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PubMed2005

Visualization of binding and transcytosis of botulinum toxin by human intestinal epithelial cells.

Botulinum toxin is an unusually potent oral poison, which means that the toxin must have an efficient mechanism for escaping the lumen of the gut to reach the general circulation. Previous work involving iodination of toxin and analysis of its movement demonstrated a specific process of transepithelial transport. In the present study, botulinum toxin labeled with Alexa Fluor 488 was used to visualize the discrete steps of binding, internalization, transcytosis, and release. The data revealed that binding sites for the toxin were distributed across the apical surface of epithelial cells, and there was no evidence of significant clustering. The amount of toxin bound to receptors at saturation was too large to be accommodated in a single wave of endocytosis. Toxin that entered epithelial cells did not remain in the vicinity of the endocytosing membrane, which is in striking contrast to events in neuronal cells. Instead, the toxin began to spread across the length of cells, eventually being released on the basolateral surface. Migration of toxin through epithelial cells required redistribution to the cell periphery. This migration pattern could be attributed to the large and centrally located nucleus, which physically displaced transport vesicles. Transcytosed toxin began to reach the contralateral surface within ca. 5 min, and transcytosis was essentially complete within 20 to 30 min.

Animals↗
PubMed2002

Ecological effects of climate fluctuations.

Climate influences a variety of ecological processes. These effects operate through local weather parameters such as temperature, wind, rain, snow, and ocean currents, as well as interactions among these. In the temperate zone, local variations in weather are often coupled over large geographic areas through the transient behavior of atmospheric planetary-scale waves. These variations drive temporally and spatially averaged exchanges of heat, momentum, and water vapor that ultimately determine growth, recruitment, and migration patterns. Recently, there have been several studies of the impact of large-scale climatic forcing on ecological systems. We review how two of the best-known climate phenomena-the North Atlantic Oscillation and the El Niño-Southern Oscillation-affect ecological patterns and processes in both marine and terrestrial systems.

Animals↗
PubMed1999

Genomic views of human history.

New tools of genomic analysis shed light on historical puzzles. Migrations of ancient peoples, differences in migration patterns of males and females, historical demography of cultures with ancient roots, and patterns of human genetic diversity are increasingly the focus of integrated analysis by historians, anthropologists, and geneticists.

Anthropology↗
PubMed1976

Genetic composition of a high-yielding influenza A virus recombinant: a vaccine strain against "Swine" influenza.

Analysis of the RNA migration pattern of a high-yielding influenza virus recombinant, X-53, used in vaccine production, reveals that only the two genes coding for hemagglutinin and neuraminidase antigens were derived from the "swine" influenza virus parent. A/New Jersey/11/76, while six were acquired from the A/PR/8/34 (HON1) parent, donor of the high yield characteristic.

Antigens, Viral↗
PubMed2006

Biodiversity of bacterial ecosystems in traditional Egyptian Domiati cheese.

Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type.

Biodiversity↗
PubMed1977

Response of microorganisms to an accidental gasoline spillage in an arctic freshwater ecosystem.

The response of microorganisms to an accidental spillage of 55,000 gallons of leaded gasoline into an Arctic freshwater lake was studied. Shifts in microbial populations were detected after the spillage, reflecting the migration pattern of the gasoline, enrichment for hydrocarbon utilizers, and selection for leaded-gasoline-tolerant microorganisms. Ratios of gasoline-tolerant/utilizing heterotrophs to "total" heterotrophs were found to be a sensitive indicator of the degree of hydrocarbon contamination. Respiration rates were elevated in the highly contaminated area, but did not reflect differences between moderately and lightly contaminated areas. Hydrocarbon biodegradation potential experiments showed that indigenous microorganisms could extensively convert hydrocarbons to CO(2). In situ measurement of gasoline degradation showed that, if untreated, sediment samples retained significant amounts of gasoline hydrocarbons including "volatile components" at the time the lake froze for the winter. Nutrient addition and bacterial inoculation resulted in enhanced biodegradative losses, significantly reducing the amount of residual hydrocarbons. Enhanced biodegradation, however, resulted in the appearance of compounds not detected in the gasoline. Since the contaminated lake serves as a drinking water supply, treatment to enhance microbial removal of much of the remaining gasoline still may be advisable.

Alaska↗
PubMed1994

A diffusion gradient chamber for studying microbial behavior and separating microorganisms.

