Inactivation of Recombinant Human Brain-Type Creatine Kinase During Denaturation by Guanidine Hydrochloride in a Macromolecular Crowding System

In this study, we quantitatively examined the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000) and dextran 70, on guanidine hydrochloride (GdnHCl)-induced denaturation of recombinant human brain-type creatine kinase (rHBCK). Our results showed that both PEG 2000 and dextran 70 had a protective effect on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in the retention of 35.33 % of rHBCK activity after 4 h of inactivation, while no rHBCK activity was observed after denaturation in the absence of macromolecular crowding agents. The presence of PEG 2000 and dextran 70 at a concentration of 100 g/L could decelerate the k ₂ value of the slow track to 21 and 33 %, respectively, in comparison to values obtained in the absence of crowding agents. Interestingly, inactivation of rHBCK in the presence of 200 g/L PEG 2000 followed first-order monophasic kinetics, with an apparent rate constant of 8?×?10^?5?s^?1. The intrinsic fluorescence results showed that PEG 2000 was better than dextran 70 at stabilizing rHBCK conformation. In addition, the results of the phase diagram indicate that more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but the protective effects of PEG 2000 on the inactivation and unfolding of rHBCK tended to increase as the ratio of PEG 2000 increased in the mixed crowding agent solution. Though it is not clear which crowding agents more accurately simulated the intracellular environment, this study could lead to a better understanding of protein unfolding in the intracellular environment.     

Use of multivariate and neutron activation analysis on studying element interrelationships in Brazilian phosphorite

Methods to determine U, Th, and other constituent elements in mineral matrices through Instrumental Neutron Activation Analysis (INAA), have been widely utilized due to their capability to extract a large quantity of information from small samples. In the present work, an experimental methodology based on INAA associated to the cluster statistics technique was used to determine the uranium concentration and its correlation with other elements present in the mineral phosphorites from the northeast region of Brazil. Data for 22 elements including rare-earth elements are presented. A good correlation between uranium and rare-earth elements is observed. For the transition metals, correlation with uranium was not noted.     

Use of a novel Hill-climbing genetic algorithm in protein folding simulations

We have developed a novel Hill-climbing genetic algorithm (GA) for simulation of protein folding. The program (written in C) builds a set of Cartesian points to represent an unfolded polypeptide’s backbone. The dihedral angles determining the chain’s configuration are stored in an array of chromosome structures that is copied and then mutated. The fitness of the mutated chain’s configuration is determined by its radius of gyration.

A four-helix bundle was used to optimise simulation conditions, and the program was compared with other, larger, genetic algorithms on a variety of structures. The program ran 50% faster than other GA programs. Overall, tests on 100 non-redundant structures gave comparable results to other genetic algorithms, with the Hill-climbing program running from between 20 and 50% faster. Examples including crambin, cytochrome c, cytochrome B and hemerythrin gave good secondary structure fits with overall alpha carbon atom rms deviations of between 5 and 5.6 Å with an optimised hydrophobic term in the fitness function.     

Use of a terbium chelate as label in spectrofluorimetric determinations of protein traces

A spectrofluorimetric method for the determination of protein traces using the terbium complex formed with diethylenetriaminepentamethanephosphonic acid and p-aminosalicyclic acid as reagents is described. The method has been applied to the trace determination of simple proteins at concentrations down to 10^?11 M in the pH range 2.5–11.     

