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PubMed2005

Preparation and in vitro stability of (n,gamma) yttrium-90 hydroxyapatite.

Yttrium-90, produced by irradiating Y2O3 (15 mg) in the Pakistan Research Reactor (PARR-I) at a flux of approximately 1.5x10(14) neutrons/cm2/s, was used to prepare yttrium-90 hydroxyapatite particles for radiosynovectomy applications. The irradiated material was dissolved in concentrated hydrochloric acid, evaporated and taken up in distilled water. The 120 h irradiation resulted in the production of approximately 12GBq (324mCi) of 90Y at the end of irradiation (EOI) and the corresponding specific activity was approximately 1017GBq/g of yttrium. Hydroxyapatite (HA) particles were synthesized by an already reported method. Labeling of HA particles with 90Y was studied without a transchelating agent. Labeling yields of approximately 100% could be achieved with 40 mg of HA and 0.4 mg of 90Y. In vitro studies showed <2% loss of 90Y activity in normal saline and 1% human serum albumin solution over a period of 8 days. The high labeling yield, good stability and ease of preparation of the 90Y-HA particles indicate that these particles may find wide application in radiation synovectomy.

Drug Stability↗
PubMed2005

Selected growth of cubic and hexagonal GaN epitaxial films on polar MgO(111).

Selected molecular beam epitaxy of zinc blende (111) or wurtzite (0001) GaN films on polar MgO(111) is achieved depending on whether N or Ga is deposited first. The cubic stacking is enabled by nitrogen-induced polar surface stabilization, which yields a metallic MgO(111)-(1 x 1)-ON surface. High-resolution transmission electron microscopy and density functional theory studies indicate that the atomically abrupt semiconducting GaN(111)/MgO(111) interface has a Mg-O-N-Ga stacking, where the N atom is bonded to O at a top site. This specific atomic arrangement at the interface allows the cubic stacking to more effectively screen the substrate and film electric dipole moment than the hexagonal stacking, thus stabilizing the zinc blende phase even though the wurtzite phase is the ground state in the bulk.

Journal Article↗
PubMed1994

Domains of tau protein and interactions with microtubules.

The role of the neuronal microtubule-associated protein tau has been studied by generating a series of tau constructs differing in one or several of its subdomains: length and composition of the repeat domains, extensions of the repeats in the N- or C-terminal direction, constructs without repeats, assembly vs projection domain, and number of N-terminal inserts. The interaction of the mutant tau proteins with microtubules was judged by several independent methods. (i) Direct binding assays between tau and taxol-stabilized microtubules yield dissociation constants and stoichiometries. (ii) Light scattering and X-ray scattering of assembling microtubule solutions reflect the capacity of tau to promote microtubule nucleation, elongation, and bundling in bulk solution. (iii) Dark field microscopy of assembling microtubules allows one to assess the efficiency of nucleation and bundling separately. The repeat region alone, the N-terminal domains alone, or the C-terminal tail alone binds only weakly to microtubules. However, binding is strongly enhanced by combinations such as the repeat region plus one or both of the flanking regions which could be viewed as "jaws" for tau on the microtubule surface (the proline-rich domain P upstream of the repeats and the "fifth" repeat R' downstream). Such combinations make tau's binding productive in terms of microtubule assembly and stabilization, while the combination of the flanking regions without repeats binds only unproductively. Efficient nucleation parallels strong binding in most cases, i.e., when a construct binds tightly to microtubules, it also nucleates them efficiently and vice versa. In addition, the proline-rich domain P in combination with the repeats R or the flanking domain R' causes pronounced bundling. This effect disappears when the N-terminal domains (acidic or basic) are added on, suggesting that the tau isoforms are not "bundling proteins" in the proper sense. In spite of the wide range of binding strength and nucleation efficiency, the stoichiometries of binding are rather reproducible (around 0.5 tau/tubulin dimer); this is in remarkable contrast to the effect of certain types of phosphorylation which can strongly reduce the stoichiometry.

Amino Acid Sequence↗
PubMed2006

Synthesis, characterization and in situ intestinal absorption of different molecular weight scutellarin-PEG conjugates.

