Magnetic alignment of rhodamine/magnetite dual-labeled microtubules probed with inverted fluorescence microscopy.

Molecular machines, as exemplified by the kinesin and microtubule system, are responsible for molecular transport in cells. The monitoring of the cellular machinery has attracted much attention in recent years, requiring sophisticated techniques such as optical tweezers, and dark field hyperspectral and fluorescence microscopies. It also demands suitable procedures for immobilization and labeling with functional agents such as dyes, plasmonic nanoparticles and quantum dots. In this work, microtubules were co-polymerized by incubating a tubulin mix consisting of 7 biotinylated tubulin to 3 rhodamine tubulin. Rhodamine provided the fluorescent tag, while biotin was the anchoring group for receiving streptavidin containing species. To control the microtubule alignment and consequently, the molecular gliding directions, functionalized iron oxide nanoparticles were employed in the presence of an external magnet field. Such iron oxide nanoparticles, (MagNPs) were previously coated with silica and (3-aminopro-pyl)triethoxysilane (APTS) and then modified with streptavidin (SA) for linking to the biotin-functionalized microtubules. In this way, the binding has been successfully performed, and the magnetic alignment probed by Inverted Fluorescence Microscopy. The proposed strategy has proved promising, as tested with one of the most important biological structures of the cellular machinery.

A Plasmonic Approach to Study Protein Interaction Kinetics through the Dimerization of Functionalized Ag Nanoparticles.

Understanding the kinetics of protein interactions plays a key role in biology with significant implications for the design of analytical methods for disease monitoring and diagnosis in medical care, research and industrial applications. Herein, we introduce a novel plasmonic approach to study the binding kinetics of protein-ligand interactions following the formation of silver nanoparticles (Ag NPs) dimers by UV-Vis spectroscopy that can be used as probes for antigen detection and quantification. To illustrate and test the method, the kinetics of the prototype biotin-streptavidin (Biot-STV) pair interaction was studied. Controlled aggregates (dimers) of STV functionalized Ag NPs were produced by adding stoichiometric quantities of gliadin-specific biotinylated antibodies (IgG-Biot). The dimerization kinetics was studied in a systematic way as a function of Ag NPs size and at different concentrations of IgG-Biot. The kinetics data have shown to be consistent with a complex reaction mechanism in which only the Ag NPs attached to the IgG-Biot located in a specific STV site are able to form dimers. These results help in elucidating a complex reaction mechanism involved in the dimerization kinetics of functionalized Ag NPs, which can serve as probes in surface plasmon resonance-based bioassays for the detection and quantification of different biomarkers or analytes of interest.

Enantioselective imine reduction catalyzed by imine reductases and artificial metalloenzymes.

Adding value to organic synthesis. Novel imine reductases enable the enantioselective reduction of imines to afford optically active amines. Likewise, novel bioinspired artificial metalloenzymes can perform the same reaction as well. Emerging proof-of-concepts are herein discussed.

Arginylated calreticulin at plasma membrane increases susceptibility of cells to apoptosis.

Post-translational modifications of proteins are important for the regulation of cell fate and functions; one of these post-translational modifications is arginylation. We have previously established that calreticulin (CRT), an endoplasmic reticulum resident, is also one of the arginylated substrates found in the cytoplasm. In the present study, we describe that arginylated CRT (R-CRT) binds to the cell membrane and identified its role as a preapoptotic signal. We also show that cells lacking arginyl-tRNA protein transferase are less susceptible to apoptosis than wild type cells. Under these conditions R-CRT is present on the cell membrane but at early stages is differently localized in stress granules. Moreover, cells induced to undergo apoptosis by arsenite show increased R-CRT on their cell surface. Exogenously applied R-CRT binds to the cell membrane and is able to both increase the number of cells undergoing apoptosis in wild type cells and overcome apoptosis resistance in cells lacking arginyl-tRNA protein transferase that express R-CRT on the cell surface. Thus, these results demonstrate the importance of surface R-CRT in the apoptotic response of cells, implying that post-translational arginylation of CRT can regulate its intracellular localization, cell function, and survival.

PEGylation, detection and chromatographic purification of site-specific PEGylated CD133-Biotin antibody in route to stem cell separation.

Recovery and purification of stem cells are determining steps in order to obtain the purity and viability required for transplantation. In this context, immunochemical techniques have been widely preferred due to their high selectivity. CD133, a glycoprotein expressed by stem cells, is a well-used marker for isolation of neural stem cells. Transplantation of neural stem cells into patients can promote neural growth and improve neuronal functions. In this study, a new method for site-specific PEGylation of CD133-Biotin antibody is performed through streptavidin-biotin conjugation. Purification was carried out by ion-exchange chromatography. The characterization of the single PEGylated CD133-Biotin antibody was confirmed using electrophoresis with silver staining and I(2)-BaCl(2) for PEG detection. Moreover, online PEG quantification directly after the chromatographic step was conducted (in each fraction) to detect exact elution times of PEG. In conclusion, the novel CD133-Biotin antibody PEGylation strategy conducted in this study could be used as a process step in route to neural stem cell recovery and purification via the modification of existing techniques such as aqueous two phase systems, PEGylated affinity columns or fluidized chromatography.

