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41.
Real-time monitoring of rolling-circle amplification using a modified molecular beacon design 总被引:2,自引:4,他引:2
We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2′-O-Me-RNA to prevent 3′ exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color. 相似文献
42.
The sequence of the mitochondrial DNA (mtDNA) molecule of the European hedgehog (Erinaceus europaeus) was determined. The length of the sequence presented is 17,442 nucleotides (nt). The molecule is thus the largest eutherian mtDNA molecule so far reported. The organization of the molecule conforms with that of other eutherians, but the control region of the molecule is exceptionally long, 1,988 nt, due to the presence of repeated motifs at two different positions in the 3 part of the control region. The length of the control region is not absolute due to pronounced heteroplasmy caused by variable numbers of the motif TACGCA in one of the repetitive regions. The sequence presented includes 46 repeats of this type. The other repeated region is composed of different AT-rich repeats. This region was identical among four clones studied. Comparison of mitochondrial peptide-coding genes identified a separate position of the hedgehog among several mammalian orders. The concatenated protein sequence of the 13 peptide-coding genes was used in a phylogenetic study using the opossum as outgroup. The position of the hedgehog sequence was basal among the other eutherian sequences included: human, rat, mouse, cow, blue whale, harbor seal, and horse. The analysis did not resolve the relationship among carnivores, perissodactyls, and artiodactyls/cetaceans, suggesting a closer relationship among these orders than acknowledged by classical approaches.
Correspondence to: U. Arnason 相似文献
43.
Berit ? Christoffersen S?ren J Jensen Trine P Ludvigsen Sara K Nilsson Anette B Grossi Peter M H Heegaard 《Comparative medicine》2015,65(4):333-341
Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP in Göttingen minipigs and to investigate the effects of age, sex, and various stimuli on these ranges. Serum concentrations of C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, pig major acute-phase protein (PMAP), albumin, and porcine α-1 acid glycoprotein (PAGP) were evaluated in 4 age groups (6, 16, 24 and 40–48 wk) of male and female Göttingen minipigs. In addition, minipigs were tested under 2 housing conditions, after acute LPS challenge, and after diet-induced obesity with and without mild diabetes. Changing the pigs to a new environment induced significant increases in CRP, PMAP, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only in obese pigs without diabetes. In conclusion, reference values for CRP, PMAP, haptoglobin, SAA, PAGP and albumin were established for male and female Göttingen minipigs of different ages. These APP were influenced by age and sex, underlining the importance of considering these factors when designing and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli. Abbreviations: APP, acute-phase proteins; APR, acute-phase response; CRP, C-reactive protein; HFD, high-fat diet; HFD+D, high fat diet + diabetes; PAGP, porcine α1 acid glycoprotein; PMAP, pig major acute-phase protein; SAA, serum amyloid AInflammation is involved in a number of important and increasingly widespread human diseases, including inflammatory bowel diseases, cancers, infections, metabolic diseases like obesity and diabetes, and cardiovascular diseases like atherosclerosis.1,5,7,11,20,41 The systemic response to inflammation is the acute-phase response (APR) which, together with innate immune responses, prevents infection, clears pathogens, and contributes to inflammation resolution and the healing process. The APR has been extensively described in humans10,22 and other mammals,8,14,29,31 and in all cases, it is regulated by cytokines including IL6 and TNFα.21,30 The APR is activated by many different stimuli, including trauma, infection, stress, neoplasia, and inflammatory stimuli, resulting in significant changes in the circulating concentrations of the so-called acute-phase proteins (APP). The APP are synthetized primarily by the liver and can be divided into positive and negative APP depending on whether their concentration in plasma increases (positive) or decreases (negative) in response to a stimulus.10 In addition, they can be divided into major and minor APP, depending on the magnitude of their concentration change after a given stimulus.22 Because the concentrations of the APP change in response to a given stimulus, their serum or plasma levels can be used diagnostically as biomarkers of disease severity and progression or to evaluate the effect of various interventions.8,14,31 The APP show different kinetics after a stimulus, with C-reactive protein (CRP) and serum amyloid A (SAA) displaying rapid increases and normalization after the stimulus has been removed, whereas haptoglobin shows a later and more prolonged response.10,31 The APR may be transient and revert to normal with recovery, or it can persist, as during chronic conditions.21 Importantly, APP and their kinetics differ somewhat between species.31To further elucidate the involvement of inflammation in human diseases, accurate animal models of inflammation, including species validated biomarkers of inflammation, are needed. Mouse models are commonly used in many research areas, but their response to several different inflammatory conditions is not comparable to that of humans, and therefore the predictive validity of these models may be limited.39 Pigs are highly comparable to humans with respect to anatomy and physiology,44 and their APR to various stimuli has been described.14,23,26 In general, the APR and the resulting changes in APP seem to be very similar in pigs compared with humans, with CRP, haptoglobin, and SAA being major positive APP and albumin being a negative APP.14 In humans, α1-acid glycoprotein (AGP) is a positive APP but has been reported to either increase,17 remain unchanged23,45 or to decrease12 in pigs, depending on the stimulus investigated. The concentrations of some of the major APP characterized in domestic pigs show significant effects of age and sex.32,34 In addition to age and sex effects, significant differences in APP between herds have been observed, most likely reflecting different pathogenic pressures in the different herds.32 Furthermore, some indications exist for possible interbreed differences in APP concentrations, although this possibility has not been investigated in detail.12Minipigs are especially relevant in biomedical research, given their smaller size and well-defined microbiology and genetics.4 Göttingen minipigs are a useful model for several conditions involving inflammation and the APR, including infection,2 obesity,19 diabetes24 and atherosclerosis,18 and different APP have already been used as biomarkers in some of these models.2 Therefore, existing data suggest that APP commonly applied in human medicine could be relevant in Göttingen minipigs as well. However, the APR and reference values of APP, including the potential influence of age and sex indicated in other studies, have not been investigated systematically in this breed.12,32,34The objective of the current study was to establish reference values of selected APP in normal Göttingen minipigs, including evaluation of the possible effects of age and sex. In addition, the effects of housing condition and acute and chronic inflammatory stimuli were assessed. 相似文献
44.