Highly water soluble esters of scutellarin with different molecular weight polyethylene glycol (PEG) were synthesized. The physicochemical properties, the stabilities under different conditions and the in situ intestinal absorption of the conjugates in rats were investigated. By PEG modification, greatly increased water solubility and a desirable partition coefficient were obtained. These compounds act as prodrugs i.e. breakdown occurrs in a predictable fashion: in vitro, the t1/2 of them in PBS buffer at pH 7.4 was above 12 h (37 degrees C), while in plasma a more rapid breakdown was observed (t1/2 1.5-3 h). PEGylation could enhance the absorption of scutellarin in rat intestine, and scutellarin, its PEG conjugates are absorbed through intestine mainly via passive transport. When the molecular weight of PEG increased from 200 to 1000 Da, the absorption of the conjugates decreased accordingly. The range of PEG molecular weight used for the PEGylation of scutellarin was about 400-1000 Da based on considerations of the yield, the stability and the absorption.

Animals↗
PubMed1993

Oxidative refolding of insulin-like growth factor 1 yields two products of similar thermodynamic stability: a bifurcating protein-folding pathway.

Can one protein sequence encode two structures? Oxidative folding of human insulin-like growth factor 1 (IGF-1), a globular protein of 70 residues, is shown to yield two products of similar thermodynamic stability. This observation is of particular interest in light of the recent demonstration that two of the three disulfide bonds in native IGF-1 rearrange in the presence of dithiothreitol [Hober, S., et al. (1992) Biochemistry 31, 1749-1756]. Kinetics of the IGF-1 folding pathway were monitored by high-performance liquid chromatography (rp-HPLC). Disulfide-pairing schemes of intermediates and products were established by peptide mapping. Two disulfide isomers were obtained as products: one with native insulin-like pairing [6-48; 18-61; 47-52] (designated native IGF-1; 60% yield) and the other with alternative pairing [6-47; 18-61; 48-52] (designated IGF-swap; 40% yield). The predominant early intermediate contains the single disulfide 18-61, which is shared in common by the two products. Relative yields of native IGF-1 and IGF-swap are independent of protein concentration under dilute conditions. In the absence of an added thiol reagent, each isomer is stable indefinitely at neutral pH; in the presence of an added thiol reagent, the two isomers interconvert with an Arrhenius activation barrier of 12 kcal/mol. Interconversion does not require complete reduction and yields the same ratio of products as initial folding, demonstrating thermodynamic control. Spectroscopic studies using circular dichroism (CD), infrared spectroscopy (FTIR), two-dimensional 1H-NMR (2D-NMR), and photochemical dynamic nuclear polarization (photo-CIDNP) suggest that IGF-1 and IGF-swap adopt similar secondary structures but distinct tertiary folds. Implications of these observations for understanding the topology of protein-folding pathways are discussed.

Amino Acid Sequence↗
PubMed1980

Isolation of lung lamellar bodies and their conversion to tubular myelin figures in vitro.

Lamellar bodies of type II alveolar epithelial cells are the intracellular storage sites of lung surfactant, while tubular myelin figures are an extracellular surfactant form found in the alveolar fluid. A refined procedure was used to isolate intact lamellar bodies from rat lung homogenates in a fraction of high purity and yield. The stability of isolated lamellar bodies under various conditions was determined by electron microscopy. Lamellar bodies were completely disrupted after incubation at 37, 24, and 0 degrees C for 0.6, 2, and 12 hr, respectively, in 0.33 M sucrose, 0.01 M HEPES (pH 7.4). In addition, they were completely disrupted after incubation for 1 hr in 0.33 M sucrose, 1 mM EGTA at 0 degrees C or 0.154 M NaCl or 0.10 M sodium phosphate (pH 7.4) at 24 degrees C. Incubation of isolated lamellar body fractions in medium containing 5 mM Ca++ or Mg++ at 37 degrees C for 1 hr resulted in the appearance of tubular myelin figures. A procedure is also presented for the isolation of tubular myelin figures from rat lung lavage fluid.

Animals↗
PubMed1993

Immobilization studies of an industrial penicillin acylase preparation on a silica carrier.

