118,609 research outputs found
Nitrogen-fixing methane-utilizing bacteria
No full textMethane occurs abundantly in nature. In the presence of oxygen this gas may be metabolized by bacteria that are able to use it as carbon and energy source. Several types of bacteria involved in the oxidation of methane have been described in literature. Methane-utilizing bacteria have in common that they can only grow on methane or methanol and not on other carbon compounds. There is much confusion in the literature about the ability of these bacteria to fix atmospheric nitrogen. The object of the investigations presented in this thesis was to obtain more information about possible nitrogen fixation by methane-utilizing bacteria.Paper I is concerned with the isolation and description of the methaneoxidizing bacterium strain 41 of the Methylosinus type. This isolate is a curved rod, motile in young cultures, but on ageing of the culture motility is lost and exospores are formed. Only methane or methanol serves as growth substrate for this bacterium. Attempts to demonstrate nitrogen fixation by this organism were only successful after it was recognized that (i) the organism was sensitive to oxygen when dependent on N 2 as nitrogen source (ii) nitrogenase activity could not be assayed by acetylene reduction when the bacterium was growing on methane.Growth of strain 41 in nitrate-containing medium with methane was little influenced by varying the oxygen pressure. But increasing the oxygen pressure when growing the bacterium in nitrogen-free medium severely reduced growth. Similar phenomena were observed when the bacterium was grown on agar plates. Colony size on nitrate-containing plates was not affected by varying the oxygen pressure but on nitrogen-free plates the effect of oxygen became apparant. Incubation in an atmosphere containing 5% oxygen allowed normal growth on nitrogen-free medium while with 20% oxygen growth was only meagre. The nitrogen-fixing colonies that developed sporadically probably were a result of locally occurring clumps of the inoculated organism.Evidence of nitrogen fixation by strain 41 when growing with methane was obtained by using 15N 2 . Excess 15N percentages of the culture were measured after the bacterium was grown at reduced oxygen pressure in nitrogen-free medium with methane and 15N 2 .Although strain 41 fixed N 2 it did not reduce acetylene when growing on methane. In papers I and II two possible explanations were considered for this erratic behaviour. It was observed that nitrogenase activity could be assayed by acetylene reduction when the bacterium was growing on methanol in nitrogen-free medium and that ethylene was co-oxidized by methanegrown cells and not by methanol-grown cells. In spite of this observation, the hypothesis that failure of the acetylene-ethylene assay was due to co-oxidation of ethylene by the methane-oxidizing bacterium was incorrect because co- oxidation of ethylene by methane-grown cells was completely prevented by acetylene.Acetylene not only completely prevented the co-oxidation of ethylene, but it also inhibited very strongly the oxidation of methane. Thus, the supply of energy and reducing power to nitrogenase, needed for the reduction of acetylene, was impeded. This explanation for the failure of the acetyleneethylene assay with methane-grown bacteria was corroborated by the observation that methanol or other substrates oxidizable in the presence of acetylene supported acetylene reduction by methane-grown cells.In paper II, experiments are reported that were undertaken to study the mechanism of inhibition of methane oxidation by acetylene. Growth of the bacterium on methanol in a nitrate-containing medium was not affected by acetylene whereas growth on methane was completely prevented in the presence of acetylene. Apparently, only the first step in the degradation route of methane, the oxidation of methane to methanol, was blocked by acetylene. This was also shown by the course of the oxygen uptake of whole-cell suspensions of strain 41. Acetylene suppressed methane-dependent oxygen uptake, whereas it did not influence methanol-dependent oxygen uptake. Interaction of acetylene with methane hydroxylase, the enzyme involved in the oxidation of methane to methanol, was further demonstrated by showing that acetylene also inhibited co-oxidation of methane hydroxylase-dependent co-substrates. Acetylene itself