Biomedical subjects

Eric A Johnson

Publications and source records attributed to Eric A Johnson.

At least 19 recordsLinked to original sources

PubMed2006

Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers.

Restriction site associated DNA (RAD) tags are a genome-wide representation of every site of a particular restriction enzyme by short DNA tags. Most organisms segregate large numbers of DNA sequence polymorphisms that disrupt restriction sites, which allows RAD tags to serve as genetic markers spread at a high density throughout the genome. Here, we demonstrate the applicability of RAD markers for both individual and bulk-segregant genotyping. First, we show that these markers can be identified and typed on pre-existing microarray formats. Second, we present a method that uses RAD marker DNA to rapidly produce a low-cost microarray genotyping resource that can be used to efficiently identify and type thousands of RAD markers. We demonstrate the utility of the former approach by using a tiling path array for the fruit fly to map a recombination breakpoint, and the latter approach by creating and using an enriched RAD marker array for the threespine stickleback. The high number of RAD markers enabled localization of a previously identified region, as well as a second region also associated with the lateral plate phenotype. Taken together, our results demonstrate that RAD markers, and the method to develop a RAD marker microarray resource, allow high-throughput, high-resolution genotyping in both model and nonmodel systems.

Animals↗

PubMed2006

Structural basis of cell surface receptor recognition by botulinum neurotoxin B.

Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking neurotransmitter release. A 'double receptor' model has been proposed in which BoNTs recognize nerve terminals via interactions with both gangliosides and protein receptors that mediate their entry. Of seven BoNTs (subtypes A-G), the putative receptors for BoNT/A, BoNT/B and BoNT/G have been identified, but the molecular details that govern recognition remain undefined. Here we report the crystal structure of full-length BoNT/B in complex with the synaptotagmin II (Syt-II) recognition domain at 2.6 A resolution. The structure of the complex reveals that Syt-II forms a short helix that binds to a hydrophobic groove within the binding domain of BoNT/B. In addition, mutagenesis of amino acid residues within this interface on Syt-II affects binding of BoNT/B. Structural and sequence analysis reveals that this hydrophobic groove is conserved in the BoNT/G and BoNT/B subtypes, but varies in other clostridial neurotoxins. Furthermore, molecular docking studies using the ganglioside G(T1b) indicate that its binding site is more extensive than previously proposed and might form contacts with both BoNT/B and synaptotagmin. The results provide structural insights into how BoNTs recognize protein receptors and reveal a promising target for blocking toxin-receptor recognition.

Animals↗

PubMed2006

Induction of the heat shock pathway during hypoxia requires regulation of heat shock factor by hypoxia-inducible factor-1.

Activation of heat shock proteins (Hsps) is critical to adaptation to low oxygen levels (hypoxia) and for enduring the oxidative stress of reoxygenation. Hsps are known to be regulated by heat shock factor (Hsf), but our results demonstrate an unexpected regulatory link between the oxygen-sensing and heat shock pathways. Hsf transcription is up-regulated during hypoxia due to direct binding by hypoxia-inducible factor-1 (HIF-1) to HIF-1 response elements in an Hsf intron. This increase in Hsf transcripts is necessary for full Hsp induction during hypoxia and reoxygenation. The HIF-1-dependent increase in Hsps has a functional impact, as reduced production of Hsps decreases viability of adult flies exposed to hypoxia and reoxygenation. Thus, HIF-1 control of Hsf transcriptional levels is a regulatory mechanism for sensitizing heat shock pathway activity in order to maximize production of protective Hsps. This cross-regulation represents a mechanism by which the low oxygen response pathway has assimilated complex new functions by regulating the key transcriptional activator of the heat shock pathway.

Animals↗

PubMed2006

Obtaining informed consent for genetic studies: The multiethnic study of atherosclerosis.