Visualizing worm micelle dynamics and phase transitions of a charged diblock copolymer in water

Assemblies of block copolymer amphiphiles are sometimes viewed as glassy, frozen, or static colloids, especially in strongly segregating solutions. Here, we visualize by fluorescence microscopy and AFM the dynamics and transitions of single cylindrical micelles and vesicles composed of a charged diblock copolymer in water. In mapping the salt- and pH-dependent phase diagrams of a near-symmetric diblock of poly(acrylic acid)-polybutadiene, low pH and high salt (NaCl, CaCl2) neutralize and screen the charged corona sufficiently to foster membrane formation and generate vesicles. Decreased salt and neutral pH increases intra-coronal repulsion and drives a transition to multi-branched cylinders and highly stable, but fluid and flexible, worm micelles. Ca2+ both stiffens cylinders and stabilizes them relative to spheres. Further increase of intra-coronal repulsion generates spherical micelles by fragmentation and pinch-off at the ends of worms. Both the transition kinetics and phase diagrams indicate divalent cation is about 5-10-fold more effective than monovalent in stabilizing all nonspherical morphologies.     

Use of microfiltration as first step in recovery of protein a from fermentation broth

The flux and transmission of protein A during microfiltration have been studied. We studied the performance of two commercial membranes: one made of nylon (Pall Ultipore Nylon₆₆, 0.2 μm) and one of polyether sulfone (Pall Omega, 0.16 μm). The Nylon₆₆ membrane had by far the best transmission of protein A although a previous study showed that bovine serum albumin (BSA), often used to characterize membranes, had much better transmission through the Omega membrane. The membrane manufacturer also states that the Omega membrane is the best membrane for this kind of application because it is a low-protein-binding membrane. The lower transmission of the Omega membrane for protein A was assumed to be owing to its smaller pores and higher charge density in combination with the larger Stokes radius for protein A. When the pH was lowered, the Nylon₆₆ membrane still had the higher transmission. It can thus be concluded that a membrane that is found suitable for the recovery process of one protein is not always the best choice for the recovery process for other proteins even though the membrane is low protein binding.     

Use of a solid-state nanopore for profiling the transferrin receptor protein and distinguishing between transferrin receptor and its ligand protein

A nanopore device is capable of providing single-molecule level information of an analyte as they translocate through the sensing aperture—a nanometer-sized through-hole—under the influence of an applied electric field. In this study, a silicon nitride (Si_xN_y)-based nanopore was used to characterize the human serum transferrin receptor protein (TfR) under various applied voltages. The presence of dimeric forms of TfR was found to decrease exponentially as the applied electric field increased. Further analysis of monomeric TfR also revealed that its unfolding behaviors were positively dependent on the applied voltage. Furthermore, a comparison between the data of monomeric TfR and its ligand protein, human serum transferrin (hSTf), showed that these two protein populations, despite their nearly identical molecular weights, could be distinguished from each other by means of a solid-state nanopore (SSN). Lastly, the excluded volumes of TfR were experimentally determined at each voltage and were found to be within error of their theoretical values. The results herein demonstrate the successful application of an SSN for accurately classifying monomeric and dimeric molecules while the two populations coexist in a heterogeneous mixture.     

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

Background  

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS) proteins of the R7 subfamily (R7-RGS). The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP), a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain.     

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17–24 kDa) for M. longa luciferase have been cloned. All the isoforms are single‐chain proteins consisting of a 17‐residue signal peptide for secretion, variable N‐terminal part and conservative C‐terminus responsible for luciferase activity. In contrast to other bioluminescent proteins containing a lot of aromatic residues which are frequently involved in light emission reaction, the C‐terminal part of MLuc contains only four Phe, two Tyr, one Trp and two His residues. To figure out whether Tyr residues influence bioluminescence, we constructed the mutants with substitution of Tyr to Phe (Y72F and Y80F). Tyrosine substitutions do not eliminate the ability of luciferase to bioluminescence albeit significantly reduce relative specific activity and change bioluminescence kinetics. In addition, the Tyr replacements have no effect on bioluminescence spectrum, thereby indicating that tyrosines are not involved in the emitter formation. However, as it was found that the intrinsic fluorescence caused by Tyr residues is quenched by a reaction substrate, coelenterazine, in concentration‐dependent manner, we infer that both tyrosine residues are located in the luciferase substrate‐binding cavity.     