Penicillin acylase (EC 3.5.1.11) was immobilized by covalent binding on a silica gel carrier activated by silanization. High immobilization yields, operational stability, enzyme loading and support reutilization capacity were obtained. The effects of several variables on the activation of the support and on the immobilization method were studied. Other supports and immobilization methods were assessed for the immobilization of penicillin acylase and compared with the basic process used. Activity versus temperature, pH, buffer molarity and penicillin concentration profiles were determined and compared for the free, crosslinked and covalently-bound silica carrier penicillin acylase preparations. The hydrolysis performance of the latter preparation was assessed in a batch basket reactor and the time course of the hydrolysis reaction modelled by a two parameter model equation. The operational stabilities of penicillin acylase coupled covalently to the silica gel support and immobilized by a crosslinking procedure using glutaraldehyde were also compared.

Biotechnology↗
PubMed2003

Influence of emulsifiers on the crystallization of solid lipid nanoparticles.

The crystallization temperature and polymorphism of tripalmitin nanoparticles in colloidal dispersions prepared by melt-homogenization and stabilized with different pharmaceutical surfactants (sodium glycocholate, sodium oleate, tyloxapol, Solutol HS 15, Cremophor EL) and their combinations with soybean phospholipid (Lipoid S100) were investigated to establish the influence of the emulsifiers on these parameters. There were no major effects on the crystallization temperature but remarkable differences in the time-course of polymorphic transitions after crystallization of the triglyceride particles indicate interaction between the surfactant layer and the triglyceride matrix. The metastable alpha-modification was most stable in dispersions solely stabilized with glycocholate. Upon fast cooling from the melt, these dispersions form an uncommon type of alpha-modification that displays only a very weak small-angle reflection indicating poor ordering between triglyceride layers. Slow crystallization of these glycocholate-stabilized nanoparticles yields the usual alpha-form. Electron microscopic investigations reveal that, in both cases, the particles in the alpha-modification are less anisometric than those of the stable beta-form. These results indicate that major rearrangements still may take place in solid lipid nanoparticles after recrystallization.

Crystallization↗
PubMed1996

Analysis of ascorbate in plant tissues by high-performance capillary zone electrophoresis.

We describe here a simple and rapid capillary electrophoresis method for the determination of ascorbic acid (L-AA) and isoascorbic acid (D-AA) in vegetative tissues. For optimal yields and stabilization, samples are extracted with cold 3% metaphosphoric acid. Hydrophobic contaminants are then removed by passage through a C18 solid-phase extraction cartridge. The analysis itself is performed on a fused silica capillary with 200 mM borate, pH 9, as the carrier electrolyte, using on-line diode array detection over the range 190-350 nm. Quantitation was performed at 260 nm, the uv-absorption maximum for ascorbate at this pH. This method has a minimum detection limit of 84 fmol/injection and linearity of detector response was observed up to at least 12 pmol/injection. We also describe the influence of electrolyte concentration, pH, and the presence of detergent on separations of L-AA, D-AA, and L-galacturonic acid-1,4-lactone. The protocol has been demonstrated to be suitable for the analysis of L-AA in Arabidopsis, parsley, and mushroom. The method has superior resolution to comparable HPLC separations, a comparable analysis time, but lower sensitivity because of the concentration limitations of the detection system.

Arabidopsis↗
PubMed2001

The importance of framework residues H6, H7 and H10 in antibody heavy chains: experimental evidence for a new structural subclassification of antibody V(H) domains.

