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E A Johnson

Publications and source records attributed to E A Johnson.

At least 37 records · Page 2Linked to original sources
PubMed2001

Prevalence of rat virus infection in progeny of acutely or persistently infected pregnant rats.

Infant rats are susceptible to persistent rat virus (RV) infection, but risk of persistent infection after prenatal exposure to virus is unclear. We examined this aspect of RV infection in the progeny of dams inoculated with virus during or prior to pregnancy. Sprague-Dawley (SD) dams were infected during pregnancy (gestation day 9) by oronasal inoculation with 10(5) TCID50 of the UMass strain of RV. SD rats were infected prior to pregnancy by oronasal inoculation of two-day-old females with 10(2) TCID50 of RV-UMass, which induced persistent infection. They were mated to non-immune males after reaching sexual maturity. Rats were assessed for RV infection by virus isolation, in situ hybridization, contact transmission, or serologic testing. The progeny of dams inoculated with virus during gestation had high prevalence of infection through postpartum week 9 (9 of 12 rats were virus positive at week 3, and 7 of 10 were virus positive at week 9). Additionally, 2 of 10 rats were virus positive at least through postpartum week 15. The progeny from persistently infected, seropositive dams had no evidence of infection and did not transmit infection to contact sentinels. However, 12 dams were virus positive at necropsy and 9 had transmitted infection to their breeding partners. These results indicate that prenatal infection in non-immune dams can lead to RV persistence in their progeny. By contrast, the progeny of persistently infected dams are protected from infection, presumably by maternal antibody, although their dams can transmit infection to their breeding partners.

Acute Disease↗
PubMed2000

brp and blh are required for synthesis of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum.

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the Deltabrp strain, bacteriorhodopsin levels are decreased approximately 4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, beta-carotene, is increased approximately 3.8-fold, whereas retinal is decreased by approximately 3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the Deltabrp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of beta-carotene increased approximately 5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of beta-carotene to retinal.

Bacteriorhodopsins↗
PubMed2000

Restraint as a stressor in mice: against the dopaminergic neurotoxicity of D-MDMA, low body weight mitigates restraint-induced hypothermia and consequent neuroprotection.

In experimental studies of stress, restraint of laboratory rodents, perceived as easy to apply and believed to be reproducible, is a commonly used manipulation. The restraint manipulation is utilized as a technique to characterize the physiological, cellular and molecular consequences of stress as well as a tool to understand the ways in which stress may interact with toxic substances. In previous work, we utilized restraint in an examination of the effect of stress on the striatal dopaminergic neurotoxicity engendered by a series of substituted amphetamines. Contrary to our expectations, and most likely due to its body temperature-reducing properties in the mouse, restraint provided total or near total protection against the neurotoxicity of these agents. During subsequent studies utilizing C57Bl6/J female mice of varying weights and ages the degree of temperature reduction and the associated ability to block (20-100%) the dopamine depletion associated with the neurotoxic amphetamine 3,4-methylendioxyamphetamine (D-MDMA, 20 mg/kg of mouse body weight, every 2 h, s.c., total of four doses) were found to vary considerably more than had been previously observed. An in-depth analysis of the role mouse weight plays in the temperature reduction induced by restraint indicates mouse weight is a primary determinant of hypothermia and subsequent neuroprotection. It suggests the induction of stress in rodents by restraint is a complex effect that may lead to unanticipated results. The restraint manipulation is not as straight-forward a procedure as is commonly believed. Our data indicate that consistent application of restraint may require an adjustment of the restrainer device to mouse body weight.

3,4-Methylenedioxyamphetamine↗
PubMed2000

Regulation of proton flow and ATP synthesis in chloroplasts.

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 micromol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the gamma and epsilon subunits of CF(1) play key roles in both regulation of activity and proton translocation.

Adenosine Triphosphate↗
PubMed2000

Cloning, nucleotide sequence, and expression of the gene encoding the bacteriocin boticin B from Clostridium botulinum strain 213B.

Boticin B is a heat-stable bacteriocin produced by Clostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18. 8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene, btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing the HindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.

Amino Acid Sequence↗
PubMed2000

Persistent rat virus infection in smooth muscle of euthymic and athymic rats.