Studies of DNA may yield important information about atherosclerosis. To determine how often study participants' consent to examine DNA is denied and the factors associated with that denial, information was collected on participants in the US Multiethnic Study of Atherosclerosis (MESA) during 2000-2004. Permission was sought for preparation of DNA, transformation of cells into cell lines, evaluation of genes related to heart and other health conditions, and access to DNA by private companies. Of the 5,494 participants at entry, 897 (16.3%) refused consent for some items and 247 (4.5%) completely denied consent. At a second examination 18 months later, 819 (15.0%) partially refused and 229 (4.2%) completely denied consent. Age among men (odds ratio per 10 years = 0.68, 95% confidence interval: 0.54, 0.85; p = 0.004), ethnicity (odds ratio for African American = 2.34, 95% confidence interval: 1.66, 3.32; p < 0.001), and field center (p < 0.001) were associated with complete denial. For those giving partial consent, the most common item refused was access to DNA by private companies (baseline: 99%; second examination: 90%); younger age, male gender, and African-American ethnicity were associated with refusal. The authors concluded that a small percentage of participants in epidemiologic studies refuse consent for DNA studies, and the majority are concerned about sharing their DNA data with industry.

Age Factors↗

PubMed2006

A structural perspective of the sequence variability within botulinum neurotoxin subtypes A1-A4.

Botulinum neurotoxin (BoNT) is a category A toxin that has been classified within seven serotypes, designated A-G. Recently, it has been discovered that sequence variability occurs in BoNTs produced by serotype A (BoNT/A) variant strains, designated as subtypes A1 and A2, which have significantly different antibody-binding properties. We have therefore made efforts to understand at the molecular level the diversity and its effects on the biological actions of the toxin, including receptor binding, substrate recognition, and catalysis. We provide the results of these studies, including the analysis of two newly sequenced BoNT/A variants, Loch Maree (A3) and 657Ba (A4), and their comparison to A1 and A2. Using sequence analysis, available functional data, molecular modeling, and comparison of models with the crystal structures of BoNT/A1 and the light chain of BoNT/A2, we conclude that these sequence differences within subtypes will impact development of broad-spectrum antibody and small ligand therapeutics, and suggest dissimilarities in binding affinity and cleavage efficiency of the SNAP-25 substrate. In particular, sequence variation in subtypes BoNT/A3 and BoNT/A4 will likely effect alpha-exosite and S1' subsite recognition, respectively.

Amino Acid Sequence↗

PubMed2006

SV2 is the protein receptor for botulinum neurotoxin A.

How the widely used botulinum neurotoxin A (BoNT/A) recognizes and enters neurons is poorly understood. We found that BoNT/A enters neurons by binding to the synaptic vesicle protein SV2 (isoforms A, B, and C). Fragments of SV2 that harbor the toxin interaction domain inhibited BoNT/A from binding to neurons. BoNT/A binding to SV2A and SV2B knockout hippocampal neurons was abolished and was restored by expressing SV2A, SV2B, or SV2C. Reduction of SV2 expression in PC12 and Neuro-2a cells also inhibited entry of BoNT/A, which could be restored by expressing SV2 isoforms. Finally, mice that lacked an SV2 isoform (SV2B) displayed reduced sensitivity to BoNT/A. Thus, SV2 acts as the protein receptor for BoNT/A.

Animals↗

PubMed2006

Identification and function of hypoxia-response genes in Drosophila melanogaster.

Hypoxia, an insufficient level of oxygen in the cell, occurs during normal activity and also in pathological conditions such as ischemia and tumorigenesis. Although many hypoxia-response genes have been identified, an understanding of the functional role for these genes in the living animal is lacking. Here we present a genome-wide study of gene expression changes during hypoxia and then functionally test a subset of these genes for roles in survival and recovery from hypoxia. We found 79 genes with increased mRNA levels when adult flies were treated with 0.5% O2 for 6 h. A subset of these genes had detectably increased levels in as short as 1 h of low-oxygen treatment. Mild hypoxia levels resulted in an increase in transcription levels for only 20 genes. Viability during hypoxia and recovery time from hypoxia-induced paralysis was examined in flies with a reduction in activity in hypoxia-response genes. The observed decreased viability and increased recovery time from paralysis in many of the lines demonstrate that the increased transcript levels seen after hypoxia are important for the response to low oxygen.