The natural habitats of most microbes are dynamic and include spatial gradients of growth substrates, electron acceptors, pH, salts, and inhibitory compounds. To mimic this diffusive aspect of nature, we developed an analytical diffusion gradient chamber (DGC) that can be used to separate, enrich for, isolate, and study the behavior of microorganisms. The chamber is a polycarbonate box containing an arena (5 by 5 by 2 cm) into which is cast a semisolid growth medium. Continuously replenished solute reservoirs positioned on each side of the arena but separated from it by a porous membrane enable the formation throughout the gel of multiple, intersecting gradients of solutes in two dimensions. With glucose as the solute, a gradient which spanned a 100-fold range in concentration was established across the arena in about 4 days. The shape of the glucose gradient was accurately predicted by a mathematical model based on Fickian diffusion. The growth and migratory behavior of Escherichia coli in response to imposed gradients of attractants (aspartate, alpha-methyl aspartate, and serine) and a repellent (valine) were examined. Cells responded in predictable ways to such gradients by forming distinctive growth and migration patterns in the DGC. This was true for wild-type E. coli as well as specific chemotaxis and motility mutants. The patterns yielded information about the threshold concentration of chemoeffectors needed to elicit a response as well as their saturating concentration. It was also evident that the metabolism of attractants significantly affected the gradients and, hence, the movement of cells. Finally, it was possible to separate E. coli and Pseudomonas fluorescens in the DGC on the basis of their differential responses to gradients of various chemoeffectors.

Journal Article↗
PubMed1996

Direct 5S rRNA Assay for Monitoring Mixed-Culture Bioprocesses.

This study demonstrates the efficacy of a direct 5S rRNA assay for the characterization of mixed microbial populations by using as an example the bacteria associated with acidic mining environments. The direct 5S rRNA assay described herein represents a nonselective, direct molecular method for monitoring and characterizing the predominant, metabolically active members of a microbial population. The foundation of the assay is high-resolution denaturing gradient gel electrophoresis (DGGE), which is used to separate 5S rRNA species extracted from collected biomass. Separation is based on the unique migration behavior of each 5S rRNA species during electrophoresis in denaturing gradient gels. With mixtures of RNA extracted from laboratory cultures, the upper practical limit for detection in the current experimental system has been estimated to be greater than 15 different species. With this method, the resolution was demonstrated to be effective at least to the species level. The strength of this approach was demonstrated by the ability to discriminate between Thiobacillus ferrooxidans ATCC 19859 and Thiobacillus thiooxidans ATCC 8085, two very closely related species. Migration patterns for the 5S rRNA from members of the genus Thiobacillus were readily distinguishable from those of the genera Acidiphilium and Leptospirillum. In conclusion, the 5S rRNA assay represents a powerful method by which the structure of a microbial population within acidic environments can be assessed.

Journal Article↗
PubMed1997

avnA, a gene encoding a cytochrome P-450 monooxygenase, is involved in the conversion of averantin to averufin in aflatoxin biosynthesis in Aspergillus parasiticus.

Recent studies have shown that at least 17 genes involved in the aflatoxin biosynthetic pathway are clustered within a 75-kb DNA fragment in the genome of Aspergillus parasiticus. Several additional transcripts have also been mapped to this gene cluster. A gene, avnA (previously named ord-1), corresponding to one of the two transcripts identified earlier between the ver-1 and omtA genes on the gene cluster was sequenced. The nucleotide sequence of the avnA gene contains a coding region for a protein of 495 amino acids with a calculated molecular mass of 56.3 kDa. The gene consists of three exons and two introns. Disruption of the avnA gene in the wild-type aflatoxigenic A. parasiticus strain (SU1-N3) resulted in a nonaflatoxigenic mutant which accumulated a bright yellow pigment. Thin-layer chromatographic studies with six different solvent systems showed that the migration patterns of the accumulated metabolite were identical to those of averantin, a known aflatoxin precursor. Precursor feeding studies with this mutant showed that norsolorinic acid and averantin were not converted to aflatoxin whereas 5'-hydroxyaverantin, averufanin, averufin, versicolorin A. sterigmatocystin, and O-methylsterigmatocystin were converted to aflatoxins. Southern blot analysis of the wild-type strain and avnA-disrupted mutant strain indicated that the avnA gene was disrupted in the mutant strain. A search of the GenBank database for similarity indicated that the avnA gene encodes a cytochrome P-450-type monooxygenase, and it has been assigned to a new P-450 gene family named CYP60A1. We have therefore concluded that the avnA gene encodes a fungal cytochrome P-450-type enzyme which is involved in the conversion of averantin to averufin in the aflatoxin biosynthetic pathway in A. parasiticus.

Aflatoxins↗
PubMed1997

Glutamate dehydrogenase activity profiles for type strains of ruminal Prevotella spp.