was only co-oxidized by methane-grown cells. Methanolgrown cells, lacking methane hydroxylase, did not co-oxidize acetylene. Experiments with whole-cell suspensions were undertaken to study the mechanism of inhibition of methane hydroxylase by acetylene. The uptake by such suspensions of methane and of dissolved oxygen, both dependent on methane concentration, was measured. From these experiments it was tentatively concluded that acetylene inhibited methane oxidation competitively. Other strains of methane-oxidizing bacteria behaved similarly to strain 41 in that growth on methane was inhibited by acetylene. Furthermore, bacteria other than the methane-oxidizing bacteria that could grow on lower hydrocarbons were inhibited by acetylene as well when growing on the alkane but not when growing on non-hydrocarbon substrates. Thus the acetylene- ethylene assay likewise could not be employed for measuring nitrogenase activity in these bacteria when the alkane was the sole energy source.The study presented in paper III surveyed the nitrogen-fixing capacity among methane-oxidizing bacteria. Nitrogen-fixing methane-oxidizing bacteria grew readily in enrichment cultures that had been inoculated with material from various habitats. Methylosinus -type bacteria were most abundant in such enrichments but Methylomonas -type organisms occurred as well. The Methylosinus -type bacteria were isolated without difficulty from the enrichments. These strains all resembled strain 41. They were motile in young cultures and formed exospores upon ageing, but the cell size and shape varied. Both straight rods and vibrioid forms were identified. The Methylomonas -type bacteria were much more difficult to isolate and to cultivate than the Methylosinus -type strains. Growth of these bacteria was enhanced in mixed culture with a small motile rod that frequently appeared as a contaminant of pure cultures. The nitrogen-fixing Methylomonas -type strain 2 possessed the type I membrane system as opposed to the type II membrane system of strain 41. This result shows that nitrogen fixation is not restricted to type II methane-oxidizing bacteria.The occurrence in nature of methane-oxidizing bacteria with the capacity to fix nitrogen was investigated by incubating dilution series of samples in nitrate-containing and nitrogen-free media. Nitrate was not found to be a significantly better nitrogen source than N 2 , indicating that the capacity to fix N 2 is common among methane-oxidizing bacteria in nature. The higher dilutions of the samples contained coccoid bacteria that differed morphologically from the isolated Methylosinus and Methylomonas -type bacteria. Apparantly, these coccoid bacteria were more abundant in the mud and soil samples investigated than were the Methylosinus and Methylomonas -type bacteria. But in enrichments they were overgrown by these faster growing organisms. Isolation of the coccoid bacteria was difficult. The first transfer from the liquid enrichment cultures to plates with nitrogen-free medium yielded colonies, but subsequent transfers from these colonies only grew in a liquid medium. One isolated coccoid organism was found to possess the type II membrane system.Paper IV comprises a study of the hydrogenase activity of strain 41. This activity was assayed by measuring the uptake of H 2 by growing cultures. There was some hydrogenase activity when the bacterium was growing in nitrate-containing medium, but the enzymic activity increased markedly when bacterial growth was dependent on the fixation of N 2 . The function of the hydrogenase-nitrogenase association was not clear. Hydrogen gas supported the reduction of acetylene, indicating that its oxidation by hydrogenase supplied reductant and energy to the nitrogenase
LONG DISTANCE RETROGRADE EFFECTSOF BOTULINUM NEUROTOXIN A (BoNT/A)