The N-terminal segment (FR-H1) of the heavy chain (V(H)) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study, we have established a causal relationship between the local sequence and the structure of this framework region and linked this relationship to important biophysical properties such as affinity, folding yield and stability. We have generated six mutants of the scFv fragment aL2, covering some of the most abundant amino acid combinations in positions H6, H7 and H10 (according to a new consensus nomenclature, Kabat H9). For the aL2 wild-type (w.t.) with the sequence 6(Q)7(P)10(A) and for two of the mutants, the X-ray structures have been determined. The structure of the triple mutant aL2-6(E)7(S)10(G) shows the FR-H1 backbone conformations predicted for this amino acid combination, which is distinctly different from the structure of the w.t, thus supporting our hypothesis that these residues determine the conformation of this segment. The mutant aL2-6(E)7(P)10(G) represents a residue combination not occurring in natural antibody sequences. It shows a completely different, unique structure in the first beta-strand of V(H), not observed in natural Fv fragments and forms a novel type of diabody. Two V(H) domains of the mutant associate by swapping the first beta-strand. Concentration-dependent changes in Trp fluorescence indicate that this dimerization also occurs in solution. The mutations in amino acids H6, H7 and H10 (Kabat H9) influence the dimerization behavior of the scFv and its thermodynamic stability. All the observations reported here have practical implications for the cloning of Fv fragments with degenerate primers, as well as for the design of new antibodies by CDR grafting or synthetic libraries.

Ampicillin↗
PubMed2002

Large-scale expression and purification of high-molecular-weight glutenin subunits.

The high-molecular-weight glutenin subunits (HMW-GSs) are considered to be one of the most important components of wheat gluten, contributing to the unique viscoelastic properties of wheat dough. The HMW-GSs are highly homologous in sequence and structure and a mixture of subunits is usually present in wheat flours. Consequently, it is difficult to purify these proteins separately in appreciable amounts. Expression in heterologous systems provides a clear opportunity to produce large amounts of single HMW-GS proteins, amounts (up to 100 mg) which are required for in vitro analysis of these proteins. However, since the first expression studies of HMW-GSs, over 10 years ago, this technology has not been widely utilized. Previous studies have been analytical or small scale (5-100 ml) and in most cases only partial purity was obtained. In the present paper, we describe in detail the expression of the HMW-GSs Glu1-Dx2, Dx5, Dy10, and Dy12 for the first time on a large scale, producing up to 100 mg of target protein from a 2-liter bacterial culture, using a Biostat fermenter. Our results include optimization of expression conditions to increase yield and stability of proteins. Results also include localization, differences between x- and y-type expression and small-scale versus large-scale expression. We also developed a large-scale purification procedure. The bacterially expressed proteins have the same molecular weight on SDS-PAGE and the same retention times on RP-HPLC as their native counterparts extracted from flour. Functionality tests, on the bacterially produced proteins, have shown a clear correlation with the equivalent native proteins from flour. These results provide a clear opportunity to produce protein in amounts necessary for more detailed studies of the structure and function of the HMW-GSs and glutenin polymers on dough development and quality.

Chromatography, High Pressure Liquid↗
PubMed1994

Does technetium-99m bicisate image local brain metabolism in late ictal temporal lobe epilepsy?

Ictal increase in regional cerebral blood flow as judged by single-photon emission tomography (SPET) is a common phenomenon during focal epileptic seizures. Up to 2 min postictally, regional hyperperfusion is a consistent finding with technetium-99m hexamethylpropylene amine oxime (HMPAO) in temporal lobe epilepsy. A new 99mTc-labelled lipophilic cerebral blood flow imaging agent, bicisate, has considerably longer radiochemical stability and yields better image quality than 99mTc-HMPAO. In this report, we present the case of a 21-year-old female patient with temporal lobe complex partial seizures. Magnetic resonance imaging revealed right hippocampal sclerosis. A dose of 550 MBq of 99mTc-bicisate was injected 35 s after the onset of a seizure during intracranial EEG-videotelemetry. At the moment of injection, subdural EEG demonstrated the beginning of late ictal discharges and postictal suppression in the right temporomesial areas. Late ictal SPET images showed marked right fronto-temporo-parietal hypoactivity. The interictal SPET study clearly showed right frontotemporal hypoactivity. These preliminary data suggest that 99mTc-bicisate shows late ictal/early postictal hypoactivity which might represent the primary change in neuronal metabolism rather than the secondary change in cerebral blood flow.

Adult↗
PubMed1994

Primary femoral shortening osteosynthesis in the management of comminuted supracondylar femoral fractures.

