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11.
Jeff W. Stevens Yasuteru Oike Christopher Handley Vincent C. Hascall Anne Hampton Bruce Caterson 《Journal of cellular biochemistry》1984,26(4):247-259
A ternary complex of hyaluronic acid-binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid-binding region has a molecular weight of ? 65,000 (HA-BR65). N-terminal amino acids in the complex were selectively l4C-carbamylated. The resulting derivatized HA-BR65 was isolated, and tryptic peptide maps were prepared and developed on two-dimensional TLC sheets. A single, labeled peptide was obtained which gave a Mr by ? 8,000 by SDS-PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA-BR65 to a polypeptide of ? 55,000 (HA-BR55) which still retains the same N-terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid-binding region. Direct SDS-PAGE analysis revealed one major intermediate with Mr ? 109,000 (HA-BR109) as well as HA-BR65. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS-PAGE gel at Mr ? 120,000 (HA-BR120) and ? 140,000 (HA-BR140). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid-binding region, and all of them contained the same N-terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid-binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus. 相似文献
12.
The oxidant hypochlorite (OCl-), a product of the myeloperoxidase system, degrades articular cartilage proteoglycan aggregate. 总被引:2,自引:0,他引:2
The myeloperoxidase-derived oxidant, hypochlorite (OCl-) was shown to be able to degrade proteoglycan aggregate prepared from bovine articular cartilage. Exposure of proteoglycan aggregate to OCl- concentrations less than 10(-4) M resulted in a decrease in the size of the constituent proteoglycan monomers, which were unable to reaggregate with hyaluronate due to the loss of the hyaluronic acid binding region as indicated by immunoblotting using the monoclonal 1-C-6 antibody. Analysis of the [35S]-labeled core proteins by SDS/polyacrylamide electrophoresis and fluorography indicated a decrease in the size of the core protein. These data suggest that concentrations of OCl- below 10(-3) M results in the cleavage of the proteoglycan core protein in or near the hyaluronic acid binding region. The physiological consequences of these data are discussed. Exposure to higher concentrations (greater than 10(-3)) of OCl- caused more extensive degradation of the core protein; however, there was no evidence to suggest that OCl- cleaves glycosaminoglycan (GAG) chains. 相似文献
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15.
Chanenath Sriaporn Kathleen A. Campbell Maeva Millan Steven W. Ruff Martin J. Van Kranendonk Kim M. Handley 《Geobiology》2020,18(5):619-640
Digitate siliceous hot spring deposits are a form of biomediated sinter that is relatively common in the Taupo Volcanic Zone (TVZ), New Zealand, and elsewhere on Earth. Such deposits have gained prominence recently because of their morphological similarity to opaline silica rocks of likely hot spring origin found by the Spirit rover on Mars and the consequent implications for potential biosignatures there. Here, we investigate the possible relationship between microbial community composition and morphological diversity among digitate structures from actively forming siliceous hot spring sinters depositing subaerially in shallow discharge channels and around pool rims at several physicochemically distinct geothermal fields in the TVZ. The TVZ digitate sinters range in morphologic subtype from knobby to spicular, and are shown to be microstromatolites that grow under varied pH ranges, temperatures, and water chemistries. Scanning electron microscopy and molecular analyses revealed that TVZ digitate sinters are intimately associated with a diverse array of bacterial, archaeal and eukaryotic micro‐organisms, and for most digitate structures the diversity and quantity of prokaryotes was higher than that of eukaryotes. However, microbial community composition was not correlated with morphologic subtypes of digitate sinter, and observations provided limited evidence that pH (acidic versus alkali) affects morphology. Instead, results suggest hydrodynamics may be an important factor influencing variations in morphology, while water chemistry, pH, and temperature are strong drivers of microbial composition and diversity. 相似文献
16.
CJ von Ruhland 《Biotechnic & histochemistry》2013,88(7):478-484
Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue. 相似文献
17.
Michael J. Wilkins Kelly C. Wrighton Carrie D. Nicora Kenneth H. Williams Lee Ann McCue Kim M. Handley Chris S. Miller Ludovic Giloteaux Alison P. Montgomery Derek R. Lovley Jillian F. Banfield Philip E. Long Mary S. Lipton 《PloS one》2013,8(3)
While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment. 相似文献
18.
The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg?1 diet) or supplemented (220 mg kg?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values. 相似文献
19.
Kim M Handley Nathan C VerBerkmoes Carl I Steefel Kenneth H Williams Itai Sharon Christopher S Miller Kyle R Frischkorn Karuna Chourey Brian C Thomas Manesh B Shah Philip E Long Robert L Hettich Jillian F Banfield 《The ISME journal》2013,7(4):800-816
Stimulation of subsurface microorganisms to induce reductive immobilization of metals is
a promising approach for bioremediation, yet the overall microbial community response is
typically poorly understood. Here we used proteogenomics to test the hypothesis that
excess input of acetate activates complex community functioning and syntrophic
interactions among autotrophs and heterotrophs. A flow-through sediment column was
incubated in a groundwater well of an acetate-amended aquifer and recovered during
microbial sulfate reduction. De novo reconstruction of community sequences
yielded near-complete genomes of Desulfobacter (Deltaproteobacteria),
Sulfurovum- and Sulfurimonas-like Epsilonproteobacteria and
Bacteroidetes. Partial genomes were obtained for Clostridiales
(Firmicutes) and Desulfuromonadales-like Deltaproteobacteria.
The majority of proteins identified by mass spectrometry corresponded to
Desulfobacter-like species, and demonstrate the role of this organism in
sulfate reduction (Dsr and APS), nitrogen fixation and acetate oxidation to CO2
during amendment. Results indicate less abundant Desulfuromonadales, and possibly
Bacteroidetes, also actively contributed to CO2 production via the
tricarboxylic acid (TCA) cycle. Proteomic data indicate that sulfide was partially
re-oxidized by Epsilonproteobacteria through nitrate-dependent sulfide oxidation
(using Nap, Nir, Nos, SQR and Sox), with CO2 fixed using the reverse TCA cycle.
We infer that high acetate concentrations, aimed at stimulating anaerobic heterotrophy,
led to the co-enrichment of, and carbon fixation in Epsilonproteobacteria.
Results give an insight into ecosystem behavior following addition of simple organic
carbon to the subsurface, and demonstrate a range of biological processes and community
interactions were stimulated. 相似文献
20.
Short-Read Assembly of Full-Length 16S Amplicons Reveals Bacterial Diversity in Subsurface Sediments
Christopher S. Miller Kim M. Handley Kelly C. Wrighton Kyle R. Frischkorn Brian C. Thomas Jillian F. Banfield 《PloS one》2013,8(2)
In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the “long tail” of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change. 相似文献