Rat virus (RV) infection can cause disease or disrupt responses that rely on cell proliferation. Therefore, persistent infection has the potential to amplify RV interference with research. As a step toward determining underlying mechanisms of persistence, we compared acute and persistent RV infections in infant euthymic and athymic rats inoculated oronasally with the University of Massachusetts strain of RV. Rats were assessed by virus isolation, in situ hybridization, and serology. Selected tissues also were analyzed by Southern blotting or immunohistochemistry. Virus was widely disseminated during acute infection in rats of both phenotypes, whereas vascular smooth muscle cells (SMC) were the primary targets during persistent infection. The prevalence of virus-positive cells remained moderate to high in athymic rats through 8 weeks but decreased in euthymic rats by 2 weeks, coincident with seroconversion and perivascular infiltration of mononuclear cells. Virus-positive pneumocytes and renal tubular epithelial cells also were detected through 8 weeks, implying that kidney and lung excrete virus during persistent infection. Viral mRNA was detected in SMC of both phenotypes through 8 weeks, indicating that persistent infection includes virus replication. However, only half of the SMC containing viral mRNA at 4 weeks stained for proliferating cell nuclear antigen, a protein expressed in cycling cells. The results demonstrate that vasculotropism is a significant feature of persistent infection, that virus replication continues during persistent infection, and that host immunity reduces, but does not eliminate, infection.

Animals↗
PubMed2000

Interferon-gamma receptor signaling is dispensable in the development of autoimmune type 1 diabetes in NOD mice.

There have been two previous conflicting reports that the development of T-cell-mediated autoimmune diabetes (type 1 diabetes) was respectively unaffected or inhibited in NOD mice genetically deficient in the T-helper (Th) 1 cytokine interferon (IFN)-gamma or the alpha-chain subunit of its receptor. Our goal was to resolve this conundrum by congenically transferring, from a 129 donor strain to the NOD background, a functionally inactivated gene for the beta-chain signaling (located on chromosome 16) rather than the alpha-chain ligand binding domain (located on chromosome 10) of the IFN-gamma receptor. These NOD.IFNgammaRBnull mice were characterized by normal patterns of leukocyte development and T-cells that produced greatly enhanced levels of the putatively type 1 diabetes-protective Th2 cytokine interleukin (IL)-4. However, despite being unable to respond to the primary Thl cytokine IFN-gamma and having T-cells that produce greatly enhanced levels of IL-4, NOD.IFNgammaRBnull mice remained highly susceptible to type 1 diabetes. This result indicated that the previously reported inhibition of type 1 diabetes in NOD mice carrying a functionally inactivated IFN-gamma receptor alpha-chain gene may have been due to a closely linked and previously unidentified diabetes resistance allele. Furthermore, our results indicate that the pathogenicity of diabetogenic T-cells in NOD mice is not dampened by an inability to respond to IFN-gamma and a concurrent shift to greatly enhanced Th2 cytokine production. This finding calls into question whether clinical protocols designed to shift beta-cell autoreactive T-cells from a Thl to Th2 cytokine production profile will truly be safe and efficacious in blocking the development of type 1 diabetes in humans.

Animals↗
PubMed1999

Monocyclic carotenoid biosynthetic pathway in the yeast Phaffia rhodozyma (Xanthophyllomyces dendrorhous).

The biosynthetic pathway of monocyclic carotenoids in the yeast Phaffia rhodozyma was studied by identifying carotenoids, applying inhibitors of carotenoid synthesis, and analyzing the carotenoids in carotenogenic mutants. Two carotenoids, torulene and 3,3'-dihydroxy-beta,phi-carotene-4,4'-dione (DCD), were identified from the yeast. Piperonyl butoxide inhibited dehydrogenation of carotenes and caused accumulation of neurosporene, lycopene, gamma-carotene, and beta-zeacarotene. Yellow mutants of P. rhodozyma produced mainly beta-carotene, which is a dicyclic carotene produced from neurosporene through lycopene. The yellow mutants grown with 0.01% triethylamine (TEA) or 2 mM 2-methylimidazole (MI) produced significantly increased amounts of torulene, which is a monocyclic carotene produced from neurosporene through beta-zeacarotene. When red strains of P. rhodozyma were grown with TEA or MI, they produced increased amounts of beta-zeacarotene, torulene, and 3-hydroxy-3',4'-didehydro-beta,phi-carotene-4-one (HDCO) (all monocyclic). DCD, accumulated especially in old cultures, was also increased in MI or TEA cultures coincident with the increases in HDCO. We propose the following monocyclic carotenoid pathway in P. rhodozyma: neurosporene-->beta-zeacarotene-->gamma-carotene-->torulene-->HDCO-->DCD.

Journal Article↗
PubMed1999

Tripeptide discriminations using circular dichroism detection.

A general spectroscopic method is described that might be applied to validating amino acid sequences in peptides and protein fragments with a view to it becoming a routine procedure with which to characterize biotechnology drug products. The tripeptides are the L-enantiomers of GGA, GGH, GGI, GGL, GGF, GHG, LGG, and YGG. The simple procedure calls for their complexation with Cu(II) ion in strong aqueous base. Binding the first three residues in the sequence, beginning at the amine terminus, completes the coordination sphere of the Cu(II) ion, so duplication of the initial sequence from peptide to peptide could be an important limiting factor in determining the extent of differentiation that is possible. The analytical focus is the selectivity associated with the chirality properties of the peptides. Detection is by circular dichroism operating in the visible range. The eight analytes were chosen as representative of a series where the sequences are most similar and therefore potentially the most difficult to discriminate spectroscopically. All have just one chiral center. Using ellipticity data at all (n = 1500) wavelengths in the measured spectra, and two novel data reduction procedures, total discrimination among all eight analytes is achieved. The method has considerable potential for use in quality control of peptide and protein biotechnological drug forms, especially their enantiomeric purities.