Nonlinear mechanical properties play an important role in numerous biological functions, for instance the locomotion strategy used by terrestrial gastropods. We discuss the progress made toward bioinspired snail-like locomotion and the pursuit of an engineered fluid that imitates the nonlinear viscoelastic properties of native gastropod pedal mucus. The rheological behavior of native pedal mucus is characterized using an oscillatory deformation protocol known as large amplitude oscillatory shear, and we review recently developed techniques for appropriately describing nonlinear viscoelastic behavior. Although materials that exhibit purely elastic and purely viscous nonlinearities are amenable to standard techniques for characterization, pedal mucus samples (and biomaterials in general) are viscoelastic, exhibiting both elastic and viscous nonlinear responses simultaneously and requiring advanced techniques for characterization. We reveal the utility of these new methods by examining trail mucus from the terrestrial slug Limax maximus using oscillatory shear rheology. Material responses which previously could only be described mathematically, with little physical insight, can now be interpreted with familiar language such as strain-stiffening/softening and shear-thickening/thinning. The new methodology is applicable to any complex material that can be tested using imposed oscillatory deformations. We have developed data-analysis software to enable wider use of this framework within and beyond the biomaterials community. The functionality of this software is outlined here. 相似文献
45.
Bonefeld CM Rasmussen AB Lauritsen JP von Essen M Ødum N Andersen PS Geisler C 《Journal of immunology (Baltimore, Md. : 1950)》2003,171(6):3003-3009
One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed. 相似文献
46.
Lavinia Paternoster David M. Evans Ellen Aagaard Nohr Claus Holst Valerie Gaborieau Paul Brennan Anette Prior Gjesing Niels Grarup Daniel R. Witte Torben J?rgensen Allan Linneberg Torsten Lauritzen Anelli Sandbaek Torben Hansen Oluf Pedersen Katherine S. Elliott John P. Kemp Beate St. Pourcain George McMahon Diana Zelenika J?rg Hager Mark Lathrop Nicholas J. Timpson George Davey Smith Thorkild I. A. S?rensen 《PloS one》2011,6(9)
47.
Depressed thermogenesis but competent brown adipose tissue recruitment in mice devoid of all hormone-binding thyroid hormone receptors 总被引:1,自引:0,他引:1
Golozoubova V Gullberg H Matthias A Cannon B Vennström B Nedergaard J 《Molecular endocrinology (Baltimore, Md.)》2004,18(2):384-401
We have examined the metabolic role of hormone-binding nuclear thyroid hormone receptors (TRs). Mice devoid of all hormone-binding TRs [TR alpha 1(-/-)beta(-/-) (TR-ablated mice)] had slightly decreased body temperature and much decreased basal metabolic rate, were still able to markedly increase metabolic rate in the cold, but were cold intolerant due to inadequate total heat production at low temperatures. A standard norepinephrine test showed that adrenergically induced thermogenesis could not be activated normally in the TR-ablated mice. This was not due to inadequate recruitment of brown adipose tissue, nor to the absence, decreased recruitment or dysfunction of the uncoupling protein-1. However, isolated brown fat cells were 10-fold desensitized, explaining the lack of response to standard adrenergic stimuli; cell culture experiments demonstrated that this desensitization was not an innate effect. Thus, the cold intolerance was probably not due to inadequate sympathetically induced nonshivering thermogenesis. Additionally, the results indicated that no metabolic effects of thyroid hormones could become manifest in the absence of nuclear TRs, that ligand-bound TRs were needed for euthermia and eumetabolism, but that TRs per se were not required for brown adipose tissue recruitment and uncoupling protein-1 gene expression. 相似文献
48.
Patrick Fischer Martina K. La Rosa Adriana Schulz Anette Preiss Anja C. Nagel 《PLoS genetics》2015,11(8)
In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B''-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila. 相似文献
49.
50.
Leisner C Loeth N Lamberth K Justesen S Sylvester-Hvid C Schmidt EG Claesson M Buus S Stryhn A 《PloS one》2008,3(2):e1678