Biocompatibility of different nanostructured TiO<Subscript>2</Subscript> scaffolds and their potential for urologic applications

Despite great efforts in tissue engineering of the ureter, urinary bladder, and urethra, further research is needed in order to improve the patient’s quality of life and minimize the economic burden of different lower urinary tract disorders. The nanostructured titanium dioxide (TiO₂) scaffolds have a wide range of clinical applications and are already widely used in orthopedic or dental medicine. The current study was conducted to synthesize TiO₂ nanotubes by the anodization method and TiO₂ nanowires and nanospheres by the chemical vapor deposition method. These scaffolds were characterized with scanning electron microscopy (SEM) and X-ray diffraction (XRD) methods. In order to test the urologic applicability of generated TiO₂ scaffolds, we seeded the normal porcine urothelial (NPU) cells on TiO₂ nanotubes, TiO₂ nanowires, TiO₂ nanospheres, and on the standard porous membrane. The viability and growth of the cells were monitored everyday, and after 3 weeks of culturing, the analysis with scanning electron microscope (SEM) was performed. Our results showed that the NPU cells were attached on all scaffolds; they were viable and formed a multilayered epithelium, i.e., urothelium. The apical plasma membrane of the majority of superficial NPU cells, grown on all three different TiO₂ scaffolds and on the porous membrane, exhibited microvilli; thus, indicating that they were at a similar differentiation stage. The maximal caliper diameter measurements of superficial NPU cells revealed significant alterations, with the largest cells being observed on nanowires and the smallest ones on the porous membrane. Our findings indicate that different nanostructured TiO₂ scaffolds, especially nanowires, have a great potential for tissue engineering and should be further investigated for various urologic applications.     

Myofilament length-dependent activation develops within 5 ms in guinea-pig myocardium

Myofilament length-dependent activation is a universal property of striated muscle, yet the molecular mechanisms that underlie this phenomenon are incompletely understood. Additionally, the rate by which sarcomere length (SL) is sensed and then transduced to form length-dependent activation is unknown. Here, using isolated guinea-pig myocardium, we employed a rapid solution-switch single myofibril technique that allows for the study of contractile action/relaxation dynamics in the virtual absence of diffusion delays. We compared contraction kinetics obtained at submaximal activation at steady-state SL with contractions observed after rapid SL ramps to that same SL just before activation. Neither the activation and relaxation kinetics nor the final submaximal force development differed significantly between the two contraction modes for SL ramps as fast as 5 ms. We conclude that the transduction of the length signal by the cardiac sarcomere to modulate thin filament activation levels occurs virtually instantaneously, possibly resulting from structural rearrangements of the contractile proteins.     

R25 is an intracellular membrane receptor for a snake venom secretory phospholipase A(2)

Ammodytoxin is a presynaptically neurotoxic (beta-neurotoxic) snake venom secretory phospholipase A(2) (sPLA(2)). We detected a 25 kDa protein which binds the toxin with very high affinity (R25) in porcine cerebral cortex. Here we show that R25 is an integral membrane protein with intracellular localisation. It is the first sPLA(2) receptor known to date that localises to intracellular membranes. Centrifugation on sucrose gradients was used to fractionate porcine cerebral cortex. The subcellular composition of the fractions was determined by following the distribution of organelle-specific markers. The distribution of R25 in the fractions matched the distribution of the mitochondrial marker succinate dehydrogenase, but not the markers for plasma membrane, lysosomes, endoplasmic reticulum, synaptic and secretory vesicles. R25 most likely resides in mitochondria, which are known to be targets for sPLA(2) neurotoxins in the nerve ending and are potentially implicated in the process of beta-neurotoxicity.     

Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane

The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.     

