Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.
High level of heterogeneity seems to be a ubiquitous feature of mammalian PrPs (prion proteins) and may be relevant to the pathogenesis of prion diseases. In the present study, we describe the heterogeneity of PrP(C) (cellular form of PrP) from porcine brain. It was disclosed and characterized by a combination of one-dimensional PAGE and two-dimensional PAGE analyses with enzymic deglycosylation and copper-affinity experiments. We found that the identified two main populations of porcine PrP(C) consist of diglycosylated forms and correspond to the full-length (molecular mass 32-36 kDa) and proteolytically modified protein (molecular mass 25-30 kDa), known as C1. The two populations were fully separated during Cu2+-loaded immobilized metal affinity chromatography, indicating different affinity for copper ions. The more basic forms, migrating as species of higher molecular mass, exhibited stronger affinity for copper ions, whereas those with more acidic pI and of lower molecular mass were low-affinity Cu2+-binding molecules and thus could represent N-terminally truncated PrP(C). Size-exclusion chromatography revealed that most of the PrP(C) molecules in porcine brain extracts exist in the form of high-molecular-mass complexes (probably with other proteins). The heterogeneity of porcine PrP(C), resulting from proteolytic modification and glycosylation, influences its ability to assemble into these complexes. N-truncated molecules dominate over full-length PrP(C) in fractions of molecular mass over the range 65-130 kDa, whereas the full-length species are the major forms of PrP(C) present in the monomeric fraction and in complexes above 130 kDa. Two-dimensional PAGE analysis indicated that the complexed PrP(C) differs in the composition of pI forms from the monomers.
This paper presents an investigation of properties of selected metallic oxides deposited at a low temperature (100 °C) by atomic layer deposition (ALD) technique, relating to their applicability as thin overlays for optical fiber sensors resistant in alkaline environments. Hafnium oxide (Hf O with y/x approx. 2.70), tantalum oxide (Ta O with y/x approx. 2.75) and zirconium oxide (Zr O with y/x approx. 2.07), which deposition was based, respectively, on tetrakis(ethylmethyl)hafnium, tantalum pentachloride and tetrakis(ethylmethyl)zirconium with deionized water, were tested as thin layers on planar Si (100) and glass substrates. Growth per cycle (GPC) in the ALD processes was 0.133-0.150 nm/cycle. Run-to-run GPC reproducibility of the ALD processes was best for Hf O (0.145 ± 0.001 nm/cycle) and the poorest for Ta O (0.133 ± 0.003 nm/cycle). Refractive indices n of the layers were 2.00-2.10 (at the wavelength λ = 632 nm), with negligible k value (at λ for 240-930 nm). The oxides examined by x-ray diffractometry proved to be amorphous, with only small addition of crystalline phases for the Zr O . The surfaces of the oxides had grainy but smooth topographies with root-mean square roughness ∼0.5 nm (at 10 × 10 μm area) according to atomic force microscopy. Ellipsometric measurements, by contrast, suggest rougher surfaces for the Zr O layers. The surfaces were also slightly rougher on the glass-based samples than on the Si-based ones. Nanohardness and Young modules were 4.90-8.64 GPa and 83.7-104.4 GPa, respectively. The tests of scratch resistance revealed better tribological properties for the Hf O and the Ta O than for the Zr O . The surfaces were hydrophilic, with wetting angles of 52.5°-62.9°. The planar oxides on Si, being resistive even to concentrated alkali (pH 14), proved to be significantly more alkali-resistive than AlO. The Ta O overlay was deposited on long-period grating sensor induced in optical fiber. Thanks to such an overlay the sensor proved to be long-lasting resistant when exposed to alkaline environment with a pH 9. Thereby, it also proved that it has a potential to be repeatedly reused as a regenerable optical fiber biosensor.
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