No full textBotulinum neurotoxins (designated BoNT/A-BoNT/G) are bacterial enzymes that block neurotransmitter release by cleaving essential components of the vesicle fusion machinery. BoNT/A, which cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa), is extensively exploited in clinical medicine to treat neuromuscular pathologies, facial wrinkles, and various types of pain. It is widely assumed that BoNT/A remains at the synaptic terminal and its effects are confined to the injection site. Here we demonstrate that catalytically active BoNT/A is retrogradely transported by central neurons and motoneurons and is then transcytosed to afferent synapses, in which it cleaves SNAP-25. SNAP-25 cleavage by BoNT/A was observed in the contralateral hemisphere after unilateral BoNT/A delivery to the hippocampus. Appearance of cleaved SNAP-25 resulted in blockade of hippocampal activity in the untreated hemisphere. Injections of BoNT/A into the optic tectum led to the appearance of BoNT/A-truncated SNAP-25 in synaptic terminals within the retina. Cleaved SNAP-25 also appeared in the facial nucleus after injection of the toxin into rat whisker muscles. Experiments excluded passive spread of the toxin and demonstrated axonal migration and neuronal transcytosis of BoNT/A. These findings reveal a novel pathway of BoNT/A trafficking in neurons and have important implications for the clinical uses of this neurotoxin
Evidence for Anterograde Transport and Transcytosis of Botulinum Neurotoxin A (BoNT/A)
No full textBotulinum neurotoxin type A (BoNT/A) is a metalloprotease that blocks synaptic transmission via the cleavage of SNAP-25 (synaptosomal-associated protein of 25 kDa). BoNT/A is successfully used in clinical neurology for the treatment of several neuromuscular pathologies and pain syndromes. Despite its widespread use, relatively little is known on BoNT/A intracellular trafficking in neurons. Using the visual pathway as a model system, here we show that catalytically active BoNT/A is capable of undergoing anterograde axonal transport and transcytosis. Following BoNT/A injection into the rat eye, significant levels of BoNT/A-cleaved SNAP-25 appeared in the retinorecipient layers of the superior colliculus (SC). Anterograde propagation of BoNT/A effects required axonal transport, ruling out a systemic spread of the toxin. Cleaved SNAP-25 was present in presynaptic structures of the tectum, but retinal terminals were devoid of the immunoreactivity, indicative of transcytosis. Experiments based on sequential administration of BoNT/A and BoNT/E showed a persistent catalytic activity of BoNT/A in tectal cells following its injection into the retina. Our findings demonstrate that catalytically active BoNT/A is anterogradely transported from the eye to the SC and transcytosed to tectal synapses. These data are important for a more complete understanding of the mechanisms of action of BoNT/A
Production and Purification of Polyclonal Antibodies Against Recombinant BoNT/A-HcC Domain for Sandwich ELISA Detection of BoNT/A: Detection of BoNT/A by Sandwich ELISA
No full textBotulinum neurotoxin (BoNT), a lethal bacterial toxin causing neuroparalytic disease, necessitates robust detection methods to prevent and manage botulism outbreaks. The receptor-binding domain of the toxin's heavy chain (Hc) has been extensively explored as a potential BoNT vaccine candidate. This study's primary goal is the swift detection of Botulinum neurotoxin type A (BoNT/A) using a sandwich ELISA method employing polyclonal antibodies. The recombinant BoNT/A-285HcC was induced with one mM IPTG at 25°C for 18 hours to reduce inclusion bodies and purified using Ni-NTA under non-denaturing conditions. Immunization of animals followed a specific regimen using purified BoNT/A-285HcC recombinant antigen and Freund's adjuvant. IgG antibodies from immunized mice serum were isolated using protein G resin. The purified antibodies' reactivity with recombinant BoNT/A-285HcC protein was assessed through western blotting. Efficient protein expression was achieved, yielding 50 mg/L. The recombinant BoNT/A-285HcC, with a molecular weight of 46 kDa, was purified with a near 90% purity level. ELISA results demonstrated a significant rise in anti-BoNT/A antibody titers following three doses. Western blot analysis confirmed the specific binding capability of the purified anti-BoNT/A IgG. Ultimately, the sandwich ELISA developed in this study exhibited the ability to detect 100 pg/ml of BoNT/A, utilizing 1.25 µg/ml of mice antibody as the capture and 0.3 µg/ml of rabbit antibody as the detection antibody. Purified polyclonal antibodies against recombinant BoNT/A-285HcC can be effectively employed in diagnostic serological tests for BoNT/A detection, with a limit of detection (LOD) of 100 pg/ml, significantly enhancing our ability to combat BoNT-related threats and ensuring the safety of medical applications
BoNT/A complex internalized faster than BoNT/A in HT-29 intestinal epithelial cells.