Animals↗

PubMed2005

Partial protection against Botulinum B neurotoxin-induced blocking of exocytosis by a potent inhibitor of its metallopeptidase activity.

Clostridium botulinum neurotoxins (BoNTs) cause botulism, which is characterized by a flaccid paralysis, through inhibition of acetylcholine release by peripheral cholinergic nerve terminals. This is due to the zinc metallopeptidase activity of the neurotoxin, cleaving one component (synaptobrevin for BoNT/B) of the exocytosis machinery. Yet, there are no specific agents able to control the peptidase-related effects of BoNT/B. We recently developed the first compounds to inhibit this enzymatic activity in the nanomolar range. Here we report that two of our best inhibitors prevent the BoNT/B-induced cleavage of native synaptobrevin on synaptic vesicles, and partially inhibit the suppression of [3H]noradrenaline release from synaptosomes that is caused by BoNT/B. These results were obtained at micromolar concentrations, consistent with the measured inhibitory potency of these inhibitors on the native toxin. These compounds provide a new way to possibly prevent and/or to control the neurotoxin effects of botulinum.

Animals↗

PubMed2005

Design and evaluation of 16S rRNA-targeted peptide nucleic acid probes for whole-cell detection of members of the genus Listeria.

Six fluorescein-labeled peptide nucleic acid oligomers targeting Listeria-specific sequences on the 16S ribosomal subunit were evaluated for their abilities to hybridize to whole cells by fluorescence in situ hybridization (FISH). Four of these probes yielded weak or no fluorescent signals after hybridization and were not investigated further. The remaining two FISH-compatible probes, LisUn-3 and LisUn-11, were evaluated for their reactivities against 22 Listeria strains and 17 other bacterial strains belonging to 10 closely related genera. Hybridization with BacUni-1, a domain-specific eubacterial probe, was used as a positive control for target accessibility in both Listeria spp. and nontarget cells. RNase T1 treatment of select cell types was used to confirm that positive fluorescence responses were rRNA dependent and to examine the extent of nonspecific staining of nontarget cells. Both LisUn-3 and LisUn-11 yielded rapid, bright, and genus-specific hybridizations at probe concentrations of approximately 100 pmol ml(-1). LisUn-11 was the brightest probe and stained all six Listeria species. LisUn-3 hybridized with all Listeria spp. except for L. grayi, for which it had two mismatched bases. A simple ethanolic fixation yielded superior results with Listeria spp. compared to fixation in 10% buffered formalin and was applicable to all cell types studied. This study highlights the advantages of peptide nucleic acid probes for FISH-based detection of gram-positive bacteria and provides new tools for the rapid detection of Listeria spp. These probes may be useful for the routine monitoring of food production environments in support of efforts to control L. monocytogenes.

Base Sequence↗

PubMed2005

Characterization of the antibody response to the receptor binding domain of botulinum neurotoxin serotypes A and E.

Clostridium botulinum neurotoxins (BoNTs) are the most toxic proteins for humans. The current clostridial-derived vaccines against BoNT intoxication have limitations including production and accessibility. Conditions were established to express the soluble receptor binding domain (heavy-chain receptor [HCR]) of BoNT serotypes A and E in Escherichia coli. Sera isolated from mice and rabbits immunized with recombinant HCR/A1 (rHCR/A1) from the classical type A-Hall strain (ATCC 3502) (BoNT/A1) and rHCR/E from BoNT serotype E Beluga (BoNT/E(B)) neutralized the homologous serotype of BoNT but displayed differences in cross-recognition and cross-protection. Enzyme-linked immunosorbent assay and Western blotting showed that alpha-rHCR/A1 recognized epitopes within the C terminus of the HCR/A and HCR/E, while alpha-rHCR/E recognized epitopes within the N terminus or interface between the N and C termini of the HCR proteins. alpha-rHCR/E(B) sera possessed detectable neutralizing capacity for BoNT/A1, while alpha-rHCR/A1 did not neutralize BoNT/E. rHCR/A was an effective immunogen against BoNT/A1 and the Kyoto F infant strain (BoNT/A2), but not BoNT serotype E Alaska (BoNT/E(A)), while rHCR/E(B) neutralized BoNT/E(A), and under hyperimmunization conditions protected against BoNT/A1 and BoNT/A2. The protection elicited by rHCR/A1 to BoNT/A1 and BoNT/A2 and by rHCR/E(B) to BoNT/E(A) indicate that immunization with receptor binding domains elicit protection within sub-serotypes of BoNT. The protection elicited by hyperimmunization with rHCR/E against BoNT/A suggests the presence of common neutralizing epitopes between the serotypes E and A. These results show that a receptor binding domain subunit vaccine protects against serotype variants of BoNTs.