Algorithms↗
PubMed1999

A convenient assay method for the quality control of peptides and proteins.

The development of a convenient and very accurate procedure with which to discriminate among subsets of structurally similar peptides and proteins, and measure enantiomeric purities with very good accuracy, has been described in a series of recent articles. A factor preventing its general application to all peptide forms is that comparisons were originally limited to closed subsets of structurally similar types, e.g., dipeptides, tripeptides, and insulin drug forms. In the most recent of these articles, a modification to the method was described which did enable the comparisons to be extended between sets, in particular the di-and tripeptides. That same modification is extended even further in this article to include additional di- and tripeptides, glycylglycine oligomers, insulin drug forms, and neuropeptides. The same principal component analysis treatment used for data reduction and statistical comparisons in prior work enables the discrimination among 49 of the total of 51 analytes investigated.

Circular Dichroism↗
PubMed1999

Pure botulinum neurotoxin is absorbed from the stomach and small intestine and produces peripheral neuromuscular blockade.

Clostridium botulinum serotype A produces a neurotoxin composed of a 100-kDa heavy chain and a 50-kDa light chain linked by a disulfide bond. This neurotoxin is part of a ca. 900-kDa complex, formed by noncovalent association with a single nontoxin, nonhemagglutinin subunit and a family of hemagglutinating proteins. Previous work has suggested, although never conclusively demonstrated, that neurotoxin alone cannot survive passage through the stomach and/or cannot be absorbed from the gut without the involvement of auxiliary proteins in the complex. Therefore, this study compared the relative absorption and toxicity of three preparations of neurotoxin in an in vivo mouse model. Equimolar amounts of serotype A complex with hemagglutinins, complex without hemagglutinins, and purified neurotoxin were surgically introduced into the stomach or into the small intestine. In some experiments, movement of neurotoxin from the site of administration was restricted by ligation of the pylorus. Comparison of relative toxicities demonstrated that at adequate doses, complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin can be absorbed from the stomach. The potency of neurotoxin in complex was greater than that of pure neurotoxin, but the magnitude of this difference diminished as the dosage of neurotoxin increased. Qualitatively similar results were obtained when complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin were placed directly into the intestine. This work establishes that pure botulinum neurotoxin serotype A is toxic when administered orally. This means that pure neurotoxin does not require hemagglutinins or other auxiliary proteins for absorption from the gastrointestinal system into the general circulation.

Absorption↗
PubMed1999

Clostridial toxins as therapeutic agents: benefits of nature's most toxic proteins.

Toxins are increasingly being used as valuable tools for analysis of cellular physiology, and some are used medicinally for treatment of human diseases. In particular, botulinum toxin, the most poisonous biological substance known, is used for treatment of a myriad of human neuromuscular disorders characterized by involuntary muscle contractions. Since approval of type-A botulinum toxin by the US Food and Drug Administration in December 1989 for three disorders (strabismus, blepharospasm, and hemifacial spasm), the number of indications being treated has increased greatly to include numerous focal dystonias, spasticity, tremors, cosmetic applications, migraine and tension headaches, and other maladies. Many of these diseases were previously refractory to pharmacological and surgical treatments. The remarkable therapeutic utility of botulinum toxin lies in its ability to specifically and potently inhibit involuntary muscle activity for an extended duration. The clostridia produce more protein toxins than any other bacterial genus and are a rich reservoir of toxins for research and medicinal uses. Research is underway to use clostridial toxins or toxin domains for drug delivery, prevention of food poisoning, and the treatment of cancer and other diseases. The remarkable success of botulinum toxin as a therapeutic agent has created a new field of investigation in microbiology.

Animals↗
PubMed1999

Survival of Listeria monocytogenes in commercial cheese brines.

The survival of Listeria monocytogenes was determined in commercial cheese brines collected from cheese factories in Wisconsin and northern Illinois. Survival of L. monocytogenes inoculated into commercial cheese brines ranged from < 7 d to over 259 d. Survival did not correlate with pH, salt content, nitrogen content, mineral content, or inherent microbial populations but was negatively associated with addition of sodium hypochlorite at the dairy plant. The L. monocytogenes generally survived longer in brines held at 4 degrees C than at 12 degrees C. Sodium hypochlorite or hydrogen peroxide inactivated L. monocytogenes when added to commercial brines in the lab at 10 to 100 ppm or 0.001% to 0.02%, respectively. Addition of 1% potassium sorbate or 1% sodium benzoate also decreased survival of L. monocytogenes. Laboratory filtration of commercial brines had a negative effect on survival in one of three brines tested. The L. monocytogenes did not grow during incubation in any of the commercial brine samples tested.

