic↗

PubMed1996

Lipid vesicle-entrapped influenza A antigen modulates the influenza A-specific human antibody response in immune reconstituted SCID-human mice.

This study was designed to investigate the capacity of purified influenza antigen in the presence and absence of adjuvant to induce human antibody responses in human-PBL-SCID mice. Non-ionic surfactant vesicles (NISV) were used as adjuvant as they have been shown to promote the development of Th1 responses in mouse studies. Human peripheral blood lymphocyte-SCID mice were inoculated with either purified influenza antigen (A/Texas, H3N2) or influenza antigen entrapped in NISV. Both vaccinated groups produced significantly higher plasma levels of influenza-specific human IgG when individually compared with non-vaccinated controls. However, similar comparisons revealed that specific IgM levels were significantly higher only in the group challenged with purified antigen. Further analysis of IgG subclasses also demonstrated an adjuvant-dependent dichotomy in the responses of the vaccine groups when compared with non-vaccinated controls. Thus, only influenza-specific IgG1 antibodies (associated with Th1 responses in humans) were significantly increased above control levels using antigen with adjuvant, while both this subclass and antigen-specific IgG4 (Th2 associated) were significantly increased with antigen alone. These results illustrate the suitability of this model for use in human vaccination studies and demonstrates that influenza antigen applied with NISV selectively promotes only Th1 responses, unlike free antigen which also promotes Th2 responses in vivo.

Adjuvants, Immunologic↗

PubMed1996

In interleukin-4-deficient mice, alum not only generates T helper 1 responses equivalent to freund's complete adjuvant, but continues to induce T helper 2 cytokine production.

The role of interleukin (IL)-4 in the activity of two frequently used vaccine adjuvants, Freund's complete adjuvant (FCA) and the aluminum hydroxide gels (alum), was studied using the standard antigen ovalbumin (OVA) in IL-4 genedisrupted mice (IL-4 -/-). In the absence of adjuvant, there was an overall reduction in antibody production to OVA in IL-4 -/- mice and significantly greater amounts of interferon (IFN)-gamma were produced following restimulation of splenocytes with antigen in vitro compared with immunocompetent controls (IL-4 +/+). FCA and alum boosted the immune response to OVA in both IL-4 -/- and IL-4 +/+ mice. In IL-4 +/+ mice, while FCA stimulated a wide-spectrum immunoglobulin response, including both Th1-associated IgG2a and Th2-associated IgG1, alum enhanced only Th2 antibody production and no OVA-specific IgG2a could be detected. In IL-4-deficient mice, however, not only was IgG2a production increased in all adjuvant-treated groups, but alum was as potent at stimulating this antibody subclass as FCA. Similarly, increased production in vitro by splenocytes of the Th1 cytokine IFN-gamma, equivalent to that produced after inoculation with FCA/OVA, was only detected in IL-4 -/- mice inoculated with alum/OVA. There was no IgE production in IL-4 -/- mice and OVA-specific IgG1 production, although still at significant levels, was reduced compared with wild-type mice irrespective of the adjuvant used. However, although production of the Th2 cytokine IL-5 was totally inhibited in IL-4-deficient mice inoculated with FCA/OVA, there was no significant difference in IL-5 production between the two strains when alum was used as adjuvant.

Adjuvants, Immunologic↗

PubMed1996

Immune responses in mice induced by HSV-1 glycoproteins presented with ISCOMs or NISV delivery systems.

The purpose of this study was to evaluate the immunogenicity of a herpes simplex virus type I (HSV-1) antigen preparation, obtained following zwitterionic detergent treatment of virus, and incorporation of the antigens into either immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) delivery systems. Using Balb/c mice the ISCOM and NISV HSV-1 vaccines were assayed for their capacity to induce and enhance both the humoral and cellular immune responses, and to elicit protection against both homologous and heterologous virus challenge. The serum from animals vaccinated with either the NISV or the ISCOM HSV-1 antigen preparation, were found to contain high levels of total IgG and IgG1 and IgG2a subclass antibodies. In addition, both preparations were found to induce high neutralizing (NT) antibody levels following a two immunization protocol and to provide some protection against homologous and heterologous HSV challenge infection. Lymphoproliferative responses were observed in cultures of splenocytes from mice immunized with both HSV-1 NISV vaccine and HSV-1 ISCOMs vaccine, following various antigenic stimuli in vitro. In general, these were most marked in animals immunized with the HSV-1 NISV preparation, and particularly so when the splenocytes were stimulated in vitro with live HSV-1. Both the NISV and ISCOM HSV-1 vaccines were found to have induced interleukin 2, interleukin 10 and interferon-gamma in spleen cell culture supernatants, although again, the highest responses in general were observed in supernatant fluids from spleen cell cultures from animals immunized with the HSV-1 NISV preparation. These results suggest that a wide range of immune activity can be elicited by HSV-1 antigens presented to the immune system of mice in these formulations.