Colloidal gold/streptavidin methods.

Biotin-avidin detection systems are widely used in both immunocytochemistry and molecular biology. They take advantage of the high affinity of biotin, a low-molecular-weight vitamin, for avidin, an egg-white protein. Because of the problem of nonspecific binding of avidin, streptavidin has largely replaced avidin for immunocytochemical procedures. Streptavidin/colloidal gold-biotin detection systems for electron microscopy are most commonly used in postembedding immunocytochemistry. Usually, the primary antibody is unlabeled, the secondary antibody is biotinylated, and the colloidal gold is conjugated to streptavidin. In certain applications, the primary antibody may be biotinylated and no bridging antibody is needed. This chapter details the use of streptavidin-gold for postembedding labeling.

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells.

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.

Clotrimazole decreases human breast cancer cells viability through alterations in cytoskeleton-associated glycolytic enzymes.

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC50 of 88.6+/-5.3 microM and a t0.5 of 89.7+/-7.2 min, with 50 microM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180 min of treatment with 50 microM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45 min treatment with 50 microM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4+/-3.6% and 41.0+/-15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.

Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus.

A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) and Western blotting results revealed that the fusion protein not only retained VEE antigen binding and specificity properties similar to those of its parent native monoclonal antibody (MAb), but also possessed streptavidin-binding activity. This experimental approach can eliminate the need for chemical biotinylation of antibodies and the risk associated of antibody denaturation and can provide a stable and reproducible reagent for rapid and efficient immunoassay of VEE when detected by horseradish peroxidase (HRP)-conjugated streptavidin.

Characterization of the cellular component of polymorphous low-grade adenocarcinoma by immunohistochemistry and electron microscopy.

In order to characterize the cellular component of the polymorphous low-grade adenocarcinoma (PLGA) of the salivary gland, a morphological and immunohistochemical study was carried out. Thirty cases of PLGA were studied by light microscopy and immunohistochemistry and five cases by transmission electron microscopy (TEM). The expression of cytokeratins (CKs) 7,8,10,13,14,18,19, vimentin and muscle-specific actin (MSA) was investigated through the streptavidin-biotin method. The majority of tumor cells stained for vimentin, CKs 8, 18 and 7. CK 14 was positive in most cells of the papillary and trabecular sub-types. Although the expression of CKs 8,18 and 14 varied among the tumors sub-types, a straight relationship between each histologic pattern and the CK expression could not be delineated. MSA was reactive in only three tumors while CKs 10 and 13 were not detected in any tumor studied. The absence of MSA and the expression of CKs 8,18 and 7, in most of the tumor cells, lead to the hypothesis that myoepithelial cells are not the major cellular component of the PLGA. TEM revealed cells exhibiting microvilli and variable amounts of secretory granules, some of them suggesting an excretory activity. The presence of CKs 8,18 and 7, added to the secretory granules, indicates that PLGA originates from cells located at the acinar-intercalated duct junction.

Extremely high thermal stability of streptavidin and avidin upon biotin binding.

The effect of biotin binding on the thermal stability of streptavidin (STV) and avidin (AVD) was evaluated using differential scanning calorimetry. Biotin binding increases the midpoint of temperature Tm of thermally induced denaturation of STV and AVD in phosphate buffer from 75 and 83 degrees C to 112 and 117 degrees C at full biotin saturation, respectively. This thermostability is the highest reported for proteins coming from either mesophilic or thermophilic organisms. In both proteins, biotin also increases the calorimetric enthalpy and the cooperativity of the unfolding. Thermal stability of STV was also evaluated in the presence of high concentrations of urea or guanidinium hydrochloride (GuHCl). In 6 M GuHCl, STV remains as a tetramer and the Tm of the STV-biotin complex is centered at 108 degrees C, a few degrees below the value obtained in phosphate buffer. On the contrary, STV under fully saturating condition remains mainly in its dimeric form in 8 M urea and the thermogram shows two endotherms. The main endotherm at a lower temperature has been ascribed to the dimeric liganded state with a Tm of 87 degrees C, and the higher temperature endotherm to the tetrameric liganded form with a Tm of 106 degrees C. As the thermostability of unliganded protein in the presence of urea is unchanged upon binding we related the extremely high thermal stability of this protein to both an increase in structural ordering and compactness with the preservation of the tetramer integrity.