A bioconjugation reaction is used to obtain fd viruses with one gold nanoparticle at the tip and gold nanowire-like structures.
Microrheology measurements were performed on suspensions of bacteriophage fd with diffusive wave spectroscopy in the concentrated regime, at different values of ionic strength. Viscosity vs. shear rate was also measured, and the effect of bacteriophage concentration and salt addition on shear thinning was determined, as well as on the peaks in the viscosity vs. shear curves corresponding to a transition from tumbling to wagging flow. The influence of concentration and salt addition on the mean square displacement of microspheres embedded in the suspensions was determined, as well as on their viscoelastic moduli up to high angular frequencies. Our results were compared with another microrheology technique previously reported where the power spectral density of thermal fluctuations of embedded micron-sized particles was evaluated. Although both results in general agree, the diffusive wave spectroscopy results are much less noisy and can reach larger frequencies. A comparison was made between measured and calculated shear modulus. Calculations were made employing the theory for highly entangled isotropic solutions of semiflexible polymers using a tube model, where various ways of calculating the needed parameters were used. Although some features are captured by the model, it is far from the experimental results mainly at high frequencies.
The structure of apolipoprotein A-I (apoA-I), the major protein of HDL, has been extensively studied in past years. Nevertheless, its corresponding three-dimensional structure has been difficult to obtain due to the frequent conformational changes observed depending on the microenvironment. Although the function of each helical segment of this protein remains unclear, it has been observed that the apoA-I amino (N) and carboxy-end (C) domains are directly involved in receptor-recognition, processes that determine the diameter for HDL particles. In addition, it has been observed that the high structural plasticity of these segments might be related to several amyloidogenic processes. In this work, we studied a series of peptides derived from the N- and C-terminal domains representing the most hydrophobic segments of apoA-I. Measurements carried out using circular dichroism in all tested peptides evidenced that the lipid environment promotes the formation of α-helical structures, whereas an aqueous environment facilitates a strong tendency to adopt β-sheet/disordered conformations. Electron microscopy observations showed the formation of amyloid-like structures similar to those found in other well-defined amyloidogenic proteins. Interestingly, when the apoA-I peptides were incubated under conditions that promote stable globular structures, two of the peptides studied were cytotoxic to microglia and mouse macrophage cells. Our findings provide an insight into the physicochemical properties of key segments contained in apoA-I which may be implicated in disorder-to-order transitions that in turn maintain the delicate equilibrium between both, native and abnormal conformations, and therefore control its propensity to become involved in pathological processes.
Lipid rafts are sphingomyelin/cholesterol-rich domains present in the plasma membrane of eukaryotic cells. In the hepatocyte, it has been shown that these domains intervene and modify a wide range of functions from which cell signaling in health and disease is of major importance. The present investigation proposes a novel strategy for the study of plasma membrane lipid rafts using surface-enhanced Raman spectroscopy (SERS) and spectra processing employing the principal component analysis (PCA) in correlation with conventional biochemical techniques. SERS has been used for artificial membranes; our approach has the advantage that allows studying purified biological membranes using small volumes of biological samples and a very simple protocol. Therefore, the use of SERS/PCA represents an important advantage for the study not only of the structure and composition of biological membranes but also for the understanding of the functions carried by the membrane-embedded proteins. Our work using SERS/PCA for the first time shows a correlation when studying the composition of lipid rafts from the cell plasma membrane and the catalytic activity of the Ca 2+ -ATPase, and its direct association with the presence of specific lipids located in membrane lipid rafts. Also, we show that changes in plasma membrane and the catalytic activity of the Ca 2+ -ATPase activity present in the plasma membrane of hepatocytes after partial depletion of membrane cholesterol carried out by methyl-beta-cyclodextrin correlate with the changes in the enzyme activity and the presence of cholesterol-rich domains of lipid rafts.
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