Use of silk protein, sericin, as a sustained-release material in the form of a gel, sponge and film

To evaluate the usability of silk protein (sericin, SC) as a sustained-release material, the physicochemical properties of SC and the release profiles of model drugs from SC gel, sponge and film were studied. Heat aids the dissolution of SC. The molecular weight of SC tended to decrease as the heating temperature and heating time increased. The gel and sponge formed by SC were moldable and consisted of high molecular weight SC polymers (250 kDa and about 400 kDa). SC film was easily broken and exhibited elastic distortion. The addition of moisture-retaining plasticizer (glycerin and sorbitol) improved the film-forming characteristics of SC. The results suggested that SC is practical as a moldable gel and sponge, and as a tensible film. To evaluate the release profiles of small molecules, fluorescein isothiocyanate-dextran ((1) FD4, 4 kDa and (2) FD70, 70 kDa) were used as two model drugs with significantly different molecular weights, and fluorescein isothiocyanate-albumin ((3) FA, 66 kDa) was used as a charged drug. Each was formulated in SC gel, sponge and film. In each preparation, the release rate of the model drugs tended to be FA相似文献   

Synthesis of a highly phosphorescent emitting iridium(III) complex and its application in OLEDs

A rigid ligand benzo[de]benzo[4,5]imidazo[2,1-α]isoquinolin-7-one (biio) was designed and conveniently synthesized, and the corresponding bis-cyclometalated iridium complex (biio)₂Ir(acac) (acac = acetylacetone) was prepared. The light emitting and electrochemical properties of this complex were studied. The complex has the characters of simply synthetic procedure and strong phosphorescence. The electroluminescent device using this complex as dopant was fabricated. The device had the structure of ITO/NPB (40 nm)/Ir complex:CBP (7%, 30 nm)/BCP (15 nm)/Alq₃ (30 nm)/LiF (1 nm)/Al (100 nm). The maximum emission of the device was at 496 nm. The maximum brightness of the device can reach 79640 cd m⁻² with an external quantum efficiency of 12.1% and a maximum current efficiency of 31.7 cd A⁻¹.     

Use of PLL-g-PEG in micro-fluidic devices for localizing selective and specific protein binding

By utilizing flow-controlled PLL-g-PEG and PLL-g-PEGbiotin modification of predefined regions of a poly(dimethylsiloxane) (PDMS) micro-fluidic device, with an intentionally chosen large (approximately 1 cm2) internal surface area, we report rapid (10 min), highly localized (6 x 10(-6) cm2), and specific surface-based protein capture from a sample volume (100 microL) containing a low amount of protein (160 attomol in pure buffer and 400 attomol in serum). The design criteria for this surface modification were achieved using QCM-D (quartz crystal microbalance with energy dissipation monitoring) of serum protein adsorption onto PLL-g-PEG-modified oxidized PDMS. Equally good, or almost as good, results were obtained for oxidized SU-8, Topas, and poly(methyl metacrylate) (PMMA), demonstrating the generic potential of PLL-g-PEG for surface modification in various micro-fluidic applications.     

Fluorescence lifetimes and emission patterns probe the 3D orientation of the emitting chromophore in a multichromophoric system

In this Communication, we report on the fluorescence behavior of an individual first generation multichromophoric dendrimer. The fact that each of the chromophores in time acts as a fluorescent trap is demonstrated by directly probing the dipole orientation of the emitting chromophore by means of defocused wide-field imaging and comparing experimental and calculated emission patterns. It is shown that in such cases the electromagnetic boundary condition effect results in discrete changes in the fluorescence lifetime as a function of time for individual dendrimers.     

Particulate matter, iron, and zinc in the atmosphere of a large Brazilian town

A Radionuclide Aerosol Sampler/Analyzer (RASA Mark 4) has been developed at PNNL for use in verifying the Comprehensive Nuclear Test Ban Treaty (CTBT). The RASA Mark 4 collects about 20,000 m³ of air per day on a 0.25 m² filter. This filter is automatically decayed for 24 hours, then advanced to a germanium detector for a 24 hour count. This system has been operated in Richland, WA for a limited period of time in a predeployment testing phase. The germanium-detector gamma-ray spectra have been analyzed by automatic spectral analysis codes to determine Minimum Detectable Concentrations (MDC) for a number of isotopes of interest. These MDC's have been compared to other atmospheric measurements in the field and in the laboratory.     