Animals↗

PubMed1995

Preparation by site-directed mutagenesis and characterization of the E211Q mutant of yeast enolase 1.

The published 'charge shuttle' mechanism of enolase (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822) assigns Glu-211 the task of orienting a water molecule that serves as the catalytic base which removes the proton from carbon-2 of the substrate. We prepared the E211Q mutant of yeast enolase 1 by site-directed mutagenesis. It appears to be folded correctly and to respond similarly to many of the normal ligands of enolase: it is stabilized against thermal denaturation by conformational Mg2+ and by Mg2+ and substrate and binds the chromophoric substrate analogue D-tartronate semialdehyde-2-phosphate (TSP) with affinity comparable to that of the native enzyme. However, it has only 0.01% (10(-4)) of the activity of native enolase under standard assay conditions and does not exhibit significantly more activity at various pH values or higher concentrations of substrate and Mg2+. Its ability to produce the form of enzyme-bound and reacted TSP that absorbs at shorter wavelengths is greatly slowed, while the longer wavelength absorbing form is produced rapidly. Overall, these observations are consistent with the hypothetical mechanism.

Amino Acid Sequence↗

PubMed1995

Calculated effects of the chemical environment of 2-phospho-D-glycerate on the pKa of its carbon-2 and correlations with the proposed mechanism of action of enolase.

The mechanism of action of enolase requires drastic lowering of the pK of the alpha carbon of the substrate to enable removal of the alpha hydrogen. Calculations suggest this is done primarily by rotation of the carboxyl ("carbon-1") of the substrate around the carbon-1 carbon-2 bond by 90 degrees so that its oxygen atoms lie in the plane formed by the three carbon atoms and secondarily by effectively neutralizing the negative charges on the carboxylate and phosphate. These predictions are in very good agreement with the proposed mechanism of action of enolase which is based on X-ray crystallographic data.

Amino Acid Sequence↗

PubMed1995

An X-ray absorption spectroscopy study of the interactions of Ni2+ with yeast enolase.

An x-ray absorption spectroscopy (XAS) study was carried out at pH 7.6 on solutions of Ni2+ and yeast enolase depleted of its physiological cofactor (Mg2+) in the presence or absence of substrate/product, the very strongly bound competitive inhibitor 2-phosphonoacetohydroxamate and Mg2+. Both "conformational" and "catalytic" Ni2+ are distorted octahedral in coordination, in agreement with several spectroscopic studies but in contrast to the coordination in the crystal at pH 6.0. The data are consistent with direct coordination of what must be the catalytic Ni2+ to the phosphate of the substrate, in agreement with some previous data but in disagreement with recent interpretations by other workers. The ligands around the metal ions obtained from the x-ray structure give simulated XAS spectra in good agreement with the observed spectra.

Binding Sites↗

PubMed1995

Antibody responses to Toxoplasma gondii antigen in human peripheral blood lymphocyte-reconstituted severe-combined immunodeficient mice reproduce the immunological status of the lymphocyte donor.

