Spin-Trapping and Chemiluminescence Studies of Neutrophils from a Holstein-Friesian Calf with Bovine Leukocyte Adhesion Deficiency

The ability of neutrophils from a Holstein-Friesian calf with bovine leukocyte adhesion deficiency (the proband with a genetic deficiency of the Mac-1 (CD11b/CD18) glycoprotein corresponding to the receptor of complement iC3b) to generate oxygen radicals was examined using electron spin resonance spectrometry (ESR) combined with a spin-trapping technique and luminol-dependent chemiluminescence spectrometry. When the neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), an ESR spectrum confirming the generation of superoxide anions (O₂^-) was clearly observed in both healthy and diseased calves. However, when the neutrophils were stimulated by opsonized zymosan, appearance of the ESR spectrum was recognized in the healthy calves but not in the diseased calf. Similar results were obtained from chemiluminescence experiments.     

Spin-Trapping and Chemiluminescence Studies of Neutrophils from a Holstein-Friesian Calf with Bovine Leukocyte Adhesion Deficiency

The ability of neutrophils from a Holstein-Friesian calf with bovine leukocyte adhesion deficiency (the proband with a genetic deficiency of the Mac-1 (CD11b/CD18) glycoprotein corresponding to the receptor of complement iC3b) to generate oxygen radicals was examined using electron spin resonance spectrometry (ESR) combined with a spin-trapping technique and luminol-dependent chemiluminescence spectrometry. When the neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), an ESR spectrum confirming the generation of superoxide anions (O₂^?) was clearly observed in both healthy and diseased calves. However, when the neutrophils were stimulated by opsonized zymosan, appearance of the ESR spectrum was recognized in the healthy calves but not in the diseased calf. Similar results were obtained from chemiluminescence experiments.     

Microbial Communities in Acid Mine Drainage and Their Interaction with Pyrite Surface

Microbes such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans have been investigated a lot, because of their important role in acid mine drainage (AMD) generation. In this article, the composition of microbial communities in two AMD samples was studied. A culture-independent 16S rDNA-based cloning approach, restriction fragment length polymorphism has been used. The interaction between microbes and natural pyrite specimen surface was researched by scanning electrode microscopy (SEM) and fluorescence in situ hybridization (FISH). The phylogenetic analysis revealed bacteria in these two samples fell into three major groups: Proteobacteria, Nitrospira, and Firmicutes. Archaea was also detected in these two samples. Thermoplasma and Ferroplasma lineages were abundant. From SEM and FISH, a number of A. ferrooxidans, a few cells of Archaea and Acidiphilium were detected adsorbed on the pyrite specimen surface. Leptospirillum sp. (hybridize with the probe LF655) has not been detected to be present on the pyrite specimen surface.     

Microbial Communities in Pre-Columbian Coprolites

The study of coprolites from earlier cultures represents a great opportunity to study an “unaltered” composition of the intestinal microbiota. To test this, pre-Columbian coprolites from two cultures, the Huecoid and Saladoid, were evaluated for the presence of DNA, proteins and lipids by cytochemical staining, human and/or dog-specific Bacteroides spp. by PCR, as well as bacteria, fungi and archaea using Terminal Restriction Fragment analyses. DNA, proteins and lipids, and human-specific Bacteroides DNA were detected in all coprolites. Multidimensional scaling analyses resulted in spatial arrangements of microbial profiles by culture, further supported by cluster analysis and ANOSIM. Differences between the microbial communities were positively correlated with culture, and SIMPER analysis indicated 68.8% dissimilarity between the Huecoid and Saladoid. Proteobacteria, Bacteroidetes and methanogens were found in all coprolite samples. Propionebacteria, Shewanella and lactic acid bacteria dominated in the Huecoid samples, while Acidobacteria, and peptococci were dominant in Saladoid samples. Yeasts, including Candida albicans and Crypotococcus spp. were found in all samples. Basidiomycetes were the most notable fungi in Huecoid samples while Ascomycetes predominated in Saladoid samples, suggesting differences in dietary habits. Our study provides an approach for the study of the microbial communities of coprolite samples from various cultures.     

