Formation of n- and p-type regions in individual Si/SiO2 core/shell nanowires by ion beam doping.

A method for cross-sectional doping of individual Si/SiO2 core/shell nanowires (NWs) is presented. P and B atoms are laterally implanted at different depths in the Si core. The healing of the implantation-related damage together with the electrical activation of the dopants takes place via solid phase epitaxy driven by millisecond-range flash lamp annealing. Electrical measurements through a bevel formed along the NW enabled us to demonstrate the concurrent formation of n- and p-type regions in individual Si/SiO2 core/shell NWs. These results might pave the way for ion beam doping of nanostructured semiconductors produced by using either top-down or bottom-up approaches.

Nasally inhaled pulsating aerosols: lung, sinus and nose deposition.

OBJECTIVES: Topical delivery of drugs to the sinuses is challenging and requires also particular administration manoeuvres from the patient. This study was conducted to investigate 1) the delivery efficiency of a pulsating aerosol (Vibrent prototype device) to the sinuses and the nose, 2) the aerosol fraction that will deposit in the lungs and 3) potential differences regarding sinus and nasal deposition ratio when comparing aerosol administration during two different administration routes. METHODS: An open label deposition study in healthy volunteers was conducted using 99mTc-DTPA radiolabeled pulsating aerosols in comparison to nasal pump sprays. Deposition and retention of pulsating aerosols was assessed by gamma camera imaging during spontaneous nasal breathing and during closed soft palate administration. RESULTS: Aerosol administration during nasal breathing vs. application with closed soft plate results in significant lung, nasal and sinus deposition. No significant differences were observed for nasal clearance. In comparison, drug delivery using nasal pump sprays resulted in non-significant sinus, 100 % nasal and non-significant lung deposition. The clearance kinetics after nasal pump spray delivery was significantly accelerated. DISCUSSION: The standard application mode of pulsation aerosols with closed soft palate results in negligible lung deposition and therefore limits drug delivery to the nasal cavity only, minimizing unwanted side effects. Administration during spontaneous nasal breathing shows only 10% lung deposition, which is tolerable during drug administration. Relevant paranasal sinus deposition is noted during both application modes and clearance kinetics remains essentially unchanged. In contrast, nasal pump sprays do not show sinus drug delivery and nasal drug residence time is shortened. CONCLUSION: Pulsating aerosols offer advantageous topical nasal and sinus drug delivery options.

Structural and chemical evolution of Au-silica core-shell nanoparticles during 20 keV helium ion irradiation: a comparison between experiment and simulation.

Au-silica core-shell nanoparticles have been irradiated with 20 keV He+ ions up to a maximum fluence of 4.7 × 1017 ions/cm2. The nanoscale structural and crystallographic evolution induced by He+ ion irradiation was followed at various stages using Transmission Electron Microscopy (TEM). During irradiation satellite Au clusters are formed around the main Au core, which remained crystalline even after the maximum He+ ion fluence. The spherical silica shell deformed into a hemisphere due to He+ ion irradiation. Three dimensional Monte-Carlo simulations, based on the binary collision approximation, have been performed on stacked infinite layers and an individual particle. The stacked layers results show that the He+ beam interacts with most of the nanoparticle and Au migrates in the direction of beam incidence agreeing with experimental findings. The individual particle results match the experiment in terms of the volume which is sputtered away however additional mechanisms, not included in the simulations, are present in the experiment during the satellite formation and silica shell deformation. These results show the ability for 20 keV He+ ions to be used for the modification of nanostructures. Furthermore, these results contribute to a quantitative understanding of the dynamic evolution of materials observed using microscopy techniques based on He+ ions.

Dosimetry and toxicology of inhaled ultrafine particles.

Both epidemiological and toxicological studies indicate that inhalation and subsequent deposition of airborne particles into the lungs have adverse health effects. Recently, the ultrafine particle (UfP) fraction (diameter < 100 nm) has received particular attention, as their small size may lead to more toxic properties. In this study we summarize the current knowledge on the dosimetry of inhaled particles (including UfPs) with a focus on recent data on translocation of UfPs into secondary target organs (such as brain and heart) suggesting that the lifetime dose of ambient UfPs in secondary target organs is about 10(11) particles. Furthermore, we highlight the main pathways of particle induced toxicity and the reasons for the potentially higher toxicity of UfPs. Finally, we discuss recent evidence indicating that (BET) surface area is the single most relevant dose metric for the toxicity of UfPs, which has important implications for regulatory measures on the toxicity of ambient and engineered particles.

Dynamic In Vivo Chest X-ray Dark-Field Imaging in Mice.

