PubMed Health⌕ Search

Biomedical subjects

E A Johnson

Publications and source records attributed to E A Johnson.

At least 19 recordsLinked to original sources
PubMed2006

Genetic diversity among Botulinum Neurotoxin-producing clostridial strains.

Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.

Botulinum Toxins↗
PubMed2006

Foliage influences forced convection heat transfer in conifer branches and buds.

Conifer foliage structures affect branch and bud temperature by altering the development and convective resistance of the thermal boundary layer. This paper examines foliage effects on forced convection in branches and buds of Picea glauca (Moench) Voss and Pinus contorta Dougl. Ex. Loud., two species that represent the range of variation in foliage structure among conifers. Forced convection is characterized by a power law relating Nusselt (heat transfer) and Reynolds (boundary layer development) numbers. Data were collected in a laminar flow wind tunnel for free stream velocities of 0.16-6.95 m s(-1). Scaling parameters were compared against literature values for silver cast branch replicas, a bed of real foliage, cylinders, and tube banks. Foliage structures reduced Nusselt numbers (heat transfer) relative to cylinders, which are typically used to approximate leafless branches and buds. Significantly different scaling relationships were observed for all foliage structures considered. Forced convection scaling relationships varied with foliage structure. The scaling relationships reported here account for variation within populations of branches and buds and can be used to characterize forced convection in a forest canopy.

Convection↗
PubMed2006

The importance of fires and floods on tree ages along mountainous gravel-bed streams.

This paper examines the commonly accepted assumption in the riparian literature that areas adjacent to streams do not burn. Using time-since-fire distributions, derived from stand-origin maps for a watershed in the front ranges of the Canadian Rocky Mountains, we found that the areas adjacent to streams and the whole study watershed have similar fire frequencies. In addition, the relative importance of fires and floods is regulated by a change in channel morphology associated with the creation of bars. The results demonstrate that fires solely control tree establishment along straight streams without bars, while the influence of floods is observed at the onset of lateral- and point-bar formation. This occurs because bars are formed in-channel and require smaller discharges in order to be flooded, compared to higher terraces. Consequently, bars are the only surfaces being flooded more frequently than they are being burned. Thus, overall the results indicate that, on this watershed, areas adjacent to streams are not less likely to burn than the uplands, except for lateral and point bars. The generality of these results to other systems should be tested as they have important implications for current forest ecological definition of "riparian zones," which typically include all fluvially derived landforms, from the channel banks to the terraces. Indeed, this study suggests that along smaller, headwater, gravel-bed mountain watersheds, the forests found on terraces are only influenced by fire and not fluvial processes and should therefore not be included in the riparian zone, while the forests on bars are the only surfaces currently being influenced by fluvial processes. Such a change in definition has implications for both ecologists and forest managers aiming to protect areas along streams as they now must take into account the effects of two disturbances on these small gravel-bed streams.

Conservation of Natural Resources↗
PubMed2005

Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization.

The botulinum neurotoxins (BoNTs) are category A biothreat agents which have been the focus of intensive efforts to develop vaccines and antibody-based prophylaxis and treatment. Such approaches must take into account the extensive BoNT sequence variability; the seven BoNT serotypes differ by up to 70% at the amino acid level. Here, we have analyzed 49 complete published sequences of BoNTs and show that all toxins also exhibit variability within serotypes ranging between 2.6 and 31.6%. To determine the impact of such sequence differences on immune recognition, we studied the binding and neutralization capacity of six BoNT serotype A (BoNT/A) monoclonal antibodies (MAbs) to BoNT/A1 and BoNT/A2, which differ by 10% at the amino acid level. While all six MAbs bound BoNT/A1 with high affinity, three of the six MAbs showed a marked reduction in binding affinity of 500- to more than 1,000-fold to BoNT/A2 toxin. Binding results predicted in vivo toxin neutralization; MAbs or MAb combinations that potently neutralized A1 toxin but did not bind A2 toxin had minimal neutralizing capacity for A2 toxin. This was most striking for a combination of three binding domain MAbs which together neutralized >40,000 mouse 50% lethal doses (LD(50)s) of A1 toxin but less than 500 LD(50)s of A2 toxin. Combining three MAbs which bound both A1 and A2 toxins potently neutralized both toxins. We conclude that sequence variability exists within all toxin serotypes, and this impacts monoclonal antibody binding and neutralization. Such subtype sequence variability must be accounted for when generating and evaluating diagnostic and therapeutic antibodies.