The glutamate dehydrogenase (GDH) activities for the type strains of Prevotella ruminicola (strain 23), Prevotella brevis (strain GA33), and Prevotella bryantii (strain B(1)4) were assessed by a combination of enzyme assays and analysis of migration patterns of GDH proteins following nondenaturing polyacrylamide gel electrophoresis. Unlike results with most other prokaryotes, but similar to results with other members of the family Bacteroidaceae, NADPH-utilizing specific activity was greatest in all species following ammonia-limited growth. Similar also to previous findings with P. bryantii, the NAD(P)H-utilizing GDH activity of P. ruminicola can be attributed to a single protein. However, P. brevis produces an additional GDH protein(s) in response to growth with peptides. These results conclusively demonstrate that all type strains of the ruminal Prevotella sp. grouping possess GDH activity.

Ammonia↗
PubMed2001

Motility of Marichromatium gracile in response to light, oxygen, and sulfide.

The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients. The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide. The observed migration patterns could be explained solely by the various phobic responses. In the dark, M. gracile formed an approximately 500-microm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at approximately 10 microM O(2). Flux calculations yielded a molar conversion ratio S(tot)/O(2) of 2.03:1 (S(tot) = [H(2)S] + [HS(-)] + [S(2-)]) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 microM O(2). The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen. A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at oxygen concentrations of >100 microM with no sulfide present the cells remained viable and motile for ca. 3 days both in light and darkness. Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.

Chromatiaceae↗
PubMed2005

Influence of inorganic nitrogen management regime on the diversity of nitrite-oxidizing bacteria in agricultural grassland soils.

To assess links between the diversity of nitrite-oxidizing bacteria (NOB) in agricultural grassland soils and inorganic N fertilizer management, NOB communities in fertilized and unfertilized soils were characterized by analysis of clone libraries and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Previously uncharacterized Nitrospira-like sequences were isolated from both long-term-fertilized and unfertilized soils, but DGGE migration patterns indicated the presence of additional sequence types in the fertilized soils. Detailed phylogenetic analysis of Nitrospira-like sequences suggests the existence of one newly described evolutionary group and of subclusters within previously described sublineages, potentially representing different ecotypes; the new group may represent a lineage of noncharacterized Nitrospira species. Clone libraries of Nitrobacter-like sequences generated from soils under different long-term N management regimes were dominated by sequences with high similarity to the rhizoplane isolate Nitrobacter sp. strain PJN1. However, the diversity of Nitrobacter communities did not differ significantly between the two soil types. This is the first cultivation-independent study of nitrite-oxidizing bacteria in soil demonstrating that nitrogen management practices influence the diversity of this bacterial functional group.

Bacteria↗
PubMed1975

Separation of viable Rickettsia typhi from yolk sac and L cell host components by renografin density gradient centrifugation.

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

Animals↗
PubMed1976

Properties of a mutant of Streptococcus mutans altered in glucosyltransferase activity.

A mutant of Streptococcus mutans GS-5 has been isolated as a smooth colonial variant on mitis salivarius agar. This mutant, designated SNG-1, adheres to glass surfaces as well as the parental organism when grown in the presence of sucrose. However, in contrast to the parental organism, glucose-grown cultures of the mutant did not adhere to smooth surfaces when incubated with sucrose under nongrowing conditions. The inability of the mutant organism to adhere to glass surfaces under the latter condition was a result to markedly reduced levels of mutant cell-associated glucosyltransferase activity. In addition, the extracellular activity of the mutant was also severely depressed relative to the parental activity. The reduced levels of mutant enzyme activity appear to be a result of a mutation in a structural gene coding for glucosyltransferase activity since (i) mutant glucosyltransferase activity is much less resistant to heat inactivation compared to the parental enzymes and (ii) the migration patterns of the mutant and parental enzymes differ on polyacrylamide gels and after isoelectric focusing on polyacrylamide gels. However, the kinetic properties of the mutant enzymes are similar to those of the comparable parental activities in terms of pH and temperature optima and Km values for sucrose. The mutant enzyme responsible for soluble glucan synthesis has been purified approximately 300-fold. These results are discussed in terms of the mechanism of glucan synthesis by S. mutans.

Binding Sites↗
PubMed1976

Enzymatic activities of cell-free extracts of Rickettsia typhi.

