Synthesis and characterization of supramolecular protein aggregates: self-assembled, molecularly-ordered, tubes from electrostatic complementation of glutamine synthetase dodecamers

Dodecameric Escherichia coli glutamine synthetase (GS) is formed from identical subunits arranged in face-to-face hexameric rings. In the presence of Zn2+ and other transition metal ions the individual dodecamers 'stack' to form protein tubes. Previous results have suggested that six binuclear intermolecular metal binding sites are generated at each dodecamer-dodecamer interface by juxtaposition of the N-terminal helices of each subunit adjacent to an analogous helix from a docked dodecamer. In principle, replacement of one of the metal binding sites within each pair of helices with charged amino acids could generate electrostatic interactions that would provide the basis for heterospecific protein-protein interactions. In turn, this would allow for ordered assembly of protein tubes with alternating, chemically distinguishable, components. This hypothesis was tested by replacement of one of the metalligating histidines (His12) with aspartic acid, arginine or cysteine. The H12C mutant was further elaborated by selective thiol modification, with either of the charged reagents 2-iodo-acetic acid or 2-chloro-acetamidine, which yield glutamate (H12C-IA) or arginine (H12C-CA) mimics at position 12. Light scattering and electron microscopy were used to monitor the 'stacking ability' of these variants in the presence of Zn2+. No, or few, GS 'tubes' were observed in solutions containing only H12D, H12R, H12C-CA or H12C-IA, in the presence or absence of Zn2+. In contrast, in mixtures containing H12C-CA and either H12D or H12C-IA, the complementary GS variants stack in the presence of 100 microM Zn2+, with apparent second order rate constants that are comparable to the wild type dodecamers. Fluorescence energy transfer experiments with fluorescein-labeled H12C-IA (donor) and rhodamine-labeled H12C-CA (acceptor) were performed and compared with the energy transfer efficiency with mixtures containing variable ratios of acceptor-labeled and donor-labeled wild type GS; the wild type mixtures provide a benchmark for the extent of energy transfer expected in random linear arrangements of donor and acceptor. The efficiency of metal-dependent energy transfer in mixtures containing the acceptor-labeled H12C-CA and the donor-labeled H12C-IA was 3.2-fold greater than expected for a random distribution of charged variants. Together, the results indicate that the charged variants provide a mechanism for heterospecific interaction between chemically distinguishable dodecamers that align in an ordered one-dimensional array.     

Synthesis and characterization of supramolecular protein aggregates: self-assembled, molecularly-ordered, tubes from electrostatic complementation of glutamine synthetase dodecamers

Dodecameric Escherichia coli glutamine synthetase (GS) is formed from identical subunits arranged in face-to-face hexameric rings. In the presence of Zn2+ and other transition metal ions the individual dodecamers 'stack' to form protein tubes. Previous results have suggested that six binuclear intermolecular metal binding sites are generated at each dodecamer-dodecamer interface by juxtaposition of the N-terminal helices of each subunit adjacent to an analogous helix from a docked dodecamer. In principle, replacement of one of the metal binding sites within each pair of helices with charged amino acids could generate electrostatic interactions that would provide the basis for heterospecific protein-protein interactions. In turn, this would allow for ordered assembly of protein tubes with alternating, chemically distinguishable, components. This hypothesis was tested by replacement of one of the metalligating histidines (His12) with aspartic acid, arginine or cysteine. The H12C mutant was further elaborated by selective thiol modification, with either of the charged reagents 2-iodo-acetic acid or 2-chloro-acetamidine, which yield glutamate (H12C-IA) or arginine (H12C-CA) mimics at position 12. Light scattering and electron microscopy were used to monitor the 'stacking ability' of these variants in the presence of Zn2+. No, or few, GS 'tubes' were observed in solutions containing only H12D, H12R, H12C-CA or H12C-IA, in the presence or absence of Zn2+. In contrast, in mixtures containing H12C-CA and either H12D or H12C-IA, the complementary GS variants stack in the presence of 100 microM Zn2+, with apparent second order rate constants that are comparable to the wild type dodecamers. Fluorescence energy transfer experiments with fluorescein-labeled H12C-IA (donor) and rhodamine-labeled H12C-CA (acceptor) were performed and compared with the energy transfer efficiency with mixtures containing variable ratios of acceptor-labeled and donor-labeled wild type GS; the wild type mixtures provide a benchmark for the extent of energy transfer expected in random linear arrangements of donor and acceptor. The efficiency of metal-dependent energy transfer in mixtures containing the acceptor-labeled H12C-CA and the donor-labeled H12C-IA was 3.2-fold greater than expected for a random distribution of charged variants. Together, the results indicate that the charged variants provide a mechanism for heterospecific interaction between chemically distinguishable dodecamers that align in an ordered one-dimensional array.      