Expression of a hepatitis A virus antigen in Lactococcus lactis and Escherichia coli and evaluation of its immunogenicity

An epidemic shift in Hepatitis A virus (HAV) infection has been observed in recent years in rapidly developing countries, with increasing numbers of severe adult cases which has led to renewed interest in vaccination. Our approach in vaccine development uses recombinant expression of the highly immunogenic HAV antigen VP1-P2a in food-grade lactic acid bacterium Lactococcus lactis and in Escherichia coli. We used genetic constructs that enable nisin-controlled expression of the antigen in L. lactis in three different forms: (a) intracellularly, (b) on the bacterial surface and (c) on the bacterial surface fused with the fragment of the E. coli flagellin molecule that can act as a molecular adjuvant. Expression of the two surface forms of the antigen was achieved in L. lactis, and the resulting antigen-displaying bacteria were administered orally to mice. Half the animals in each of the two groups developed specific IgGs, with titers increasing over time and reaching 1:422 without flagellin and 1:320 with flagellin. A much higher titer 1:25,803 was observed with the parenterally administered antigen, which was purified from E. coli. With the latter, a significant mucosal IgA response was also observed. Despite significant titers, the IgGs elicited with oral or parenteral administration could not prevent HAV from infecting cells in a virus neutralization assay, suggesting that the antibodies cannot recognize viral surface epitopes. Nevertheless, orally administered HAV antigen expressed in L. lactis elicited significant systemic humoral immune response showing the feasibility for development of effective HAV vaccine for mucosal delivery.     

Physiopathologic dynamics of vesicle traffic in astrocytes

The view of how astrocytes, a type of glial cells, contribute to the functioning of the central nervous system (CNS) has changed greatly in the last decade. Although glial cells outnumber neurons in the mammalian brain, it was considered for over a century that they played a subservient role to neurons. This view changed. Functions thought to be exclusively present in neurons, i.e. excitability mediated release of chemical messengers, has also been demonstrated in astrocytes. In this process, following an increase in cytosolic calcium activity, membrane bound vesicles, storing chemical messengers (gliotransmitters), fuse with the plasma membrane, a process known as exocytosis, permitting the exit of vesicle cargo into the extracellular space. Vesicles are delivered to and are removed from the site of exocytosis by an amazingly complex set of processes that we have only started to learn about recently. In this paper we review vesicle traffic, which is subject to physiological regulation and may be changed under pathological conditions.     

Zinc-decorated silica-coated magnetic nanoparticles for protein binding and controlled release

The aim of this study was to be able to reversibly bind histidine-rich proteins to the surface of maghemite magnetic nanoparticles via coordinative bonding using Zn ions as the anchoring points. We showed that in order to adsorb Zn ions on the maghemite, the surface of the latter needs to be modified. As silica is known to strongly adsorb zinc ions, we chose to modify the maghemite nanoparticles with a nanometre-thick silica layer. This layer appeared to be thin enough for the maghemite nanoparticles to preserve their superparamagnetic nature. As a model the histidine-rich protein bovine serum albumin (BSA) was used. The release of the BSA bound to Zn-decorated silica-coated maghemite nanoparticles was analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrated that the bonding of the BSA to such modified magnetic nanoparticles is highly reversible and can be controlled by an appropriate change of the external conditions, such as a pH decrease or the presence/supply of other chelating compounds.     

Different role of intracellular loops of glucagon-like peptide-1 receptor in G-protein coupling

Previous studies revealed the importance of the third intracellular loop of glucagon-like peptide-1 receptor (GLP-1R) in coupling to G(s) and G(i1) proteins. In order to further study the signaling mechanisms of GLP-1R, we tested three peptides, corresponding to the sequences of the first (IC(1)), the second (IC(2)), and the third (IC(3)) intracellular loop of GLP-1R, for their interactions with heterotrimeric G-proteins of different types (G(alphas), G(alphao), G(alphai1), and G(alpha11) plus G(beta1gamma2)) overexpressed in sf9 cells. IC(3) peptide powerfully stimulates all types of tested G-proteins, whereas IC(1) and IC(2) peptides show differential effects on G-proteins. Both IC(1) and IC(2) peptides activate G(s) and cooperate with IC(3) peptide in its stimulation. G(o) is not affected by IC(1) and IC(2). G(i1) and G(11) are not affected by IC(1), but are activated by IC(2), which in activation cooperates with IC(3). We suggest that GLP-1R is not coupled only to G(s) and G(i1), as shown previously, but also to G(o) and G(11). IC(3) loop is the main switch that mediates signaling via GLP-1R to G-proteins, while IC(1) and IC(2) loops are important in discrimination between different types of G-proteins.