Supracondylar fractures are most often the result of high-energy vehicular trauma in the young population, and the result of low-energy injuries in the elderly. Therefore, these fractures are generally associated with comminution or osteoporosis. Anatomic reconstruction with stable medial buttressing is often not possible. Utilization of AO principles and techniques with anatomic reduction and internal fixation often fails to yield sufficient stability. We have developed a technique for the treatment of these distal femoral fractures which involves shortening by impaction. Rather than achieving anatomic reduction, the femur is shortened by telescoping the fragments to reestablish inherent stability of the bone prior to internal fixation. Over a 20-year period we treated 25 comminuted supracondylar femoral fractures with primary shortening osteosynthesis in 24 patients. Sixteen patients were available for review at an average follow-up of 10 years (range 4-24 years). Ninety-four percent of the patients demonstrated complete radiographic consolidation by 14 weeks after the operative intervention. One patient required secondary bone grafting and reached consolidation at 30 weeks. Primary cancellous grafting was utilized in only 25% of the patients. One-third of the patients in review were given a heel lift; the average shortening in these patients was 2.6 cm (range 2.1-5 cm). Two-thirds of the patients did not require a heel lift, the average shortening in this group of patients being 1.2 cm (range 0.8-2.0 cm). At follow-up the average range of motion of the knee was 114 degrees (range 90 degrees-130 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

The role of accuracy for early stages of the origin of life.

The role of accuracy in developing stages in the origin of life is investigated by mathematical models. Errors yield difficult stability problems in a description of a cooperative system with information-carriers (genes) and catalytic polymers (enzymes) that are related to each other. This problem is highly relevant for a proper understanding of the RNA world. The model that is treated takes two relevant error effects, which can be coupled, into account. One is the occurrence of parasites, inactive genes that are efficiently reproduced by the catalytic support of other polymers but which do not give rise to any catalytic activity. The other is an error propagation due to the fact that erroneous catalysts will provide an increased error level and thus more erroneous catalysts which may lead to still increasing error levels, eventually an error catastrophe.

Evolution, Chemical↗
PubMed1984

Lipid composition and protoplast-forming capacity of Streptomyces antibioticus.

The lipid and fatty acid composition of a strain of Streptomyces antibioticus has been studied as a function of culture age and glycine concentration in the growth medium. Under all conditions, the main polar lipids were phosphatidylethanolamine, cardiolipin and phosphomannoinositides in order of decreasing abundance; no ornithinolipids were detected. Acylglucoses and menaquinones were found among the nonpolar lipids. The main fatty acids present were anteiso 15:0 and anteiso 17:0. The lipid composition of the cells varied with the age of the culture, but no uniform pattern of variation was found in the cultures grown on different amounts of glycine. Among the cells harvested at the end of the exponential phase of growth, those grown on 2% glycine give the highest yield of protoplast formation. These cells were found to contain low amounts of nonpolar lipids and of phosphatidylethanolamine, and high proportions of anteiso fatty acids. We propose that the membrane bilayer of these cells, because of its peculiar lipid composition, is particularly stable and fluid. As a consequence, the yield and stability of derived protoplasts should be increased.

Fatty Acids↗
PubMed1999

[Fractures of the proximal humerus].