Iatrogenic esophageal injuries and their treatment

In our Department 22 patients were treated due to esophagus perforations in the years 1990-1996. Most of them (16) occurred after endoscopic examinations: diagnostic-3, after strictures dilatation-10, endoscopic intubation-2, sclerotherapy-1, transesophageal cardioversion-1. There were 5 intraoperative perforations: 3-after cardiomyotomy, 2-after intubation. The following treatment was performed: oesophageal suture and drainage-5 patients, subtotal oesophagectomy with oesophagogastrostomy-3 patients, oesophagectomy with salivary fistula and oesophago-gastro or -colostomy in second operation--9 patients, perforation covered with gastric fundus--3, intubation--1 patient, conservative treatment--1 patient. Four (18%) patient died.     

Failure mechanisms of ceramic membrane reactors in partial oxidation of methane to synthesis gas

In the course of generating synthesis gas (H₂, CO) from methane, we have observed two types of fractures occurring on the Sr(Co, Fe)O_x-type oxygen membrane reactors. The first type occurred shortly after the reaction started and the second type often occurred days after the reaction. To determine the causes of these fractures, we have examined the starting material and fractured membranes using a combination of X-ray diffraction and thermogravimetric analyses. We found that the first type of fracture was the consequence of an oxygen gradient in the membrane, pointing from the reaction side to the air side. This causes a lattice mismatch inside the membrane, leading to fracture. The second type of fracture, however, was the result of a chemical decomposition. We found that the Sr(Co, Fe)O_x-type membrane had been reduced to SrCO₃, and elemental Co and Fe by the synthesis gas generated in the reaction. The decomposition causes enormous expansion leading to a large crack along the axis of tube.     

The study of the interactions between graphene and Ge(001)/Si(001)

The interaction between graphene and germanium surfaces was investigated using a combination of microscopic and macroscopic experimental techniques and complementary theoretical calculations.Density functional theory (DFT) calculations for different reconstructions of the Ge(001) surface showed that the interactions between graphene and the Ge(001) surface introduce additional peaks in the density of states,superimposed on the graphene valence and conduction energy bands.The growth of graphene induces nanofaceting of the Ge(001) surface,which exhibits well-organized hill and valley structures.The graphene regions covered by hills are of high quality and exhibit an almost linear dispersion relation,which indicates weak graphene-germanium interactions.On the other hand,the graphene component occupying valley regions is significantly perturbed by the interaction with germanium.It was also found that the stronger graphene-germanium interaction observed in the valley regions is connected with a lower local electrical conductivity.Annealing of graphene/Ge(001)/Si(001) was performed to obtain a more uniform surface.This process results in a surface characterized by negligible hill and valley structures;however,the graphene properties unexpectedly deteriorated with increasing uniformity of the Ge(001) surface.To sum up,it was shown that the mechanism responsible for the formation of local conductivity inhomogeneities in graphene covering the Ge(001) surface is related to the different strength of graphene-germanium interactions.The present results indicate that,in order to obtain high-quality graphene,the experimental efforts should focus on limiting the interactions between germanium and graphene,which can be achieved by adjusting the growth conditions.     

Fast Switching Liquid Crystals for Color-Sequential LCDs

High birefringence (0.3 < Deltan < 0.4) and relatively low-viscosity liquid crystal mixtures containing isothiocyanato tolane and isothiocyanato terphenyl liquid crystals are developed. A twisted-nematic (TN) 1.6-mum thin cell for color-sequential liquid crystal display with ~1-ms response time is demonstrated.     