No full textCells grown on coverslips were incubated with 300 nM BoNT/A-488 (A) or BoNT/A complex-488 (B) for 30 min, washed with HBSS and labeled plasma membrane with WGA-594 (red), washed, fixed and mounted for confocal microscopy as described in methods. Images obtained using a 63X oil immersion objective. Z-stack images showed BoNT/A only on the cell surface at 30 min, while at the same time, BoNT/A complex showed internalization into the cell compartment. Optical slices were viewed from basal to apical side of the cells.</p
A smart home system is like a " Mother"!: The effects of product metaphor on consumers' comprehension of really new products (RNPs)
No full textReally new products (RNPs) are often difficult to comprehend, which may hinder consumers’ adoption. It is generally believed that designers can stimulate consumers’ comprehension by embodying RNPs in the form of product metaphors. However, empirical evidence for this is lacking. This study empirically examines the effects of product metaphors on consumers’ comprehension of RNPs. The findings of an experiment (N= 114) demonstrated an interaction effect of the presence of a product metaphor and a textual clue that explains the product metaphor on consumers’ comprehension of RNPs. Specifically, embodying a RNP in the form of a product metaphor will confuse consumers and reduce comprehension, unless the product metaphor is also explained through a textual clue.Marketing and Consumer Researc
Morbidity after IT BoNT-B injection.
No full text<p>Table shows summary of clinical observations following intrathecal injection of BoNT-B in mouse. Maximal tolerable dose was determined to be BoNT-B 0.5 U/5 µL.</p><p>*Clinical observation was made at time of sacrifice.</p
Plasma levels of oxidative stress markers, before and after BoNT/A Treatment, in chronic migraine
No full textThe pathophysiological mechanisms of migraine transformation are debated. Modifications of plasma oxidative stress biomarkers have been described in chronic migraine. OnabotulintoxinA (BoNT/A) treatment, approved for chronic migraine prophylaxis, possibly reduces pain neurotransmitters release and oxidative stress products. Aims of our study were to investigate differences in the levels of selected plasmatic oxidative stress biomarkers (Advanced Oxidation Protein Products (AOPP), Ferric Reducing Antioxidant Power (FRAP), Thiolic Groups (SH)) comparing chronic migraineurs (CM) and healthy controls (HC). We also explored possible clinical and biochemical modifications in the CM group after six months of treatment with BoNT/A. At the baseline, we found higher values of AOPP (p < 0.001), and lower values of SH (p < 0.001) and FRAP (p = 0.005) in the CM group. At the six-month follow-up we found a reduction of AOPP (p < 0.001) and an increase of FRAP (p < 0.001) and SH (p = 0.023) within the CM group. BoNT/A treatment improved migraine symptoms in the CM group. We confirmed previous reports of imbalanced antioxidant mechanisms in chronic migraine showing lower antioxidant capacities in patients than controls. BoNT/A improved the levels of plasma oxidative stress biomarkers and confirmed its role as an effective prophylactic treatment for CM. Other studies should investigate the potential antioxidant properties of BoNT/A treatment
Effect of BoNT/C on Syntaxin1A Immunofluorescence
No full text<p>Sperm were incubated with 100 nM BoNT/C (15 min at 37 °C) as explained in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#pbio-0030323-g004" target="_blank">Figure 4</a>. The cells were then fixed and triple-stained with an anti-syntaxin1A antibody that recognizes an epitope trimmed by the toxin (red; [A, D, G, and J]), FITC-PSA to differentiate between reacted and intact sperm (green; [B, E, H, and K]), and Hoechst 33258 to visualize all cells in the field (blue; [C, F, I, and L]). Notice that spontaneously reacted sperm were negative for syntaxin1A staining (arrowheads in [D] and [E]). BoNT/C had no effect on resting sperm (compare [A–C] with [D–F]). However, labeling in sperm stimulated with 10 μM Ca<sup>2+</sup> in the presence of BAPTA-AM to prevent exocytosis (observe that PSA staining is not affected) was significantly reduced by the toxin (asterisks, [G]). In contrast, the same experimental condition in the presence of the protease-inactive toxin (BoNT/C-EA) had no effect (J–L). Bars = 5 μm.</p
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