Effect of Concentration of Organic Chemicals on Their Biodegradation by Natural Microbial Communities

The effect of concentration on the biodegradation of synthetic organic chemicals by natural microbial communities was investigated by adding individual ¹⁴C-labeled organic compounds to stream water at various initial concentrations and measuring the formation of ¹⁴CO₂. The rate of degradation of p-chlorobenzoate and chloroacetate at initial concentrations of 47 pg/ml to 47 μg/ml fell markedly with lower initial concentrations, although half or more of the compound was converted to CO₂ in 8 days or less. On the other hand, little mineralization of 2,4-dichlorophenoxyacetate and 1-naphthyl-N-methylcarbamate, or the naphthol formed from the latter, occurred when these compounds were present at initial concentrations of 2 to 3 ng/ml or less, although 60% or more of the chemical initially present at higher concentrations was converted to CO₂ in 6 days. It is concluded that laboratory tests of biodegradation involving chemical concentrations greater than those in nature may not correctly assess the rate of biodegradation in natural ecosystems and that low substrate concentration may be important in limiting biodegradation in natural waters.     

Disordered Microbial Communities in Asthmatic Airways

Background

A rich microbial environment in infancy protects against asthma [1], [2] and infections precipitate asthma exacerbations [3]. We compared the airway microbiota at three levels in adult patients with asthma, the related condition of COPD, and controls. We also studied bronchial lavage from asthmatic children and controls.

Principal Findings

We identified 5,054 16S rRNA bacterial sequences from 43 subjects, detecting >70% of species present. The bronchial tree was not sterile, and contained a mean of 2,000 bacterial genomes per cm² surface sampled. Pathogenic Proteobacteria, particularly Haemophilus spp., were much more frequent in bronchi of adult asthmatics or patients with COPD than controls. We found similar highly significant increases in Proteobacteria in asthmatic children. Conversely, Bacteroidetes, particularly Prevotella spp., were more frequent in controls than adult or child asthmatics or COPD patients.

Significance

The results show the bronchial tree to contain a characteristic microbiota, and suggest that this microbiota is disturbed in asthmatic airways.     

Unravelling Microbial Communities with DNA-Microarrays: Challenges and Future Directions

High-throughput technologies are urgently needed for monitoring the formidable biodiversity and functional capabilities of microorganisms in the environment. Ten years ago, DNA microarrays, miniaturized platforms for highly parallel hybridization reactions, found their way into environmental microbiology and raised great expectations among researchers in the field. In this article, we briefly summarize the state-of-the-art of microarray approaches in microbial ecology research and discuss in more detail crucial problems and promising solutions. Finally, we outline scenarios for an innovative combination of microarrays with other molecular tools for structure-function analysis of complex microbial communities.     

Stereoselectivity of Isoflurane in Adhesion Molecule Leukocyte Function-Associated Antigen-1

Background

Isoflurane in clinical use is a racemate of S- and R-isoflurane. Previous studies have demonstrated that the effects of S-isoflurane on relevant anesthetic targets might be modestly stronger (less than 2-fold) than R-isoflurane. The X-ray crystallographic structure of the immunological target, leukocyte function-associated antigen-1 (LFA-1) with racemic isoflurane suggested that only S-isoflurane bound specifically to this protein. If so, the use of specific isoflurane enantiomers may have advantage in the surgical settings where a wide range of inflammatory responses is expected to occur. Here, we have further tested the hypothesis that isoflurane enantioselectivity is apparent in solution binding and functional studies.

Methods

First, binding of isoflurane enantiomers to LFA-1 was studied using 1-aminoanthracene (1-AMA) displacement assays. The binding site of each enantiomer on LFA-1 was studied using the docking program GLIDE. Functional studies employed the flow-cytometry based ICAM binding assay.

Results

Both enantiomers decreased 1-AMA fluorescence signal (at 520 nm), indicating that both competed with 1-AMA and bound to the αL I domain. The docking simulation demonstrated that both enantiomers bound to the LFA-1 “lovastatin site.” ICAM binding assays showed that S-isoflurane inhibited more potently than R-isoflurane, consistent with the result of 1-AMA competition assay.

Conclusions

In contrast with the x-ray crystallography, both enantiomers bound to and inhibited LFA-1. S-isoflurane showed slight preference over R-isoflurane.     

P-选择素糖蛋白配体1与白细胞的起始黏附

P-选择素糖蛋白配体1（P—selectin glycoprotein ligand 1,PSGL-1）是20世纪90年代初期发现的一种具同源二聚体结构的跨膜糖蛋白,表达于几乎所有白细胞表面,是迄今为止阐述得最为详尽的选择素配体。PSGL-1是以P-选择素为亲和探针分离得到的,与P-选择素有高度的亲和性。近年来,越来越多的研究证明了PSGL-1同时也是L-选择素和E-选择素的生理配体。通过PSGL-1与选择素分子间的相互作用,白细胞在血管内皮细胞上产生滚动（即起始黏附）,进而使白细胞逐步活化并稳定黏附于血管内皮。现从PSGL-1的结构、分布、表达调控、信号转导、生理病理角色、临床应用等方面进行综述。     