Animals↗
PubMed2004

Comparison of the pH-induced conformational change of different clostridial neurotoxins.

Clostridial neurotoxins are internalized inside acidic compartments, wherefrom the catalytic chain translocates across the membrane into the cytosol in a low pH-driven process, reaching its proteolytic substrates. The pH range in which the structural rearrangement of clostridial neurotoxins takes place was determined by 8-anilinonaphthalene-1-sulfonate and tryptophan fluorescence measurements. Half conformational change was attained at pH 4.55, 4.50, 4.40, 4.60, 4.40, and 4.40 for tetanus neurotoxin and botulinum neurotoxin serotypes /A, /B, /C, /E, and /F, respectively. This similarity indicates the key residues for the conformation transition are strongly conserved. Acidic liposomes support the conformational rearrangement shifting the effect versus higher pH values, whereas zwitterionic liposomes do not. The disulfide bridge linking the light and the heavy chains together needs to be oxidized to allow toxin membrane insertion, indicating that in vivo its reduction follows exposure to the cytosol after penetration of the endosomal membrane.

Anilino Naphthalenesulfonates↗
PubMed2004

Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease.

Botulinum neurotoxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X-ray crystal structures suggest that the active sites of the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate-induced conformational changes for enzyme activation, we synthesized a series of transition-state analog inhibitors in which the dipeptide cleavage site is replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. In this paper, we report our efforts to design inhibitors of BoNT/A metalloprotease. We confirm that an effective substrate sequence for BoNT/A metalloprotease is a 17-mer peptide corresponding to residues 187-203 of SNAP-25. A more stable substrate, Nle202SNAP-25 [187-203] was synthesized in order to develop an assay for proteolytic activity of BoNT/A metalloprotease that can be used to monitor time-dependent inhibition. Alpha-thiol amide analogs of Gln-197 were incorporated via solid-phase peptide synthesis into both 17-mer minimal peptide substrate sequences. The synthesis, characterization and inhibition kinetics for the alpha-thiol amide analogs of holotoxin A substrate are described. These substrate-derived inhibitors were shown to be submicromolar inhibitors of BoNT/A catalytic activity.

Amino Acid Sequence↗
PubMed2001

Site-directed mutagenesis identifies active-site residues of the light chain of botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are metalloproteases which block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such proteolysis accounts for the extreme toxicity of these neurotoxins and of their prolonged effect. The recently determined structures of BoNT/A and/B allows one to design active-site mutants to probe the role of specific residues in the proteolysis of SNARE proteins. Here we present the results of mutations of the second glutamyl residue involved in zinc coordination and of a tyrosine and a phenylalanine residues that occupy critical positions within the active site of BoNT/A. The spectroscopic properties of the purified mutants are closely similar to those of the wild-type molecule indicating the acquisition of a correct tertiary structure. Mutation of the Glu-262* nearly abolishes SNAP-25 hydrolysis as expected for a residue involved in zinc coordination. The Phe-266 and Tyr-366 mutants have reduced proteolytic activity indicating a direct participation in the proteolytic reaction, and their possible role in catalysis is discussed.

Amino Acid Sequence↗
PubMed2001

Transgenic rescue implicates beta2-microglobulin as a diabetes susceptibility gene in nonobese diabetic (NOD) mice.

Type 1 diabetes in both humans and nonobese diabetic (NOD) mice results from T-cell-mediated autoimmune destruction of insulin-producing pancreatic beta cells. Linkage studies have shown that type 1 diabetes in NOD mice is a polygenic disease involving more than 15 chromosomal susceptibility regions. Despite extensive investigation, the identification of individual susceptibility genes either within or outside the major histocompatibility complex region has proven problematic because of the limitations of linkage analysis. In this paper, we provide evidence implicating a single diabetes susceptibility gene, which lies outside the major histocompatibility complex region. Using allelic reconstitution by transgenic rescue, we show that NOD mice expressing the beta(2) microglobulin (beta(2)M)(a) allele develop diabetes, whereas NOD mice expressing a murine beta(2)M(b) or human allele are protected. The murine beta(2)M(a) allele differs from the beta(2)M(b) allele only at a single amino acid. Mechanistic studies indicate that the absence of the NOD beta(2)M(a) isoform on nonhematopoietic cells inhibits the development or activation of diabetogenic T cells.

