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91.
We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in 13C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of 13C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the 13C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the 13C-labeled compounds. Similar results were obtained with the 13C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the 13C analysis of the captured SSU rRNA. 相似文献
92.
Persson E 《Biochemical and biophysical research communications》2006,341(1):28-32
The initial recognition and binding of macromolecular substrates by factor VIIa (FVIIa) in complex with tissue factor has been shown to be mediated by areas distinct from the active site (so-called exosites). The present aim was to shed light on whether the N-terminal tail of the protease domain of FVIIa influences factor X (FX) binding, and whether the zymogen-like conformation of free FVIIa has a decreased affinity for FX compared to the active conformation. Two derivatives of FVIIa, one (FFR-FVIIa) with a stably buried N-terminus representing the active conformation of FVIIa and one (V154G-FVIIa) with a fully exposed N-terminus representing the zymogen-like conformation, were used as inhibitors of FVIIa-catalyzed FX activation. Their inhibitory capacities were very similar, with K(i) values not significantly different from the K(m) for FX. This indicates that the conformational state of the N-terminus does not affect FX binding or, alternatively, that the activation domain including the N-terminal insertion site is easily shifted to the stable conformation ensuing FX docking to the zymogen-like conformation. The net outcome is that FX binding to the zymogen-like form of FVIIa does not appear to be impaired. 相似文献
93.
Baldal EA Brakefield PM Zwaan BJ 《Evolution; international journal of organic evolution》2006,60(7):1435-1444
Starvation resistance is a trait often associated with longevity. Animals with increased longevity frequently show elevated starvation resistance and vice versa. Consequently, both life-history traits are thought to share genetic and physiological mechanisms, such as increased fat content and lowered metabolic rate. Here, we present results from 20 generations of selection on Drosophila melanogaster for increased starvation resistance at the time of adult eclosion. We observe that starvation resistance can be the result of more than one mechanism, all associated with an increase in fat resources. In general, metabolic rate is lowered under starved conditions relative to fed conditions. Metabolic rate in the starvation resistant lines is generally higher than in control lines under starved conditions. Starvation resistant flies are able to sustain a higher metabolic rate for a longer period of time when food is unavailable. This implies depletion of the increased fat reserves. However, longevity was not consistently affected by selection for increased starvation resistance. Similarly, paraquat resistance differed between selection lines and did not associate with starvation resistance, but rather with longevity. The results are discussed in relation to previous reported results on starvation resistance and its relation with mechanisms of aging and longevity. 相似文献
94.
Calpain 2 regulates membrane protrusion during cell migration. However, relevant substrates that mediate the effects of calpain on protrusion have not been identified. One potential candidate substrate is the actin binding protein cortactin. Cortactin is a Src substrate that drives actin polymerization by activating the Arp2/3 complex and also stabilizes the cortical actin network. We now provide evidence that proteolysis of cortactin by calpain 2 regulates membrane protrusion dynamics during cell migration. We show that cortactin is a calpain 2 substrate in fibroblasts and that the preferred cleavage site occurs in a region between the actin binding repeats and the alpha-helical domain. We have generated a mutant cortactin that is resistant to calpain proteolysis but retains other biochemical properties of cortactin. Expression of the calpain-resistant cortactin, but not wild-type cortactin, impairs cell migration and increases transient membrane protrusion, suggesting that calpain proteolysis of cortactin limits membrane protrusions and regulates migration in fibroblasts. Furthermore, the enhanced protrusion observed with the calpain-resistant cortactin requires both the Arp2/3 binding site and the Src homology 3 domain of cortactin. Together, these findings suggest a novel role for calpain-mediated proteolysis of cortactin in regulating membrane protrusion dynamics during cell migration. 相似文献
95.
Coenen M Cieza A Stamm TA Amann E Kollerits B Stucki G 《Arthritis research & therapy》2006,8(4):R84-14
Functioning is recognized as an important study outcome in rheumatoid arthritis (RA). The Comprehensive ICF Core Set for RA
is an application of the International Classification of Functioning, Disability and Health (ICF) of the World Health Organisation
with the purpose of representing the typical spectrum of functioning of patients with RA. To strengthen the patient perspective,
persons with RA were explicitly involved in the validation of the Comprehensive ICF Core Set for RA using qualitative methodology.
The objective of the study was twofold: to come forward with a proposal for the most appropriate methodology to validate Comprehensive
ICF Core Sets from the patient perspective; and to add evidence to the validation of the Comprehensive ICF Core Set for RA
from the perspective of patients. The specific aims were to explore the aspects of functioning and health important to patients
with RA using two different focus group approaches (open approach and ICF-based approach) and to examine to what extent these
aspects are represented by the current version of the Comprehensive ICF Core Set for RA. The sampling of patients followed
the maximum variation strategy. Sample size was determined by saturation. The focus groups were digitally recorded and transcribed
verbatim. The meaning condensation procedure was used for the data analysis. After qualitative data analysis, the resulting
concepts were linked to ICF categories according to established linking rules. Forty-nine patients participated in ten focus
groups (five in each approach). Of the 76 ICF categories contained in the Comprehensive ICF Core Set for RA, 65 were reported
by the patients based on the open approach and 71 based on the ICF-based approach. Sixty-six additional categories (open approach,
41; ICF-based approach, 57) that are not covered in the Comprehensive ICF Core Set for RA were raised. The existing version
of the Comprehensive ICF Core Set for RA could be confirmed almost entirely by the two different focus group approaches applied.