Submarine Hydrothermal Vents of the Aeolian Islands: Relationship Between Microbial Communities and Thermal Fluids

The relationship between fluid geochemistry and microbial communities was investigated for shallow (< 25 m) submarine hydrothermal vents in the Aeolian Islands (Southern Italy). Thermal waters, gases, and deposits of white filamentous material were collected from 11 sites. The geochemical analyses showed a magmatic component was present in all sampled fluids. The total microbial abundances, evaluated as direct counts of picoplanktonic cells (ranging from 0.2 to 2 mu m in diameter), were between 1.55 107 and 4.18 108 cells per liter. Picophytoplankton (total autofluorescent cells) ranged from 9.6 105 to 7.88 106 cells per liter. Yellow-orange autofluorescent prokaryotes belonging to the cyanobacteria were more abundant than red autofluorescent eukaryotic cells. Chemolithoautotrophic, sulfur-oxidizing, rod-shaped Bacteria were isolated from venting water samples and identified as Thiobacillus -like. Microscopic examination of the white mat deposits showed the presence of filamentous microorganisms.     

Microbial Communities and Exopolysaccharides from Polynesian Mats

Microbial mats present in two shallow atolls of French Polynesia were characterized by high amounts of exopolysaccharides associated with cyanobacteria as the predominating species. Cyanobacteria were found in the first centimeters of the gelatinous mats, whereas deeper layers showing the occurrence of the sulfate reducers Desulfovibrio and Desulfobacter species as determined by the presence of specific biomarkers. Exopolysaccharides were extracted from these mats and partially characterized. All fractions contained both neutral sugars and uronic acids with a predominance of the former. The large diversity in monosaccharides can be interpreted as the result of exopolymer biosynthesis by either different or unidentified cyanobacterial species. Received July 25, 2000; accepted October 21, 2000     

Effect of Boron Deficiency on Photosynthesis and Reductant Sources and Their Relationship with Nitrogenase Activity in Anabaena PCC 7119

Nitrogenase activity of Anabaena PCC 7119 is inhibited under conditions of boron deficiency. To elucidate the mechanisms of this inhibition, this study examined how the deficiency of boron affected photosynthesis, photosynthetic pigments, the enzymes of the oxidative pentose phosphate pathway, and respiration of Anabaena PCC 7119 cultures. After 24 to 48 hours of boron deficiency, reductions in photosynthetic O₂ evolution and in CO₂ fixation were observed. At the same time, the activities of oxidative pentose phosphate pathway enzymes and respiration increased significantly with boron deficiency. No change was observed in these processes when assays were performed after 4 to 6 hours of deficiency, a time at which nitrogenase activity was severely inhibited. These results suggest that the requirement for boron in N₂ fixation is independent of its effects on photosynthesis and reductant supply.     

Prevalence of Bacteria of Division TM7 in Human Subgingival Plaque and Their Association with Disease

Members of the uncultivated bacterial division TM7 have been detected in the human mouth, but little information is available regarding their prevalence and diversity at this site. Human subgingival plaque samples from healthy sites and sites exhibiting various stages of periodontal disease were analyzed for the presence of TM7 bacteria. TM7 ribosomal DNA (rDNA) was found in 96% of the samples, and it accounted for approximately 0.3%, on average, of all bacterial rDNA in the samples as determined by real-time quantitative PCR. Two new phylotypes of this division were identified, and members of the division were found to exhibit filamentous morphology by fluorescence in situ hybridization. The abundance of TM7 rDNA relative to total bacterial rDNA was higher in sites with mild periodontitis (0.54% ± 0.1%) than in either healthy sites (0.21% ± 0.05%, P < 0.01) or sites with severe periodontitis (0.29% ± 0.06%, P < 0.05). One division subgroup, the I025 phylotype, was detected in 1 of 18 healthy samples and 38 of 58 disease samples. These data suggest that this phylotype, and the TM7 bacterial division in general, may play a role in the multifactorial process leading to periodontitis.     

Pyrosequencing Reveals the Microbial Communities in the Red Sea Sponge Carteriospongia foliascens and Their Impressive Shifts in Abnormal Tissues