Animals↗
PubMed2001

Inhibition of autoimmune diabetes in nonobese diabetic mice by transgenic restoration of H2-E MHC class II expression: additive, but unequal, involvement of multiple APC subtypes.

Transgenic restoration of normally absent H2-E MHC class II molecules on APC dominantly inhibits T cell-mediated autoimmune diabetes (IDDM) in nonobese diabetic (NOD) mice. We analyzed the minimal requirements for transgenic H2-E expression on APC subtypes (B lymphocytes vs macrophages/dendritic cells (DC)) to inhibit IDDM. This issue was addressed through the use of NOD stocks transgenically expressing high levels of H2-E and/or made genetically deficient in B lymphocytes in a series of genetic intercross and bone marrow/lymphocyte chimera experiments. Standard (H2-E(null)) NOD B lymphocytes exert a pathogenic function(s) necessary for IDDM. However, IDDM was inhibited in mixed chimeras where H2-E was solely expressed on all B lymphocytes. Interestingly, this resistance was abrogated when even a minority of standard NOD H2-E(null) B lymphocytes were also present. In contrast, in NOD chimeras where H2-E expression was solely limited to approximately half the macrophages/DC, an active immunoregulatory process was induced that inhibited IDDM. Introduction of a disrupted IL-4 gene into the NOD-H2-E transgenic stock demonstrated that induction of this Th2 cytokine does not represent the IDDM protective immunoregulatory process mediated by H2-E expression. In conclusion, high numbers of multiple subtypes of APC must express H2-E MHC class II molecules to additively inhibit IDDM in NOD mice. This raises a high threshold for success in future intervention protocols designed to inhibit IDDM by introduction of putatively protective MHC molecules into hemopoietic precursors of APC.

Animals↗
PubMed2001

Resonance energy transfer between tryptophan 57 in the epsilon subunit and pyrene maleimide labeled gamma subunit of the chloroplast ATP synthase.

The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the gamma subunit, is specifically labeled with pyrene maleimide (PM). The epsilon subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1. CF1 deficient in the epsilon subunit (CF1-epsilon) was reconstituted with mutant epsilon subunits in which phenylalanine replaced tryptophan at position 15 (epsilonW15F) and position 57 (epsilonW15/57F). CF1(epsilonW15F) containing a single tryptophan, epsilonW57, was labeled with PM at gammaC322. Resonance energy transfer (RET) from epsilonW57 to PM on gammaC322 occurred with an efficiency of energy transfer of 20%. RET was also observed from epsilonW57 to PM attached to the disulfide thiols of the gamma subunit (gammaC199,205) with an efficiency of approximately 45%. The R(o) (the distance at which the efficiency of energy transfer is 50%) for the epsilonW57 and PM donor/acceptor pair is 30 A, indicating that both gammaC322 and gammaC199,205 must be within 40 A of epsilonW57. These RET measurements show that both gammaC322 and gammaC199,205 are located near the base of the alpha/beta hexamer. This places the C-terminus of CF1 gamma much closer to epsilon than hypothesized based on homology to crystal structures of mitochondrial F1. These new RET measurements also allow the alignment of the predicted epsilon subunit structure. The orientation is similar to that predicted from cross-linking and mutational studies for the epsilon subunit of Escherichia coli F1.

Chloroplasts↗
PubMed2001

Restraint stress applied prior to chemical sensitization modulates the development of allergic contact dermatitis differently than restraint prior to challenge.

BALB/c mice were sensitized and challenged on the skin with the contact sensitizer, dinitroflourobenzene. Acute restraint applied before sensitization diminished, whereas restraint administered before challenge enhanced, chemical-induced ear swelling and leukocytic infiltration in the dermis. Administration of RU486, a glucocorticoid receptor antagonist, partially reversed restraint modulation of the ear swelling response in both restraint paradigms. Restraint did not modulate significantly the concentration of TNF-alpha and IL-1 beta in ear tissue homogenates. These data show that acute restraint modulates cutaneous sensitization differently than challenge, but the changes are not reflected in TNF-alpha or IL-l beta production.