Amino Acid Sequence↗

PubMed2005

Characterization of Clostridium botulinum strains associated with an infant botulism case in the United Kingdom.

The sixth case of infant botulism in the United Kingdom was reported in 2001. The case was caused by a type B strain of Clostridium botulinum. Strains of C. botulinum were isolated from the baby's feces and from foodstuffs in the household in an attempt to document transmission. The aims of this study were to characterize the strains of C. botulinum associated with the botulism case. This was performed using a variety of techniques, including C. botulinum culture phenotypic properties, neurotoxin characterization, and pulsed-field gel electrophoresis (PFGE) banding patterns. Cultures associated with this case as well as isolates from stored and historical samples were analyzed and compared. C. botulinum type B PFGE patterns from the infant and from an opened container of infant formula were indistinguishable, while the PFGE profile of a strain presumably isolated from an unopened archival container was unique. The results suggest that the unopened brand of formula was not the source for transmission of spores to the infant and that the strain was distinct from previous botulism cases in the United Kingdom. Since environmental testing was not performed, it is not possible to deduce other sources of transmission.

Botulinum Toxins↗

PubMed2004

Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells.

Botulinum neurotoxins (BoNTs) act as zinc-dependent endopeptidases that cleave proteins required for neurotransmitter release. To detect toxin activity, fragments of the toxin substrate proteins, synaptobrevin (Syb) or synaptosome-associated protein of 25 kDa (SNAP-25), were used to link cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP). Cleavage of these fusion proteins by BoNTs abolished fluorescence resonance energy transfer between the CFP and YFP, providing a sensitive means to detect toxin activity in real-time in vitro. Furthermore, using full-length SNAP-25 and Syb as the linkers, we report two fluorescent biosensors that can detect toxin activity within living cells. Cleavage of the SNAP-25 fusion protein abolished fluorescence resonance energy transfer between CFP and YFP, and cleavage of Syb resulted in spatial redistribution of YFP fluorescence in cells. This approach provides a means to carry out cell-based screening of toxin inhibitors and to study toxin activity in situ. By using these biosensors, we found that the subcellular localizations of SNAP-25 and Syb are critical for efficient cleavage by BoNT/A and B, respectively.

Animals↗

PubMed2004

Nucleotide sequence and transcriptional analysis of the type A2 neurotoxin gene cluster in Clostridium botulinum.

The nucleotide sequences of the upstream regions of the botulinum neurotoxin type A1 (BoNT/A1) cluster of Clostridium botulinum strain NCTC 2916 and the BoNT/A2 cluster of strain Kyoto-F were determined. A novel gene, designated orfx3, was identified following the orfx2 gene in both clusters. ORF-X2 and ORF-X3 exhibit similarity to the BoNT cluster associated P-47 protein. The BoNT/A1 and BoNT/A2 clusters share a similar gene arrangement, but exhibit differences in the spacing between certain genes. Sequences with similarity to transposases were identified in these intergenic regions, suggesting that these differences arose from an ancestral insertion event. Transcriptional analysis of the BoNT/A2 cluster revealed that the genes of the cluster are primarily synthesized as three polycistronic transcripts. Two divergent polycistronic transcripts, one encoding the orfx1, orfx2, and orfx3 genes, the second encoding the p47, ntnh, and bont/a2 genes, are transcribed from conserved BoNT cluster promoters. The third polycistronic transcript, expressed at low levels, encodes the positive regulatory botR gene and the orfx genes. This is the first complete analysis of a botulinum toxin A2 cluster.

