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S J Harris

Publications and source records attributed to S J Harris.

At least 55 records · Page 3Linked to original sources
PubMed1995

Self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme.

The extent of self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme has been investigated by velocity sedimentation as a function of both NaCl and protein concentrations. The core enzyme, existing as essentially monomeric species having a sedimentation coefficient of 13.1 S at NaCl concentrations greater than 0.2 M, undergoes reversible self-association at lower salt concentrations. Estimates for the stoichiometry of association and equilibrium constants of reaction were determined from the effect of protein concentration on the weight-average sedimentation coefficient measured at different NaCl concentrations. Data analysis by a nonlinear curve-fitting procedure indicated that protein self-association is best described by a sequential model characterized by weaker association constants for each additional step of oligomerization, and any model that involves cooperative formation of oligomeric species can be excluded. These findings are at variance with the conclusion of a previous study [Shaner, S. L., Platt, D. M., Wensley, C. G., Yu, H., Burgess, R. R., & Record, M. T. (1982) Biochemistry 26, 5539-5551] which suggested that core RNA polymerase exists in equilibrium between monomeric and tetrameric forms of the enzyme and excluded the existence of intermediate species. Simulation of sedimentation velocity boundary and gradient profiles are used to assess the validity of both models of association of core protein. It was clear that had the core enzyme undergone a cooperative monomer<-->tetramer mode of association, then bimodality would have been observed in the derivative tracings of the sedimentation boundary under these experimental conditions. Nevertheless, no such observation was reported by Shaner et al. and this study. The sequential model favored by the results of this study is consistent with the proposed model resulted from a small-angle X-ray study [Heumann, H, Meisenberger O., & Pilz, I. (1982) FEBS Lett. 138, 273-276]. Further analysis of the data by the Wyman linked-function relationship [Wyman, J. (1964) Adv. Protein Chem. 19, 223-286] implies that core enzyme monomer loses approximately three counterions per contact upon association to higher oligomeric species.

DNA-Directed RNA Polymerases↗
PubMed1995

The rectangular hyperbolic binding equation for multivalent ligands.

An improved procedure for the characterization of interactions involving multivalent ligands which eliminates a previous requirement that interaction parameters for independent and equivalent binding be evaluated from a general counterpart of the Scatchard analysis is described. Binding data for the interaction of aldolase with muscle myofibrils are then used to illustrate application of the procedure, which allows the stoichiometry and intrinsic binding constant to be evaluated by standard nonlinear regression analysis in terms of a rectangular hyperbolic relationship.

Fructose-Bisphosphate Aldolase↗
PubMed1995

A simple in vivo model of collagen degradation using collagen-gelled cotton buds: the effects of collagenase inhibitors and other agents.

A simple in vivo model of collagen degradation has been developed, and the effects of various agents have been tested. Type I collagen was prepared from rat skin and acetylated with either [3H]- or [14C] acetic anhydride. The radiolabelled collagen was added to sterile cotton buds and incubated at 37 degrees C to allow the collagen to form native fibrils that were firmly adsorbed to the cotton matrix. After subcutaneous implantation of the collagen-gelled cotton buds into rats, the radiolabelled collagen was progressively removed over a period of weeks by an infiltrating granuloma. Of the agents that were administered directly into the cotton buds using subcutaneously implanted osmotic mini-pumps, only the synthetic collagenase inhibitors CI-A (containing a hydroxamate moiety as a zinc ligand) and CI-C (containing a thiol moiety as a zinc ligand) were able to prevent the removal of collagen: their efficacy correlated with the level of collagenase inhibitory activity assayed in the exudate fluid sequestered within the cotton bud granuloma. Of the agents that were administered systemically, including anti-inflammatory drugs and other compounds used as therapies for arthritis, only hydrocortisone was able to inhibit the removal of radiolabelled collagen. These results suggest that, in this model, interstitial collagenase, a member of the matrix metalloproteinase family, comprised the major degradative pathway for collagen. The collagen-gelled cotton bud model is a useful test system for delineating those processes that result in collagen catabolism. In addition, the model can be used for testing agents, including those of limited or unknown systemic bioavailability, in order to discover novel therapeutic agents for preventing collagen degradation in connective tissue diseases such as arthritis.