Animals↗
PubMed2001

Th1 to Th2 cytokine shifts in nonobese diabetic mice: sometimes an outcome, rather than the cause, of diabetes resistance elicited by immunostimulation.

Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model. Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guérin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic beta-cell autoreactive T cells from a Th1 (IFN-gamma) to a Th2 (IL-4 and IL-10) profile. However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship. To partially address this issue we produced NOD mice genetically deficient in IFN-gamma, IL-4, or IL-10. Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development. Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-gamma to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance. Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-gamma. Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among beta-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.

Adjuvants, Immunologic↗
PubMed2001

Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century.

Microbial enzymes used in food processing are typically sold as enzyme preparations that contain not only a desired enzyme activity but also other metabolites of the production strain, as well as added materials such as preservatives and stabilizers. The added materials must be food grade and meet applicable regulatory standards. The purpose of this report is to present guidelines that can be used to evaluate the safety of the metabolites of the production strain that are also present in the enzyme preparation, including of course, but not limited to, the desired enzyme activity itself. This discussion builds on previously published decision tree mechanisms and includes consideration of new genetic modification technologies, for example, modifying the primary structure of enzymes to enhance specific properties that are commercially useful. The safety of the production strain remains the primary consideration in evaluating enzyme safety, in particular, the toxigenic potential of the production strain. Thoroughly characterized nonpathogenic, nontoxigenic microbial strains, particularly those with a history of safe use in food enzyme manufacture, are logical candidates for generating a safe strain lineage, through which improved strains may be derived via genetic modification by using either traditional/classical or rDNA strain improvement strategies. The elements needed to establish a safe strain lineage include thoroughly characterizing the host organism, determining the safety of all new DNA that has been introduced into the host organism, and ensuring that the procedure(s) that have been used to modify the host organism are appropriate for food use. Enzyme function may be changed by intentionally altering the amino acid sequence (e.g., protein engineering). It may be asked if such modifications might also affect the safety of an otherwise safe enzyme. We consider this question in light of what is known about the natural variation in enzyme structure and function and conclude that it is unlikely that changes which improve upon desired enzyme function will result in the creation of a toxic protein. It is prudent to assess such very small theoretical risks by conducting limited toxicological tests on engineered enzymes. The centerpiece of this report is a decision tree mechanism that updates previous enzyme safety evaluation mechanisms to accommodate advances in enzymology. We have concluded that separate mutagenicity testing is not needed if this decision tree is used to evaluate enzyme safety. Under the criteria of the decision tree, no new food enzyme can enter the market without critical evaluation of its safety.

Animals↗
PubMed2001

Clostridium botulinum and its neurotoxins: a metabolic and cellular perspective.

Clostridium botulinum comprises a diverse assemblage of clostridia that have the common property of producing a distinctive protein neurotoxin (BoNT) of similar pharmacological activity and extraordinary potency. BoNTs are produced in culture as molecular complexes consisting of BoNT, hemagglutinin (HA) and associated subcomponent proteins, nontoxic nonhemagglutinin (NTNH), and RNA. The genes encoding the protein components reside as a cluster on the chromosome, on bacteriophages, or on plasmids depending on the C. botulinum serotype. A gene BotR coding for a regulatory protein has been detected in toxin gene clusters from certain strains, as well as ORFs coding for uncharacterized components. The gene encoding TeNT is located on a large plasmid, and expression of the structural gene is controlled by the regulatory gene, TetR, located immediately upstream of the TeNT structural gene. TeNT is not known to be assembled into a protein/nucleic acid complex in culture. Cellular synthesis of BoNT and TeNT have been demonstrated to be positively regulated by the homologous proteins, BotR/A and TetR. Evidence suggests that negative regulatory factors and general control cascades such as those involved in nitrogen regulation and carbon catabolite repression also regulate synthesis of BoNTs. Neurotoxigenic clostridia have attracted considerable attention from scientists and clinicians during the past decade, and many excellent reviews are available on various aspects of these organisms and their neurotoxins. However, certain areas have not been well-studied, including metabolic regulation of toxin formation and genetic tools to study neurotoxigenic clostridia. These topics are the focus of this review.