Cell-free extracts of Rickettsia typhi were tested for activities of enzymes of the tricarboxylic acid cycle, of glutamate catabolism, and of glycolysis. The organisms were grown in the yolk sacs of chicken embryos, harvested shortly before the time of embryo death, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The following enzymatic activities were demonstrated: high levels of malate dehydrogenase (MDH), moderate levels of glutamate-oxaloacetate transaminase, glutamate, succinate, and isocitrate dehydrogenases, and citrate synthase, and low levels of glutamate-pyruvate transaminase. The specific activities of some of these enzymes were higher when the rickettsiae were harvested at a time of active proliferation, 3 to 4 days prior to embryo death. Rickettsial MDH was differentiated from host MDH by its migration pattern on polyacrylamide gel electrophoresis. The activities of MDH and two other dehydrogenases, demonstrable after the cells had been disrupted, were absent from purified, intact rickettsial preparations. No activity was detected for glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, lactate dehydrogenases, phosphoglucose isomerase, fructoaldolase, or pyruvate kinase. Our results suggest that extracts of R. typhi that contain demonstrable enzymes involved in the catabolism of glutamate and tricarboxylic acid cycle intermediates, unlike Coxiella burnetti, lack detectable glycolytic activity.

Alanine Transaminase↗
PubMed1977

Angiostrongylus cantonensis: phospholipase in nonsensitized and sensitized rats after challenge.

Rats given an initial infection with Angiostrongylus cantonensis had moderately elevated phospholipase B activity in the lungs at 8 and 15 days after challenge, and greatly elevated levels were evident at 35, 43, and 49 days. In the brain, the values were elevated at 15 through 35 days. These periods of increased activity in the lungs and brain coincided with the migration patterns of the third stage larvae and the adult worms in this host. The elevated enzyme levels also were were correlated with increased numbers of eosinophils in the bone marrow at 8 and 15 days and again at 36, 43, and 49 days after infection. Similarly infected rats exhibited leukocytosis at 1 through 10 weeks of observation after challenge, and striking eosinophilia at 1, 7, 8, and 9 weeks. Rats reinfected after removal of the worms of the initial infection by thiabendazole treatment showed an anamnestic response characterized by (i) elevated enzyme values in both the lungs and brain at 1 day after reinfection and (ii) eosinophilia in the bone marrow by day 4. These accelerated responses were accompanied by a significant reduction in the worm burden of the rats. The results, which support our hypothesis that inflammation, elevated phospholipase B activity, and reduction in worm burden are causally related, are discussed in light of similar findings reported earlier from our studies with Trichinella spiralis and Nippostrongylus brasiliensis.

Animals↗
PubMed1977

Radioimmunoassay for the antigenic determinants of cholera toxin and its components.

A radioimmunoassay procedure is described for the detection of cholera toxin and its component polypeptide chains. Cholera toxin, A subunit, B subunit, alpha chain, and gamma chain were iodinated by the chloramine T procedure. Radiolabeling did not significantly alter the polyacrylamide electrophoretic migration patterns of the toxin or its components. Moreover, radiolabeled toxin, B subunit, and alpha chain preparations retained substantial ability to bind to intestinal mucosal homogenates. The minimal amount of antitoxin detectable with radiolabeled toxin was 0.04 antitoxin units/ml. Substitution of radiolabeled B subunit, A subunit, and alpha chain for radiolabeled toxin decreased the sensitivity of the test. Radiolabeled gamma chain did not bind to the antitoxin preparation. Competitive inhibition studies, with titrated anti-choleragen serum and radiolabeled toxin or components, indicated that the minimum concentration of toxin detectable was 7.0 x 10(-8) mumol/ml at a 90% inhibition level. The A subunit and alpha chain preparations inhibited the binding of the radiolabeled B subunit to antitoxin sites. Conversely, B subunit inhibited the binding of radiolabeled A subunit and alpha chain to antitoxin. The gamma chain did not show any reaction with antitoxin or cross-reaction with either whole toxin or its components. These results strongly suggest that the A subunit and the alpha chain contain antigenic determinant(s) that are common to the B subunit. The B subunit (beta chain) and the alpha chain of cholera toxin may therefore contain region(s) of chemical similarity.

Antigens, Bacterial↗
PubMed1977

Rabbit intestinal fluid stimulation by an enterotoxigenic factor of Staphylococcus aureus.

An exoprotein of Staphylococcus aureus 100 that elicited a positive ileal loop response in the rabbit model was investigated in this study. The protein, as it occurred in the culture supernatant fluid, could be detected initially in the late log phase of growth under aerobic and anaerobic conditions. It was stable under acidic conditions to pH 2.0 after 24 h at 4 degrees C. Thirty-minute treatments at 80 degrees C destroyed the ileal loop activity whereas similar trials at 70 degrees C had no effect. Although preparations of staphylococcal enterotoxins purified by other investigators did not produce positive ileal loops, the enterotoxigenic activity of the S. aureus 100 supernatant fluid could be neutralized by antisera prepared against enterotoxins A and B. Throughout purification studies, the active moiety reacted serologically with antiserum A. Polyacrylamide gel electrophoresis of the partially purified protein produced migration patterns nearly identical to those of enterotoxin A.

Animals↗