Animals↗
PubMed1995

Effects of heterogeneity and cooperativity on the forms of binding curves for multivalent ligands.

An exploratory investigation is made of the binding behavior that is likely to be encountered with multivalent ligands under circumstances where a single intrinsic binding constant does not suffice to describe all acceptor-ligand interactions. Numerical simulations of theoretical binding behavior have established that current criteria for recognizing heterogeneity and cooperativity of acceptor sites on the basis of the deviation of the binding curve from rectangular hyperbolic form for univalent ligands also apply to the interpretation of the corresponding binding curves for multivalent ligands. However, for systems in which the source of the departure from equivalence and independence of binding sites resides in the ligand, these criteria are reversed. On the basis of these observations a case is then made for attributing results of an experimental binding study of the interaction between pyruvate kinase and muscle myofibrils to positive cooperativity of enzyme sites rather than to heterogeneity or negative cooperativity of the myofibrillar sites.

Kinetics↗
PubMed1995

The molecular mechanism of platelet adhesion.

One of the most primitive of host-defence mechanisms is haemostasis, the ability to control blood loss. In response to vascular trauma, platelets rapidly adhere to the exposed subendothelial matrix, a process that ultimately results in the sealing of the vessel by a plug of platelets stabilised by fibrin. Paradoxically, it is the same cascade of events that leads to thrombosis and vessel occlusion, resulting in heart attack and stroke. The molecular events involved in platelet adhesion have therefore been the subject of intense investigation. In all but the largest blood vessels, the initial contact adhesion of platelets is mediated by subendothelial matrix bound von Willebrand Factor (vWF) and a specific vWF receptor on platelets, the glycoprotein (GP) Ib-V-IX complex. Our understanding of this process arose from analysis of two congenital bleeding disorders, von Willebrand's disease and the Bernard-Soulier syndrome, in which vWF or the GP Ib-V-IX, respectively, are either absent or dysfunctional. This overview discusses our current molecular understanding of platelet adhesion and how engagement of vWF by the GP Ib-V-IX complex on platelets initiates the subsequent events in platelet activation leading to either haemostasis or thrombosis.

Animals↗
PubMed1995

Properties of an identified dopamine-containing neurone in culture from the snail Helisoma.

The giant neurone in the left pedal ganglion of the snail Helisoma trivolvis is homologous with the giant dopaminergic neurone of Planorbis corneus, because the neurones have a very similar location and morphology, and react similarly with glyoxylic acid to produce an intense blue fluorescence, indicating the presence of dopamine. Each of these neurones is therefore referred to as a giant dopaminergic neurone, or GDN. Conditions for the extension of neurites and formation of chemical junctions in culture have been determined for the H. trivolvis GDN, and compared with other neurones from this species. The pattern of neurites that extended from the neurone was indistinguishable from that of another identified aminergic neurone, the large serotonergic neurone (LSN), but differed markedly from many other central neurones. However, the type of substrate also greatly affected the pattern of the neurites observed. Some of the electrical properties of the GDN in culture differed from those recorded in situ: peak spike amplitude was increased, spike half-width reduced and the firing pattern of the neurone was altered. However, the resting membrane potential was very similar. The GDN formed chemical and electrical junctions in culture. The chemical junctions formed were of the same type as those found in situ. They formed rapidly, within 18 h after plating, but were not stable and were lost within 48 h, to be replaced by a non-rectifying electrical junction. A chemical junction may form in either direction between the GDN and the LSN, but only rarely did such junctions allow transmission in both directions, as observed in situ. Experiments in which neurones were plated out at different times suggested that the direction of formation of the chemical junction was not dependent on the degree or state of neurite extension.

Animals↗
PubMed1994

Association of a phospholipase A2 (14-3-3 protein) with the platelet glycoprotein Ib-IX complex.