Animals↗
PubMed2001

Botulinum toxin therapy for pain and inflammatory disorders: mechanisms and therapeutic effects.

Botulinum toxin (BTX) injections are a well-recognised therapeutic modality for the treatment of regional involuntary muscle disorders and recently BTX has been used for treatment of pain and inflammatory disorders. The primary purpose of this review is to discuss the mechanism of action of therapeutic BTX in light of both the traditional understanding of BTX pharmacological effects as well as new observations. The review will deal with clinical observations and relevant animal experimentation. The data and hypotheses presented are not only relevant to botulinum toxin technology but will certainly prove important in the basic mechanisms of some of the diseases where botulinum toxin has been successfully applied. BTX used clinically comprises botulinum neurotoxin (BoNT) complexed with non-toxic proteins. The non-toxic components of the BTX complexes stabilise the labile BoNT during purification and formulation as a therapeutic. The complex proteins may also have unrecognised clinical significance such as slowing diffusion in tissues or imparting stability. The mechanisms of BTX formulations acting on SNARE proteins are briefly reviewed providing a basis for BTX clinical applications. The potential for design of improved botulinum toxins and formulations is addressed.

Animals↗
PubMed2001

Adenovirus early region 3(E3) immunomodulatory genes decrease the incidence of autoimmune diabetes in NOD mice.

The early three (E3) region of the adenovirus (Ad) encodes a number of immunomodulatory proteins that interfere with class I major histocompatibility-mediated antigen presentation and confer resistance to cytokine-induced apoptosis in cells infected by the virus. Transgenic expression of Ad E3 genes under the rat insulin II promoter (RIP-E3) in beta-cells in nonobese diabetic (NOD) mice decreases the incidence and delays the onset of autoimmune diabetes. The immune effector cells of RIP-E3/NOD mice maintain the ability to infiltrate the islets and transfer diabetes into NOD-scid recipients, although at a significantly reduced rate compared with wild-type littermates. The islets of RIP-E3/ NOD mice can be destroyed by adoptive transfer of splenocytes from wild-type NOD mice; however, the time to onset of hyperglycemia is delayed significantly, and 40% of these recipients were not diabetic at the end of the experiment. These findings suggest that expression of E3 genes in beta-cells affects both the activation of immune effector cells and the intrinsic resistance of beta-cells to autoimmune destruction.

Adenoviridae↗
PubMed2001

Autoreactive diabetogenic T-cells in NOD mice can efficiently expand from a greatly reduced precursor pool.

A broad repertoire of pancreatic beta-cell autoreactive T-cells normally contributes to the development of type 1 diabetes in NOD mice. However, it has been unknown if a large reduction in the precursor pool from which autoreactive T-cells are drawn would inhibit the development of type 1 diabetes. To address this issue, we reduced the precursor frequency of autoreactive T-cells in NOD mice through allelic exclusion induced by transgenic expression of an H2-Db class I-restricted T-cell receptor (TCR) specific for a pathologically irrelevant lymphocytic choriomeningitis virus (LCMV) peptide. TCR allelic exclusion greatly reduced the pool of T-cells from which diabetogenic effectors could be derived in these NODxLCMV TCR Tg mice. Surprisingly, this did not impair their type 1 diabetes susceptibility. Furthermore, a diabetogenic CD8 T-cell population that is prevalent in standard NOD mice was present at essentially equivalent levels in pancreatic islets of NODxLCMV TCR Tg mice. Other data indicated that the antigenic specificity of these CD8 T-cells is primarily the function of a shared TCR-alpha chain. Although the percentage of TCR transgenic T-cells decreased in NOD versus B6,D2 control mice, much higher total numbers of both the TCR transgenic and the nontransgenic T-cells accumulated in the NOD strain. This transgenic T-cell accumulation in the absence of the cognate peptide indicated that the NOD genetic background preferentially promotes a highly efficient antigen-independent T-cell expansion. This might allow diabetogenic T-cells in NOD mice to undergo an efficient expansion before encountering antigen, which would represent an important and previously unconsidered aspect of pathogenesis.

Alleles↗