Base Sequence↗

PubMed2004

Microfluidic tectonics platform: A colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay system.

A fabrication platform for realizing integrated microfluidic devices is discussed. The platform allows for creating specific microsystems for multistep assays in an ad hoc manner as the components that perform the assay steps can be created at any location inside the device via in situ fabrication. The platform was utilized to create a prototype microsystem for detecting botulinum neurotoxin directly from whole blood. Process steps such as sample preparation by filtration, mixing and incubation with reagents was carried out on the device. Various microfluidic components such as channel network, valves and porous filter were fabricated from prepolymer mixture consisting of monomer, cross-linker and a photoinitiator. For detection of the toxoid, biotinylated antibodies were immobilized on streptavidin-functionalized agarose gel beads. The gel beads were introduced into the device and were used as readouts. Enzymatic reaction between alkaline phosphatase (on secondary antibody) and substrate produced an insoluble, colored precipitate that coated the beads thus making the readout visible to the naked eye. Clinically relevant amounts of the toxin can be detected from whole blood using the portable enzyme-linked immunosorbent assay (ELISA) system. Multiple layers can be realized for effective space utilization and creating a three-dimensional (3-D) chaotic mixer. In addition, external materials such as membranes can be incorporated into the device as components. Individual components that were necessary to perform these steps were characterized, and their mutual compatibility is also discussed.

Antibodies↗

PubMed2004

Regulation of neurotoxin complex expression in Clostridium botulinum strains 62A, Hall A-hyper, and NCTC 2916.

The kinetics of botulinum toxin gene expression have been investigated in Clostridium botulinum type A strains 62A, Hall A-hyper, and type A(B) strain NCTC 2916 during the growth cycle. The analyses were performed in TPGY and type A Toxin Production Media (TPM). The mRNA transcript levels encoding the proteins of the neurotoxin complex were determined using Northern analyses. Neurotoxin concentrations in culture supernatants and lysed cell pellets were assayed using ELISA, Western blots, and mouse bioassay. Proteolytic activation of botulinum neurotoxin during the growth cycle was evaluated by Western blots. For all three strains, mRNA transcripts for the toxin complex genes were initially detected in early log phase, reached peak levels in early stationary phase, and rapidly decreased in mid-to-late stationary phase and during lysis. Toxin expression varied depending on the strain and growth medium. Toxin production was highest in strain Hall A-hyper, followed by NCTC 2916 and 62A. For C. botulinum strain Hall A-hyper, cell lysis and toxin release into the supernatant occurred rapidly for cells grown in TPM, while cells grown in TPGY remained in stationary phase with minimal lysis and toxin release through 96 h of growth. In contrast, strains 62A and NCTC 2916 lysed more extensively than Hall A-hyper in TPGY. TPM supported higher toxin production and activation than TPGY in strains 62A and Hall A-hyper. These data support that the genes of the botulinum neurotoxin complex are temporally expressed during late-log and early stationary phase and that toxin complex formation depends on the strain and growth medium. Botulinum toxin synthesis and activation appears to be a complex process that is highly regulated by nutritional and environmental conditions. Further research is needed to elucidate the sensing mechanisms and genetic regulatory factors controlling these processes.

Journal Article↗

PubMed2004

The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability.