These studies describe the production of specific antibodies in human peripheral blood lymphocyte-reconstituted severe-combined immunodeficient (PBL-SCID) mice following vaccination with antigen from the protozoan parasite Toxoplasma gondii. To determine the effect of previous exposure of the lymphocyte donor to antigen, human-PBL-SCID animals were created by transferring peripheral blood lymphocytes from either a single T. gondii-seronegative or a single seropositive donor. These reconstituted animals were subsequently inoculated with T. gondii soluble tachyzoite antigen (STAg) entrapped within non-ionic surfactant vesicles as an immunological adjuvant. Animals were bled at pre-determined time points post-vaccination and the expression of human anti-STAg antibodies in the plasma determined by enzyme-linked immunosorbent assay. Human antibodies specific for STAg were readily inducible in both groups of reconstituted animals, although the pattern of isotype production differed markedly between groups. The response in animals reconstituted with lymphocytes from the T. gondii-seronegative donor consisted primarily of IgM and subsequently of IgG (predominantly IgG1). In animals reconstituted with lymphocytes from the seropositive donor, no parasite-specific IgM could be demonstrated. The detectable response to STAg consisted entirely of human antibodies of the IgG isotype (IgG1), indicative of a memory-type response. These results mimicked exactly the antibody responses that would be expected had the lymphocyte donors been directly challenged with either the antigen or the live infectious agent, demonstrating that the immune system within these animals is functional and reproducible with regard to both the primary and secondary responses of the human donors.

Animals↗

PubMed1995

Accurate determination of adjuvant-associated protein or peptide by ninhydrin assay.

Modern peptide and subunit vaccines are increasingly having to rely on the use of immunological adjuvants to achieve effective immunity. However, the only adjuvant currently approved for use in humans is aluminium hydroxide, although many adjuvants are currently under preclinical development. Determining immunogen concentration in the presence of adjuvants such as aluminium hydroxide gel, liposomes or NISV has proved to be problematic. One approach has been to use radiolabelled antigens to extrapolate concentration to a preparation using native immunogen. However, the use of a colorimetric assay would allow greater flexibility in terms of immunogen used and would reduce costs and remove safety problems. Of the colorimetric methods we have examined thus far, only the manual ninhydrin assay has produced consistent results with detection of microgram quantities of protein or peptide in the presence of NISV or Alhydrogel, but not liposomes. As the assay relies on the detection of free amino groups after protein hydrolysis, peptides as well as proteins may be effectively determined irrespective of amino acid composition, a considerable advantage over other colorimetric assay systems.

Adjuvants, Immunologic↗

PubMed1995

Percutaneous transluminal coronary angioplasty failures in patients with multivessel disease. Is there an increased risk?

In recent years, there has been a nationwide trend toward performing percutaneous transluminal coronary angioplasty in patients with multivessel coronary artery disease. The clinical course of 57 consecutive patients who required emergency first-time coronary artery bypass grafting operations were reviewed to assess for difference in outcome between the 28 patients (49%) with single-vessel disease and the 29 patients (51%) with multivessel disease. The two groups were similar in preoperative characteristics except for a higher proportion of chronic obstructive pulmonary disease in the patients with multivessel disease (p = 0.03). Twice as many patients with multivessel disease were in shock (single-vessel disease = 4 [14%], multivessel disease = 8 [28%], p = not significant) en route to the operating room and significantly more patients with multivessel disease required on-going cardiopulmonary resuscitation (single-vessel disease = 0 [0%], multivessel disease = 5 [17%], p = 0.03). Significantly more coronary artery bypass grafts were placed in the patients with multivessel disease (single-vessel disease = 1.5 +/- 0.6, multivessel disease = 2.9 +/- 0.7, p < 0.01), which required longer aortic clamping time (p = 0.02) and cardiopulmonary bypass time (p < 0.01). There were seven postoperative deaths; all but one occurred in patients with multivessel disease (single-vessel disease = 1 [4%], multivessel disease = 6 [21%], p = 0.05). According to multivariate analysis, incremental risk factors of mortality were preoperative shock (p < 0.01), urgent or emergency percutaneous transluminal coronary angioplasty (p = 0.06), and multivessel disease (p = 0.12). Despite a similar incidence of myocardial infarction (single-vessel disease = 8 [29%], multivessel disease = 12 [41%], p = not significant), patients with multivessel disease had a higher incidence of cardiac morbidity (single-vessel disease = 4 [14%], multivessel disease = 11 [38%], p = 0.04) and noncardiac morbidity (single-vessel disease = 4 [14%], multivessel disease = 12 [41%], p = 0.02). By multivariate analysis, incremental risk factors of morbidity were preoperative shock (p < 0.01), multivessel disease (p = 0.02), and ejection fraction < 50% (p = 0.07). In the subset of patients with multivessel disease, preoperative shock, ejection fraction < 50, and an age of 60 years or greater were associated with higher morbidity and mortality. In conclusion, the risk of percutaneous transluminal coronary angioplasty failure is considerably higher in patients with multivessel disease. In certain subsets of patients with multivessel disease, coronary artery bypass grafting would be a safer procedure when compared with percutaneous transluminal coronary angioplasty for initial myocardial revascularization.