Cheese↗
PubMed1999

Toxin production by Clostridium botulinum in pasteurized milk treated with carbon dioxide.

The addition of carbon dioxide to milk at levels of <20 mM inhibits the growth of selected spoilage organisms and extends refrigerated shelf life. Our objective was to determine if the addition of CO2 influenced the risk of botulism from milk. Carbon dioxide was added to pasteurized 2% fat milk at approximately 0, 9.1, or 18.2 mM using a commercial gas-injection system. The milk was inoculated with a 10-strain mixture of proteolytic and nonproteolytic Clostridium botulinum spore strains to yield 10(1) to 10(2) spores/ml. Milk was stored at 6.1 or 21 degrees C for 60 or 6 days, respectively, in sealed glass jars or high-density polyethylene plastic bottles. Milk stored at 21 degrees C curdled and exhibited a yogurt-like odor at 2 days and was putrid at 4 days. Botulinal toxin was detected in 9.1 mM CO2 milk at 4 days and in all treatments after 6 days of storage at 21 degrees C. All toxic samples were grossly spoiled based on sensory evaluation at the time toxin was detected. Although botulinal toxin appeared earlier in milk treated with 9.1 mM CO2 compared to both the 18.2 mM and untreated milk, gross spoilage would act as a deterrent to consumption of toxic milk. No botulinal toxin was detected in any treatment stored at 6.1 degrees C for 60 days. At 6.1 degrees C, the standard plate counts (SPCs) were generally lower in the CO2-treated samples than in controls, with 18.2 mM CO2 milk having the lowest SPC. These data indicate that the low-level addition of CO2 retards spoilage of pasteurized milk at refrigeration temperatures and does not increase the risk of botulism from treated milk stored at refrigeration or abuse temperatures.

Animals↗
PubMed1999

Evaluation of botulinal toxin production in packaged fresh-cut cantaloupe and honeydew melons.

The ability of Clostridium botulinum to produce toxin on cubed, packaged melons was investigated relative to microbial spoilage at various incubation temperatures and in different packaging systems. Freshly cut cubes (approximately 2.5 cm3) of cantaloupe and honeydew melons were surface inoculated with a 10 strain mixture of proteolytic and nonproteolytic spores of C. botulinum (10 to 15 cubes per package; approximately 100 total spores per package). To initially evaluate toxin production and spoilage in a passively modified atmosphere, melon cubes were loosely packaged in air in polyethylene pouches, sealed, and incubated at 7 or 15 degrees C for up to 21 days. At various sampling intervals, samples were tested for headspace oxygen and carbon dioxide levels, pH, presence of botulinal toxin, aerobic and anaerobic plate counts, and counts of yeasts and molds. During incubation, headspace oxygen levels decreased, headspace carbon dioxide levels increased, aerobic and anaerobic plate counts increased, and the pH remained constant or decreased slightly. Botulinal toxin was not detected in any cantaloupe samples or in honeydew samples incubated at 7 degrees C. Botulinal toxin was detected in some honeydew samples at 15 degrees C after 9 days of incubation, but the toxic honeydews were severely spoiled and considered organoleptically unacceptable. A similar second experiment was performed in which half of the melon cubes were treated with UV light to inactivate vegetative organisms before packaging, and these were incubated at 7, 15, or 27 degrees C. In this second experiment, toxin production occurred in the UV-treated samples at 15 degrees C with gross spoilage and at 27 degrees C with only marginal spoilage. These data indicate that inhibition of spoilage organisms with UV light could result in botulinal toxin formation in packaged melons before overt spoilage.

Bacteria, Aerobic↗
PubMed1998

Conjugative transfer of the Escherichia coli-Clostridium perfringens shuttle vector pJIR1457 to Clostridium botulinum type A strains.

An RP4-oriT shuttle vector pJIR1457 originally developed for Clostridium perfringens was successfully transferred by conjugation from Escherichia coli to Clostridium botulinum type A strains and to a nontoxigenic C. botulinum type A-transposon Tn916 mutant strain lacking the entire toxin gene cluster. The light chain (LC) of botulinum toxin was highly expressed in the toxin deletion mutant strain from a pJIR1457 construct containing the recombinant botulinal gene for LC. This shuttle vector system will be valuable for genetic analysis of C. botulinum and will enable genetic manipulation and recombinant expression studies of botulinum neurotoxins as pharmaceutical agents.

Botulinum Toxins↗