Botulinum neurotoxin type A (BoNT/A) is the etiological agent responsible for botulism, a disease characterized by peripheral neuromuscular blockade. BoNT/A is produced by Clostridium botulinum as a single chain protein that is activated by proteolytic cleavage to form a 50 kDa light chain (LC, 448 amino acids) and a disulfide bond-linked 100 kDa heavy chain (HC, 847 amino acids). Whilst HC comprises the receptor binding and translocation domains, LC is a Zn2+-endopeptidase that cleaves at a single glutaminyl-arginine bond corresponding to residues 197 and 198 at the C-terminus of SNAP25. Cleavage of SNAP25 uncouples the neural exocytosis docking/fusion machinery. LC/A (LC 1-448) and several C-terminal deletion proteins of LC/A were engineered and expressed as His-tagged fusion proteins in Escherichia coli. LC 1-448 was purified, but precipitated upon storage. Approximately 40% of LC 1-448 was a covalent dimer due to the formation of inter-chain disulfide bond formation at Cys430. Conversion of Cys430 to Ser abolished dimer formation of LC 1-448, but did not improve solubility. Three C-terminal deletion peptides were engineered; LC 1-425 and LC 1-418 were expressed and could be purified as soluble and stable proteins, whilst LC 1-398 was soluble, but not stable to storage. Kinetic studies showed that LC 1-448 and LC 1-425 efficiently cleaved GST-SNAP25 and the fluorescent substrate SNAPtide, while LC 1-418 catalyzed the cleavage of both the SNAP25 and the fluorescent substrate SNAPtide with a similar Km, but at a 10-fold slower kcat. Thus, regions within the C-terminus of LC/A contribute to solubility, stability, and catalysis.

Amino Acid Sequence↗

PubMed2004

Stimulation of astaxanthin formation in the yeast Xanthophyllomyces dendrorhous by the fungus Epicoccum nigrum.

A fungal contaminant on an agar plate containing colonies of Xanthophyllomyces dendrorhous markedly increased carotenoid production by yeast colonies near to the fungal growth. Spent-culture filtrate from growth of the fungus in yeast-malt medium also stimulated carotenoid production by X. dendrorhous. Four X. dendrorhous strains including the wild-type UCD 67-385 (ATCC 24230), AF-1 (albino mutant, ATCC 96816), Yan-1 (beta-carotene mutant, ATCC 96815) and CAX (astaxanthin overproducer mutant) exposed to fungal concentrate extract enhanced astaxanthin up to approximately 40% per unit dry cell weight in the wild-type strain and in CAX. Interestingly, the fungal extract restored astaxanthin biosynthesis in non-astaxanthin-producing mutants previously isolated in our laboratory, including the albino and the beta-carotene mutant. The fungus was identified as Epicoccum nigrum by morphology of sporulating cultures, and the identity confirmed by genetic characterization including rDNA sequencing analysis of the large-subunit (LSU), the internal transcribed spacer, and the D1/D2 region of the LSU. These E. nigrum rDNA sequences were deposited in GenBank under accesssion numbers AF338443, AY093413 and AY093414. Systematic rDNA homology alignments were performed to identify fungi related to E. nigrum. Stimulation of carotenogenesis by E. nigrum and potentially other fungi could provide a novel method to enhance astaxanthin formation in industrial fermentations of X. dendrorhous and Phaffia rhodozyma.

Base Sequence↗

PubMed2004

Single-cell microbiology: tools, technologies, and applications.

The field of microbiology has traditionally been concerned with and focused on studies at the population level. Information on how cells respond to their environment, interact with each other, or undergo complex processes such as cellular differentiation or gene expression has been obtained mostly by inference from population-level data. Individual microorganisms, even those in supposedly "clonal" populations, may differ widely from each other in terms of their genetic composition, physiology, biochemistry, or behavior. This genetic and phenotypic heterogeneity has important practical consequences for a number of human interests, including antibiotic or biocide resistance, the productivity and stability of industrial fermentations, the efficacy of food preservatives, and the potential of pathogens to cause disease. New appreciation of the importance of cellular heterogeneity, coupled with recent advances in technology, has driven the development of new tools and techniques for the study of individual microbial cells. Because observations made at the single-cell level are not subject to the "averaging" effects characteristic of bulk-phase, population-level methods, they offer the unique capacity to observe discrete microbiological phenomena unavailable using traditional approaches. As a result, scientists have been able to characterize microorganisms, their activities, and their interactions at unprecedented levels of detail.

Animals↗