Focus groups are a highly useful qualitative method to validate the Comprehensive ICF Core Set for RA from the patient perspective.
The ICF-based approach seems to be the most appropriate technique. 相似文献
96.
Juretschko S Schönhuber W Kulakauskas S Ehrlich DS Schleifer KH Amann R 《Systematic and applied microbiology》1999,22(1):1-8
A method is described for the in situ detection of individual whole fixed cells of Escherichia coli containing ColE1-related plasmids. It makes use of fluorescence in situ hybridization (FISH) and the regulatory RNA II as a target molecule for both, Cy3- and HRP-labeled olinucleotide probes. Various methods for signal amplification were compared. Probes targeting the regulatory RNA I did not result in the in situ detection of plasmid-bearing cells. 相似文献
97.
Almost one thousand 16S rRNA sequences of Gram-positive bacteria with a low DNA G + C content from public databases were analyzed using the ARB software package. A signature region was identified between positions 354 and 371 (E. coli numbering) for the Bacillus sub-branch of the Gram-positive bacteria with a low DNA G + C content, the former orders Bacillales and Lactobacillales. Three oligonucleotide probes, namely LGC354A, LGC354B, and LGC354C, were designed to target this diagnostic site. Their fluorescent derivatives were suitable for whole cell detection by fluorescence in situ hybridization (FISH). Hybridization conditions were adjusted for differentiation of target and related non-target reference species. When applying FISH to whole bacterial cells in a sample of activated sludge from a communal wastewater treatment plant, members of the Bacillus sub-branch were detected at levels from 0.01% of cells in samples fixed with paraformaldehyde to over 8 percent in the same samples fixed with ethanol and treated with lysozyme. The problems of quantitative in situ analysis of Gram-positive bacteria with a low DNA G + C content in biofilm flocs are discussed and recommendations made. Members of the Bacillus sub-branch were detected in different abundances in activated sludge samples from different wastewater plants. 相似文献
98.
The response of the microbial community of marine sediments to organic carbon input under anaerobic conditions. 总被引:2,自引:0,他引:2
R Rosselló-Mora B Thamdrup H Sch?fer R Weller R Amann 《Systematic and applied microbiology》1999,22(2):237-248
Cyanobacterial biomass was added to anaerobic sediment to simulate the natural input of complex organic substrate that occurs in nature after algae blooms. Sediments were incubated at 0 degree C, 8 degrees C and 24 degrees C for 13 days. Community dynamics were measured by fluorescence in situ hybridisation (FISH), denaturing gradient gel electrophoresis (DGGE), and sequencing of 16S rDNA PCR products. Metabolic changes were followed by the analysis of total carbon mineralisation, sulfate reduction, and ammonium production rates. The addition of organic material resulted in significant changes in the composition of the microbial community at all temperatures tested. Sulfate reduction was the main mineralisation process detected. However, not sulfate-reducers but rather members of the Cytophaga-Flavobacterium phylogenetic cluster showed the highest increase in the bacterial cells as detected by FISH. We conclude that these organisms play an important role in the anaerobic decomposition of complex organic material perhaps because they are the main catalysts of macromolecule hydrolysis and fermentation. The molecular methods also indicated a stimulation of ribosome synthesis. The detection of a large number of rRNA-rich cells belonging to the Cytophaga-Flavobacterium phylogenetic cluster further supports the importance of their role in the degradation of complex organic material in anaerobic marine sediments. Their detection in high numbers in the field may indicate recent deposition events. 相似文献
99.
Fluorescence in situ hybridisation (FISH) with rRNA-targeted oligonucleotide probes facilitates the rapid and specific identification of individual microbial cells in their natural environments. Over the past year there have been a number of methodological developments in this area and new applications of FISH in microbial ecology and biotechnology have been reported. 相似文献
100.
Venturelli M Amann M McDaniel J Trinity JD Fjeldstad AS Richardson RS 《American journal of physiology. Heart and circulatory physiology》2012,302(1):H333-H339
The exact role of arousal in central and peripheral hemodynamic responses to passive limb movement in humans is unclear but has been proposed as a potential contributor. Thus, we used a human model with no lower limb afferent feedback to determine the role of arousal on the hemodynamic response to passive leg movement. In nine people with a spinal cord injury, we compared central and peripheral hemodynamic and ventilatory responses to one-leg passive knee extension with and without visual feedback (M+VF and M-VF, respectively) as well as in a third trial with no movement or visual feedback but the perception of movement (F). Ventilation (Ve), heart rate, stroke volume, cardiac output, mean arterial pressure, and leg blood flow (LBF) were evaluated during the three protocols. Ve increased rapidly from baseline in M+VF (55 ± 11%), M-VF (63 ± 13%), and F (48 ± 12%) trials. Central hemodynamics (heart rate, stroke volume, cardiac output, and mean arterial pressure) were unchanged in all trials. LBF increased from baseline by 126 ± 18 ml/min in the M+VF protocol and 109 ± 23 ml/min in the M-VF protocol but was unchanged in the F protocol. Therefore, with the use of model that is devoid of afferent feedback from the legs, the results of this study reveal that, although arousal is invoked by passive movement or the thought of passive movement, as evidenced by the increase in Ve, there is no central or peripheral hemodynamic impact of this increased neural activity. Additionally, this study revealed that a central hemodynamic response is not an obligatory component of movement-induced LBF. 相似文献