Platelet adhesion to subendothelial von Willebrand factor involves receptor recognition by the platelet glycoprotein (GP) Ib-IX and initiates activation signals that contribute to primary hemostasis. We show here that GPIb-IX is specifically associated with an intracellular 29-kDa protein. The physicochemical characteristics and amino acid sequence of this protein indicate that it is identical to the human zeta-isoform 14-3-3 protein, previously characterized as a platelet phospholipase A2 (PLA2). As activation of PLA2 is an early event in GPIb-IX-mediated signaling, this result suggests that ligand occupancy of GPIb-IX may directly activate PLA2, leading to platelet activation.

14-3-3 Proteins↗
PubMed1993

Induction of HIV-specific cytotoxic T lymphocytes in vivo with hybrid HIV-1 V3:Ty-virus-like particles.

In general, it has proven difficult to induce CTL responses using simple proteins or peptides without resorting to specialized adjuvants. In this study we show that particulate polymeric Ag in the form of hybrid Ty virus-like particles carrying the V3 region of HIV-1 gp120/160 envelope protein (V3:Ty-VLP) induce V3-specific CTL in BALB/c mice in the absence of adjuvant or lipid vehicle. In vitro restimulation of splenocytes with V3 peptide was necessary in order to generate effector CTL. Th cell activation was not required for this in vitro restimulation phase. The CTL induced by the V3:Ty-VLP were CD8+ve, H-2d-restricted, and HIV-1 isolate-specific (IIIB or MN). Co-administration of IIIB V3:Ty-VLP and MN V3:Ty-VLP primed both IIIB and MN V3-specific CTL. However, only IIIB V3-specific CTL were primed by hybrid Ty-VLP carrying IIIB, MN, and RF V3 loop sequences on the same particle indicating that there is intra- but not intermolecular competition between CTL epitopes. In direct comparisons, V3:Ty-VLP were substantially more potent than rgp120. Rgp160 and a 40mer IIIB V3 peptide both failed to prime V3-specific CTL. These data suggest that the particulate nature of hybrid Ty-VLP facilitates uptake into APC with subsequent access to the MHC class I processing pathway and that they may be useful vaccine vehicles for inducing cytolytic immunity against HIV-1 and other intracellular pathogens.

Amino Acid Sequence↗
PubMed1993

Influence of an anti-tumor necrosis factor monoclonal antibody on cytokine levels in patients with sepsis. The CB0006 Sepsis Syndrome Study Group.

OBJECTIVES: To determine the safety, pharmacokinetics, and activity of an anti-tumor necrosis factor (TNF)-alpha monoclonal antibody in severe sepsis. DESIGN: Open-label, prospective, phase II multicenter trial with escalating doses of a murine monoclonal antibody (CB0006). SETTING: Twelve academic medical center intensive care units in the United States and Europe. PATIENTS: Eighty patients with severe sepsis or septic shock who received standard supportive care and antimicrobial therapy in addition to the anti-TNF antibody. INTERVENTIONS: Patients were treated intravenously with one of four dosing regimens with CB0006: 0.1 mg/kg, 1.0 mg/kg, 10 mg/kg or two doses of 1 mg/kg 24 hrs apart. MEASUREMENTS AND MAIN RESULTS: The murine monoclonal anti-TNF antibody was well tolerated despite the development of anti-murine antibodies in 98% of patients. No survival benefit was found for the total study population, but patients with increased circulating TNF concentrations at study entry appeared to benefit by the high dose anti-TNF antibody treatment. Increased interleukin (IL)-6 levels predicted a fatal outcome (p = .003), but TNF levels were not found to be a prognostic indicator. TNF levels were higher (206.7 +/- 60.7 vs. 85.9 +/- 26.1 pg/mL; p < .001) and outcome was poor (41% vs. 71% survival; p = .007) in patients who were in shock at study entry when compared with septic patients not in shock. CONCLUSIONS: The murine anti-TNF-alpha monoclonal antibody CB0006 has proven to be safe in this clinical trial and may prove to be useful in septic patients with increased circulating TNF concentrations. Further studies are needed to determine efficacy and the ultimate clinical utility of this immunotherapeutic agent in sepsis.