Proximal humeral fractures are common particularly in the elderly. The decision of the optimal treatment is dependent on many factors. On the one hand the biological age of the patient and the bone structure plays a key-role, on the other hand the living conditions and individual needs are of importance. Most fractures with minimal displacement respond satisfactorily to simple conservative treatment including short sling immobilisation and functional aftertreatment under supervision of the physiotherapist. Most recently there is a trend towards more aggressive surgical intervention with percutaneous insertion of cannulated screws also in the slightly displaced fracture situation. This protocol allows for earlier functional and less painful aftertreatment, less risk of displacement of the fracture fragments and better outcome. In severely unstable fractures with marked displacement of the fragments an operative stabilisation is advocated by most surgeons. Again there is a trend from plating towards cannulated screw fixation combined with tension absorbing (resorbable) sutures. In special cases which are described in detail a minimal invasive percutaneous screw technique with less stripping of bone and therefore preservation of the crucial blood supply of the humeral head is recommended. Instead of percutaneous pinning using K-wires only, cannulated screws are inserted today. Plating of proximal humerus joint fractures is the exception in our days, only the subcapital unstable fracture of the elderly would be an indication. LC-condylar plating seems to yield better stability than the conventional T-plate-system. In the most severe fractures of the proximal humerus (4-segment-fractures and dislocation fractures according to Neer, respectively C-2- and C-3-fractures according to the AO-classification) there is still controversy on the best management. Most authors prefer hemiarthroplasty in this situation whereas the other group of orthopaedic surgeons try open reduction and internal fixation particularly in the younger individuals. This stabilisation provides the orthopaedic surgeons with a formidable challenge and requires a lot of experience in this field. Also the understanding of the fracture morphology is needed for optimal results. In spite of good stabilisation techniques often partial or total humeral head necrosis occurs in the most severe fractures. Surprisingly enough results with reasonable function can be obtained even with partial avascular necrosis of the humeral head. A crucial part of the management is team work with the physiotherapist and an individual program for each fracture situation, depending on the stability of the fixation. Close contact between these two professions is of utmost importance. Finally it can be stated that the management of proximal humeral fractures is fairly standardised but it is always dependent on the experience and resources of the attending surgeon and must be tailored to the individual needs of the patient.

Humans↗
PubMed2004

Treatment strategies for proximal femur fractures in osteoporotic patients.

Fractures of the proximal end of the femur, together with vertebral fractures, are greatly exacerbated by osteoporosis and can be regarded as the most typical and most serious complications of this disease. The demand for prompt mobilisation with full loading of the affected limb, combined with a desire for the gentlest of treatments, becomes increasingly difficult to meet in ageing patients with advanced osteoporosis. The advantages of osteosynthesis in respect to these demands when operating on elderly patients with fractures do not apply due to the inability of the osteoporotic bone to hold the osteosynthetic components sufficiently until fracture healing occurs. This inability is related to the anatomy of the proximal end of the femur and its loading patterns. Under eccentric loading, high bending loads occur, leading to failure of the osteosynthetic anchorage at the center of the femoral head. This leads subsequently to stressful revision operations for the patient. The prosthetic replacement is a good option in cases of dislocated intracapsular fractures, but in cases of trochanteric fractures it is still debated. Therefore, it is vital for the trauma surgeon to have specific knowledge of the patient's bone quality in order to optimise the result of the preferred procedure. With reference to our own experimental research and a study of the current literature, this knowledge can be summarised as follows: the most stable anchorage for the implant is achieved by placing the implant through the midpoint of the femoral head (highest bone mineral density) or just below ("best backing"). Anchoring femoral head implants so that they are stable in rotation within the head-neck fragment will significantly raise their load bearing capacity. This is also true for intramedullary load bearing devices in trochanteric fractures. The distance between the load-bearing device in the femoral neck and the articular surface is inversely correlated to the stability at yield, as other studies have already shown. There seems to be a limit for a successful realisation of an osteosynthesis that lies at a femoral head bone mineral density of 250 mg/cm(3) calcium-hydroxyapatite (CaHAp). Nevertheless, high precision surgery in regard to fracture reduction and implant placement is a essential requirement for a successful osteosynthesis. Reproducible local measurements of bone mineral density and trabecular alterations, as well as quick screening methods, are very much desired by the authors.

Aged↗
PubMed1983

A simple quantitative method for the determination of 3-fluorotyrosine substitution in proteins.

A rapid quantitative method is described for determining 3-fluorotyrosine incorporation into proteins. Derivatives of tyrosine and 3-fluorotyrosine with o-phthalaldehyde are well separated from one another by a reverse-phase high-performance liquid chromatography system used for routine analyses of o-phthalaldehyde-amino acid derivatives. Since both amino acids are well resolved from all other derivatized amino acids, the method is useful for amino acid analyses of proteins. Determination of the fluorotyrosine content of proteins by this method involves a single separation step, is reproducible, and requires no corrections for stability or yield. Further, the o-phthalaldehyde derivatives of 5-fluorotryptophan, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine can also be resolved. The method may be generally applicable to fluorinated aromatic amino acid-labeled proteins that are studied structurally and dynamically by nuclear magnetic resonance.

Chromatography, High Pressure Liquid↗