Bridged polysilsequioxane adsorption materials containing phosphonic acid residues

Reactions of hydrolytic polycondensation of bis(triethoxysilane) [(C₂H₅O)₃Si]₂C₂H₄ (or [(C₂H₅O)₃Si]₂C₆H₄) and functional agent (C₂H₅O)₃Si(CH₂)₂P(O)(OC₂H₅)₂ (alkoxysilanes molar ratio of 2: 1 and 4: 1, fluoride ion catalyst and ethanol solvent) yielded powder-like xerogels that contained phosphonic acid residues in the surface layer. Their treatment with boiling concentrated hydrochloric acid resulted in transformation of functional groups ≡Si(CH₂)₂P(O)(OC₂H₅)₂ into acid groups ≡Si(CH₂)₂P(O)(OH)₂. The methods of IR, ¹H MAS NMRm and ¹³C, ²⁹Si, ³¹P MAS NMR spectroscopy showed the following (1) The surface layer in the initial xerogels contains not only phosphorus functional groups, but also some non-hydrolyzed ethoxysilyl groups as well as silanol groups. (2) The hydrochloric acid treatment of the initial xerogels causes the hydrolysis of not only ethoxy groups in the phosphonic acid residues, but also most residual ethoxysilyl groups. (3) Vacuum drying of xerogels after acid treatment forms ≡Si(CH₂)₂P(O)(OH)-OSi≡ links in their surface layer (not more than 20% of phosphorus-containing groups). (4) According to ²⁹Si CP MAS NMR spectroscopic data, boiling acid treatment relatively enriches the xerogel structure T² and T³ units and accounts for the higher rigidity of the hybrid framework. These units also account for retention of the porous structure in these xerogels over time, while most initial xerogels have porous structures that collapse in 12–18 months of storage. The acid-treated xerogels were attributed to microporous adsorbents (having specific surface area of 620 to 760 m²/g). According to the AFM data, both initial and acid-treated xerogels contain almost spherical aggregates of the primary particles (globules).     

Application of Comparative Method for Estimating Microporosity of Porous Solids in Adsorption from Solutions

The concept of a comparative method in terms of the liquid adsorption on solids is presented. This method is based on comparison of the excess adsorption isotherm on a porous solid with that measured on a reference nonporous adsorbent. It is proved that proposed approach gives the possibility for separating a total excess adsorption isotherm into partial isotherms, proper for micro- and mesopores. Several experimental systems are analyzed to illustrate the usefulness of the comparative method for evaluating the surface phase capacity, which presents an important quantity in the study of adsorption from solution in the whole concentration region. The comparative method examined for adsorption systems of microporous active carbons seems to be useful for investigating other porous materials as silica gels, zeolites or metal oxides.     

Influence of selected alkoxysilanes on dispersive properties and surface chemistry of spherical silica precipitated in emulsion media

The process of silicas formation in emulsion system in the reaction of precipitation from water solutions of sodium silicate and hydrochloric acid has been studied. The effects of silica surface modifications with silane coupling agents from the group of alkoxysilanes with different functional groups have been determined. Both unmodified and modified silicas have been thoroughly characterised by scanning electron microscopy (SEM) and dynamic light scattering (DLS) and other methods.The effect of alkoxysilanes on the diameter distribution of the modified silica is insignificant; the presence of silane coupling agents causes a small increase in the particle diameter and a small increase in the tendency towards agglomeration, moreover the particles of silicon dioxide precipitated are characterised by spherical shape. The course of the electrokinetic curves depends mainly on the proton affinity of the modifiers. Significant changes in the stability of silica dispersions have been found as a result of modification with N-2-(aminoethyl)-3-aminopropyltrimethoxysilane. This compound causes the greatest increase in the tendency towards agglomeration of the primary particles. The modification mechanism has been established to be chemisorption, as indicated by the ¹³C and ²⁹Si CP MAS NMR spectra. The products have been shown to have typical mesoporous structure. The surface area of the unmodified silica is 133 m²/g, while those for the modified silicas vary from 115 to 182 m²/g.