Age Factors↗

PubMed1994

Catalytic metal ion binding in enolase: the crystal structure of an enolase-Mn2+-phosphonoacetohydroxamate complex at 2.4-A resolution.

Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phospho-D-glycerate (PGA) to form phosphoenolpyruvate (PEP), requires two divalent metal ions per active site for activity. The first metal ion, traditionally referred to as "conformational", binds in a high-affinity site I. The second metal ion, "catalytic", binds in site II only in the presence of a substrate or substrate analogue and with much lower affinity for the physiological cofactor Mg2+. While the high-affinity site has been well characterized, the position of the lower affinity site has not been established so far. Here, we report the structure of the quaternary complex between enolase, the transition-state analogue phosphonoacetohydroxamate (PhAH), and two Mn2+ ions. The structure has been refined by using 16 561 reflections with F/sigma (F) > or = 3 to an R = 0.165 with average deviations of bond lengths and bond angles from ideal values of 0.013 A and 3.1 degrees, respectively. The "catalytic" metal ion is coordinated to two oxygen atoms of the phosphono moiety of PhAH and to the carbonyl oxygen of Gly37. Most likely, disordered water molecules complement its coordination sphere. The interaction with the site II metal ion must stabilize negative charge on the phosphate group and produce electron withdrawal from carbon 2 of the substrate, facilitating proton abstraction from carbon 2, the rate-limiting step in the catalytic process. The Gly37 residue is located in the flexible loop Ser36-His43, which assumes an "open" conformation in the absence of substrate and a "closed" conformation in the presence of a substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1994

Crystallization at low salt concentration and alkaline pH and preliminary crystallographic data for a monoclinic form of yeast enolase.

Yeast enolase (2-phospho-D-glycerate hydrolyase, E.C. 4.2.1.11) has been crystallized by vapor diffusion from a solution containing 22% PEG 4000, 100 mM Tris buffer pH = 9.3, 200 mM Li(2)SO(4). The crystals are monoclinic with a = 122.5, b = 111.8, c = 63.7 A, beta = 95.6 degrees, space group P2(1) and two dimeric molecules are present in an asymmetric part of the unit cell. Crystals have been successfully transferred to an artificial mother liquor, pH = 7.8, 20 mM in Mg(2+) and 5 mM in 2-phospho-D-glycerate. We believe that under these lower salt concentration and more alkaline conditions we should be able to localize the two metal ions that participate in catalysis as well as examine binding of high-affinity inhibitors.

Journal Article↗

PubMed1994

Congenital toxoplasmosis in the Balb/c mouse: prevention of vertical disease transmission and fetal death by vaccination.

Vertical disease transmission only occurs in Balb/c mice infected with Toxoplasma gondii for the first time during pregnancy. This is similar to the situation in humans, where a previous infection with T. gondii tends to give life-long immunity against reinfection and fetal disease transmission. The Balb/c mouse therefore provides a suitable model to study the effectiveness of T. gondii vaccine candidates. A soluble tachyzoite antigen (STAg) preparation was used to vaccinate female Balb/c mice. STAg was inoculated subcutaneously into Balb/c mice in phosphate-buffered saline (PBS), emulsified in Freund's complete adjuvant (FCA), or entrapped within non-ionic surfactant vesicles (NISV). While all inocula induced cellular immunity as measured by parasite-specific spleen cell proliferation in vitro, the highest mean proliferative values were observed in spleens from mice where NISV had been used as the adjuvant and the lowest values were observed where FCA had been used. More importantly, cultures from the NISV/STAg vaccinated mice produced significantly more gamma-interferon than the other experimental groups. This vaccine formulation was therefore identified as that most likely to induce protective immunity against toxoplasmosis. Mice were inoculated subcutaneously with either NISV/STAg or STAg in PBS 4 and 2 weeks before mating and infected orally with 20 tissue cysts of T. gondii on day 12 of pregnancy. The incidence of fetal infection and death in these mice and non-vaccinated infected dams was compared. Of 84 pups born to 14 non-vaccinated dams 45 were viable, of which 18 were found to be infected on reaching maturity.(ABSTRACT TRUNCATED AT 250 WORDS)

Abortion, Veterinary↗

PubMed1994

Studies on the adjuvant activity of non-ionic surfactant vesicles: adjuvant-driven IgG2a production independent of MHC control.

