We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.
Abstract. The substrate specificities of dynein, kinesin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein from Tetrahymena cilia and the/~-heavy chain-containing particle from the outer-ann dynein of sea urchin. flagella, could be distinguished by their substrate specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate specificities, kinesin-induced microtubule translocation varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold. With both kinesin and heavy meromyosin, the relative velocities of filament translocation did not correlate well with the relative filament-activated substrate turnover rates. Furthermore, some ATP analogues that did not support the filament translocation exhibited filamentactivated substrate turnover rates. Filament-activated substrate turnover and power production, therefore, appear to become uncoupled with certain substrates.In conclusion, the substrate specificities and coupling to motility are distinct for different types of molecular motor proteins. Such nucleotide "fingerprints" of enzymatic activities of motor proteins may prove useful as a tool for identifying what type of motor is involved in powering a motility-related event that can be reconstituted in vitro.
Accurate quantification of plasma glucagon levels in humans is necessary for understanding the physiological and pathological importance of glucagon. Although several immunoassays for glucagon are available, they provide inconsistent glucagon values owing to cross-reactivity of the antibodies with peptides other than glucagon. To overcome this limitation, we developed a novel method to measure glucagon levels by a liquid chromatography (LC)-high-resolution mass spectrometry (HRMS) assay via parallel reaction monitoring (PRM) without immunoaffinity enrichment. Using stable isotope-labeled glucagon as an internal standard and 200 μL of plasma, the lower limit of quantification was 0.5 pM. This method was applied to measure plasma glucagon levels during the oral glucose tolerance test (OGTT) and meal tolerance test (MTT) in healthy volunteers, and its results were compared with those of sandwich enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). During the OGTT, this method showed significant suppression of plasma glucagon levels, and similar patterns were observed with sandwich ELISA and RIA. In contrast, during the MTT, plasma glucagon levels were slightly elevated according to the LC-MS/MS and sandwich ELISA results and were reduced according to RIA results. Our newly developed LC-MS/MS method overcomes a lack of specificity among currently available immunoassays for glucagon and may contribute to a better understanding of the importance of glucagon. Graphical abstract Flowchart for the extraction and quantification of glucagon in human plasma, and plasma glucagon responses in healthy volunteers quantified by the present LC-MS/MS, sandwich ELISA, and RIA during OGTT and MTT.
It was more than 50 years ago that an appearance of birefringence in alginate gels prepared under cation flow was reported for the first time, however, the anisotropic structure of the alginate gel has not been studied in detail. In the present study, anisotropic Ca-alginate gels were prepared within dialysis tubing in a high Ca(2+)-concentration external bath, and optical and small-angle X-ray scattering (SAXS) measurements were performed to characterize the structure of the gel. The observations of the gel with crossed polarizers and with circular polarizers revealed the molecular orientation perpendicular to the direction of Ca(2+) flow. Analyses of the SAXS intensity profiles indicated the formation of rod-like fibrils consisting of a few tens of alginate molecules and that the anisotropy of the gel was caused by the circumferential orientation of the large fibrils. From the observed asymmetric SAXS pattern, it was found that the axis of rotational symmetry of the anisotropic structure was parallel to the direction of Ca(2+) flow. The alignment factor (A(f)) calculated from the SAXS intensity data confirmed that the orientation of the fibrils was perpendicular to the direction of Ca(2+) flow.
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