Antibodies, Monoclonal↗
PubMed1993

Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion.

In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that the particles elicited a strong anti-Gag antibody response and a weak antibody response to the V3 region. A strong anti-V3 cytolytic T-cell response was elicited in immunized mice. These data show that retroviral Gag particles can be used as antigen presentation vehicles.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Pupillometer for clinical applications using dual 256-element linear CCD arrays.

A new pupillometer for measuring the size of the pupil of the human eye is described. It uses two inexpensive 256-element charge coupled device (CCD) arrays in an orthogonal configuration. The system is controlled and data are analysed by means of a Z80A microcomputer. Software allows the position and diameter of the pupil to be displayed on a monochrome video monitor. Infra-red diodes irradiate the eye so that measurements can be made in darkness. The accuracy is better than +/- 0.1 mm, over the range 3.2-9.5 mm. Dynamic measurements of the pupil light reflex response can be made with sampling periods as short as 20 ms.

Electronics, Medical↗
PubMed1992

Idiopathic bone cavity (traumatic bone cyst) with the radiographic appearance of a fibro-osseous lesion.

This article presents an unusual case of idiopathic bone cavity that originally was either not observed or diagnosed as a fibro-osseous condition. When a differential diagnosis was established, it included fibro-osseous conditions, odontogenic cysts and neoplasms, and central giant cell granuloma but not the definitive diagnosis. The patient's demographic data, history, symptoms, and clinical and radiographic appearance all conspired to obscure the true nature of the condition, which was not revealed until a biopsy was attempted. A more thorough initial radiographic examination might have led to an earlier diagnosis.

Adult↗
PubMed1992

Enhanced proliferative cellular responses to HIV-1 V3 peptide and gp120 following immunization with V3:Ty virus-like particles.

The induction of CD4+ T-helper (Th) cell responses is likely to be an important requirement of vaccine candidates designed to prevent or moderate human immunodeficiency virus-1 (HIV-1) infection. We have investigated the ability of hybrid Ty virus-like particles carrying the V3 loop region of the HIV-1 IIIB envelope gp120 (V3:Ty-VLP) to elicit V3-specific proliferative responses. Significant proliferation in response to stimulation in vitro with homologous IIIB V3 peptide was observed following immunization of mice with V3:Ty-VLP either as an aluminium hydroxide precipitate or without adjuvant. Responses to MN V3 peptide were also observed in certain mouse haplotypes. To assess the effect of presenting the V3 loop in this particulate form, we compared the responses induced by V3:Ty-VLP with those obtained with two non-particulate immunogens, recombinant gp120 (rgp120) and V3 peptide conjugated to albumin. V3-specific responses to V3 peptide in vitro were reproducibly higher following immunization with V3:Ty-VLP than with either rgp120 or V3-albumin coagulate (V3-alb). The data indicate that immunization with the V3 loop as a hybrid Ty-VLP results in enhanced proliferative responses to V3 peptide and recognition of rgp120 in vitro. Some cross-reactivity of Th cells for V3 sequences from different isolates was also observed.

Adjuvants, Immunologic↗
PubMed1991

Induction of high-titer neutralizing antibodies, using hybrid human immunodeficiency virus V3-Ty viruslike particles in a clinically relevant adjuvant.

The localization of neutralization determinants within the envelope glycoproteins of human immunodeficiency virus (HIV) has been largely achieved by immunizing small animals in conjunction with Freund's adjuvant. However, for eventual use in humans, candidate HIV vaccine components must also be efficacious in a nontoxic formulation. We describe here the production of hybrid Ty viruslike particles carrying the major neutralizing domain of HIV and demonstrate the induction of high-titer virus-neutralizing antibodies and an HIV-specific T-cell proliferative response after immunization in conjunction with aluminum hydroxide. As aluminum hydroxide and aluminum phosphate are the only adjuvants currently licensed for use in humans, these observations have implications for the development of an effective vaccine against HIV.

Amino Acid Sequence↗
PubMed1990

Interactions of glycolytic enzymes with erythrocyte membranes.

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.

Animals↗