The ability of non-ionic surfactant vesicles (NISV) to stimulate humoral responses to bovine serum albumin (BSA) in H-2b, H-2d and H-2k congenic mice on the Balb genetic background was compared with that of Freund's complete adjuvant (FCA). After two subcutaneous inoculations of BSA formulated in each adjuvant, the NISV preparation was found to stimulate significantly higher total antibody production than FCA in mice carrying the H-2b haplotype at all time points measured after secondary inoculation (2, 5 and 10 weeks) and in Balb/c mice (H-2d) at two weeks after inoculation. Both adjuvants were found to overcome the apparent non-responsiveness of Balb/B mice (H-2b) to BSA alone. Analysis of the IgG subclass responses to BSA revealed a pattern of IgG1 but not IgG2a production similar to that for whole immunoglobulin. IgG1 responses invariably differed significantly, not only between adjuvant formulations but also between different H-2 haplotypes receiving the same inoculation. On the other hand, IgG2a responses did not differ significantly between H-2 haplotypes in animals given the same adjuvant preparations, although they did differ significantly in mice given BSA alone. Therefore, these results suggest that adjuvants cannot only circumvent antigen-specific non-responsiveness or low responsiveness, but also can induce antibody isotype switching independent of major histocompatibility complex controls.

Adjuvants, Immunologic↗

PubMed1994

The demonstration of an essential role for macrophages in the in vivo generation of IgG2a antibodies.

BALB/c mice were depleted of macrophages by intravenous inoculation of dichloromethylene diphosphonate entrapped in liposomes 24 h before primary and 24 h before secondary sensitization intravenously with 100 micrograms bovine serum albumin (BSA) or ovalbumin (OVA). The effectiveness of macrophage depletion was confirmed by immunocytochemistry. Five days and 14 days after secondary challenge with BSA, plasma samples from these and control mice inoculated with empty liposomes were examined for the production of BSA-specific IgG1 and IgG2a antibodies. Macrophage depletion resulted in a significantly increased production of the Th2 lymphocyte-associated IgG1 isotype, while the production of specific IgG2a antibodies, produced under the influence of Th1 cells, was totally ablated. Similar results were obtained when OVA was used as the test antigen. Furthermore, analysis of interferon-gamma (IFN-gamma) production after antigen or concanavalin A (Con A) restimulation in vitro indicated that macrophage depletion in vivo significantly reduced production of this Th1 cell-associated cytokine. These results provide strong in vivo and in vitro evidence for the macrophage being the antigen-presenting cell population responsible for Th1 cell activation.

Animals↗

PubMed1993

Effects of various salts on the steady-state enzymatic activity of E. coli alkaline phosphatase.

Seventeen salts were tested at various concentrations for their effects on E. coli alkaline phosphatase steady-state activity. Three effects were distinguished: a general ionic strength effect, and weaker cation and anion effects. 1. All salts tested, including those with "noninteracting" cations and anions, stimulate alkaline phosphatase activity usually ca. 100% at moderate (0.05-0.3 M) concentrations. 2. Cations such as Na+ and Li+ produce further increases in activity at concentrations up to 1 M. The noninteracting cations tetramethylammonium and tetrapropylammonium produce lower activities at these concentrations. These do not provide the secondary stimulatory effect of cations such as Na+ or Li+. 3. Anions associated with greater "salting in" effectiveness such as thiocyanate also reduce activity at ca. 1 M concentrations. These latter effects are not dependent on protein concentration so they probably do not involve subunit dissociation. There is little effect on the fluorescence or fluorescence-polarization spectrum of the enzyme so there is no general effect of 1 M salts on the conformation of the protein. The Michaelis constant for the substrate, p-nitrophenylphosphate, and inhibition constant for inorganic phosphate are increased to some extent by salts, but the increase in activity is due to an increase in Vmax. Our working hypothesis is that increased ionic strength weakens electrostatic interactions, enabling noncovalently bound phosphate to dissociate more rapidly.

Alkaline Phosphatase↗