The effect of miR-7 on behavior and global protein expression in glioma cell lines

Malignant glioma is a common cancer of the nervous system. Despite recent research efforts in cancer therapy, the prognosis of patients with malignant glioma has remained dismal. MicroRNAs are noncoding RNAs that inhibit the expression of their targets in a sequence-specific manner, and a few have been shown to act as oncogenes or tumor suppressors. Here, we aimed at exploring the precise biological role of microRNA-7 (miR-7) and the global protein changes in glioma cell lines transiently transfected with miR-7. Transfection of miR-7 into glioma cell lines causes inhibition of cell migration and invasion and suppression of tumorigenesis. Moreover, ectopic expression of miR-7 inhibits lung metastases of glioma in vivo. Among 65 protein spots with differential expression separated by 2-DE, 37 proteins were successfully identified by MS/MS analysis. Of those, the 25 downregulated proteins, which include 14-3-3ζ, eukaryotic translation initiation factor 5A (EIF5A), and annexin A4, may be downstream targets of miR-7, a finding that could elucidate some aspects of the behavior of glioma cells at the protein level. In conclusion, the absence of miR-7 function could cause downstream molecules to switch on or off, resulting in glioma development, invasion, and metastases. MiR-7-based gene treatment may be a novel anti-invasion therapeutic strategy for malignant glioma.     

Use of infrared spectroscopy for controlling protein concentration in lacrimal fluid and diagnosing corneal diseases

It was shown that changes in the absorption band intensities of peptide groups in infrared spectra of proteins contained in lacrimal fluid can be used for controlling their concentrations and diagnosing corneal diseases.     

Preparation of in-house reference material of benzylpenicillin in milk and results of a Brazilian proficiency testing scheme

Two milk test materials containing benzylpenicillin and a benzylpenicillin-free material were prepared and used for the operation of a Brazilian proficiency testing (PT) scheme according to the requirements of ISO/IEC 17043. The PT scheme was designed to include laboratories in charge of confirmatory analysis as well as screening analysis. Sets of sample vials, benzylpenicillin-free and spiked items containing 0.2 g of lyophilized milk, were distributed to each participant. Of 20 participants who reported screening data, 18 accomplished satisfying results, providing evidence of the capability of Brazilian laboratories to produce reliable qualitative information. The assigned value (robust average calculated from 6 results) and its uncertainty were (8.28 ± 0.52) μg/L. Participants’ performance was evaluated using z-scores. A small number of participants were able to report quantitative benzylpenicillin results, and consequently care should be taken in the interpretation of the laboratory’s performance, considering the statistical nature of z-scores. Long-term stability testing on remaining PT items showed that a stable benzylpenicillin quality control material was produced and can be used for internal quality control or validation purposes.     

Synthesis and properties of a solid solution formed in the CrVMoO7 - AlVMoO7 system

It has been shown by the methods of X-ray powder diffraction (XRD), differential thermal analysis (DTA) and infrared spectroscopy (IR) that solid solutions of a formula Cr_1−xAl_xVMoO₇, where xε (0−0.65), are formed in the system CrVMoO₇-AlVMoO₇. The obtained research results have proven that the ions Al3+ are incorporated into the crystal lattice of CrVMoO₇ instead of Cr³⁺, which causes a contraction of the lattice and a shift of IR absorption bands towards higher values of wavenumbers. The phases Cr_1−xAl_xVMoO₇ melt incongruently in the temperature range from 710°C (for x=0.65) to ∼820°C in the case of x close to zero This revised version was published online in July 2006 with corrections to the Cover Date.