Abnormality and disease in sponges have been widely reported, yet how sponge-associated microbes respond correspondingly remains inconclusive. Here, individuals of the sponge Carteriospongia foliascens under abnormal status were collected from the Rabigh Bay along the Red Sea coast. Microbial communities in both healthy and abnormal sponge tissues and adjacent seawater were compared to check the influences of these abnormalities on sponge-associated microbes. In healthy tissues, we revealed low microbial diversity with less than 100 operational taxonomic units (OTUs) per sample. Cyanobacteria, affiliated mainly with the sponge-specific species “Candidatus Synechococcus spongiarum,” were the dominant bacteria, followed by Bacteroidetes and Proteobacteria. Intraspecies dynamics of microbial communities in healthy tissues were observed among sponge individuals, and potential anoxygenic phototrophic bacteria were found. In comparison with healthy tissues and the adjacent seawater, abnormal tissues showed dramatic increase in microbial diversity and decrease in the abundance of sponge-specific microbial clusters. The dominated cyanobacterial species Candidatus Synechococcus spongiarum decreased and shifted to unspecific cyanobacterial clades. OTUs that showed high similarity to sequences derived from diseased corals, such as Leptolyngbya sp., were found to be abundant in abnormal tissues. Heterotrophic Planctomycetes were also specifically enriched in abnormal tissues. Overall, we revealed the microbial communities of the cyanobacteria-rich sponge, C. foliascens, and their impressive shifts under abnormality.     

Microbial Communities Associated with Anaerobic Benzene Degradation in a Petroleum-Contaminated Aquifer

Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or Geobacteraceae-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the Geobacter cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number–PCR enumeration, which indicated that members of the family Geobacteraceae were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated Geobacter spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer Geothrix fermentans and the aerobe Variovorax paradoxus were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither G. fermentans nor V. paradoxus is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that Geobacter spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site’s capacity for anaerobic benzene degradation.     

Syntrophy and Interspecies Electron Transfer in Methanogenic Microbial Communities

Microbiology - Anaerobic digestion of municipal and other organic waste is a microbial process for conversion of complex organic substances to biogas (a renewable energy source) comprising a...     

Endothelial Glycocalyx Layer Properties and Its Ability to Limit Leukocyte Adhesion

The endothelial glycocalyx layer (EGL), which consists of long proteoglycans protruding from the endothelium, acts as a regulator of inflammation by preventing leukocyte engagement with adhesion molecules on the endothelial surface. The amount of resistance to adhesive events the EGL provides is the result of two properties: EGL thickness and stiffness. To determine these, we used an atomic force microscope to indent the surfaces of cultured endothelial cells with a glass bead and evaluated two different approaches for interpreting the resulting force-indentation curves. In one, we treat the EGL as a molecular brush, and in the other, we treat it as a thin elastic layer on an elastic half-space. The latter approach proved more robust in our hands and yielded a thickness of 110 nm and a modulus of 0.025 kPa. Neither value showed significant dependence on indentation rate. The brush model indicated a larger layer thickness (∼350 nm) but tended to result in larger uncertainties in the fitted parameters. The modulus of the endothelial cell was determined to be 3.0–6.5 kPa (1.5–2.5 kPa for the brush model), with a significant increase in modulus with increasing indentation rates. For forces and leukocyte properties in the physiological range, a model of a leukocyte interacting with the endothelium predicts that the number of molecules within bonding range should decrease by an order of magnitude because of the presence of a 110-nm-thick layer and even further for a glycocalyx with larger thickness. Consistent with these predictions, neutrophil adhesion increased for endothelial cells with reduced EGL thickness because they were grown in the absence of fluid shear stress. These studies establish a framework for understanding how glycocalyx layers with different thickness and stiffness limit adhesive events under homeostatic conditions and how glycocalyx damage or removal will increase leukocyte adhesion potential during inflammation.     

Microbial Communities in Chesapeake & Ohio Canal National Historical Park and Their Function as Indicators of Water Quality

Abstract

The objective of this study was to establish meaningful relationships between prokaryotic community profiles and water quality parameters in different water bodies (spring, stream, cave, and mine) in the middle reach of the Chesapeake & Ohio Canal National Historic Park (C&O), Maryland. The microbial profiles in the water samples were determined using metagenomic analysis. The relationships between microbial phylogenetic profiles and water quality parameters were investigated using principal component analysis (PCA) and redundancy analysis (RDA). The most abundant phyla identified in most samples were Proteobacteria (55.4%), Bacteroidetes (12.3%), Actinobacteria (10.6%), Firmicutes (2.4%), Planktomycetes (1.8%), Verrucomicrobia (1.5%), Chloroflexi (1.5%), and Acidobacteria (1.3%), which are major bacterial and archaeal groups typically observed in natural freshwater environments. PCA showed that water chemistry was determined primarily by the geology of the site and the type of water source (i.e., spring, stream, cave, or mine). Most samples located in carbonate formations correlated with high alkalinity, inorganic carbon, and calcium, representing the typical karstic geochemistry. RDA shows that pH, electrical conductivity, temperature and nutrients including nitrate, phosphate, and sulfate, were significant determinants of the microbial ecology.