X-ray grating interferometry is a powerful emerging tool in biomedical imaging, providing access to three complementary image modalities. In addition to the conventional attenuation modality, interferometry provides a phase modality, which visualizes soft tissue structures, and a dark-field modality, which relates to the number and size of sub-resolution scattering objects. A particularly strong dark-field signal originates from the alveoli or air sacs in the lung. Dark-field lung radiographs in animal models have already shown increased sensitivity in diagnosing lung diseases, such as lung cancer or emphysema, compared to conventional X-ray chest radiography. However, to date, X-ray dark-field lung imaging has either averaged information over several breaths or has been captured during a breath hold. In this paper, we demonstrate the first time-resolved dark-field imaging of a breath cycle in a mechanically ventilated mouse, in vivo, which was obtained using a grating interferometer. We achieved a time resolution of 0.1 s, visualizing the changes in the dark-field, phase, and attenuation images during inhalation and exhalation. These measurements show that the dark-field signal depends on the air volume and, hence, the alveolar dimensions of the lung. Conducting this type of scan with animal disease models would help to locate the optimum breath point for single-image diagnostic dark-field imaging and could indicate if the changes in the dark-field signal during breath provide a diagnostically useful complementary measure.

Particle clearance from the airways of subjects with bronchial hyperresponsiveness and with chronic obstructive pulmonary disease.

The aim of this study was to determine particle clearance and retention from non-alveolated airways of 14 healthy subjects (HS), 10 subjects with asymptomatic bronchial hyperresponsiveness (BHR), and 23 patients with chronic obstructive pulmonary disease (COPD). Monodisperse iron oxide particles of 1.6 micro m geometric and 3.5 micro m aerodynamic diameter labeled with (99m)Tc were delivered to the airways by inspiration of small aerosol boli into shallow volumetric lung depths. In each subject the penetration front depth of the aerosol boli was adjusted to 55% of the Fowler dead space of the airways. Particle deposition was enhanced by about 7 seconds of breath-holding after bolus inhalation. Retention of the particles in the airways during the 48 hours after their administration was assessed by measuring the decline in lung activity with a sensitive gamma counter. Particle deposition was not significantly different among study groups. Twenty-four hour particle retention in the airways was not different among study groups. Sixty-one percent of the particles were retained at 24 hours in HS, 58% in BHR, and 64% in COPD. However, subjects with BHR showed accelerated mucociliary clearance compared to healthy subjects, whereas clearance was retarded in COPD patients. This long-term particle retention in the airways has to be taken into account in aerosol toxicology risk assessment and aerosol therapy dose evaluation.

Distribution pattern of inhaled ultrafine gold particles in the rat lung.

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 microg/m3 (4 x 10(6)/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma-mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5-8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma-mass spectrometry analysis of the lung, 1945 +/- 57 ng (mean +/- SD) and 1512 +/- 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 +/- 67 ng and 96 +/- 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

The solution structure of the guanine nucleotide exchange domain of human elongation factor 1beta reveals a striking resemblance to that of EF-Ts from Escherichia coli.

BACKGROUND: In eukaryotic protein synthesis, the multi-subunit elongation factor 1 (EF-1) plays an important role in ensuring the fidelity and regulating the rate of translation. EF-1alpha, which transports the aminoacyl tRNA to the ribosome, is a member of the G-protein superfamily. EF-1beta regulates the activity of EF-1alpha by catalyzing the exchange of GDP for GTP and thereby regenerating the active form of EF-1alpha. The structure of the bacterial analog of EF-1alpha, EF-Tu has been solved in complex with its GDP exchange factor, EF-Ts. These structures indicate a mechanism for GDP-GTP exchange in prokaryotes. Although there is good sequence conservation between EF-1alpha and EF-Tu, there is essentially no sequence similarity between EF-1beta and EF-Ts. We wished to explore whether the prokaryotic exchange mechanism could shed any light on the mechanism of eukaryotic translation elongation. RESULTS: Here, we report the structure of the guanine-nucleotide exchange factor (GEF) domain of human EF-1beta (hEF-1beta, residues 135-224); hEF-1beta[135-224], determined by nuclear magnetic resonance spectroscopy. Sequence conservation analysis of the GEF domains of EF-1 subunits beta and delta from widely divergent organisms indicates that the most highly conserved residues are in two loop regions. Intriguingly, hEF-1beta[135-224] shares structural homology with the GEF domain of EF-Ts despite their different primary sequences. CONCLUSIONS: On the basis of both the structural homology between EF-Ts and hEF-1beta[135-224] and the sequence conservation analysis, we propose that the mechanism of guanine-nucleotide exchange in protein synthesis has been conserved in prokaryotes and eukaryotes. In particular, Tyr181 of hEF-1beta[135-224] appears to be analogous to Phe81 of Escherichia coli EF-Ts.

Elongation factor EF-1 alpha from Artemia is specifically labeled at Lys-63 by the guanine nucleotide analog pyridoxal-5'-diphospho-5'-guanosine.

We have synthesized the guanine nucleotide analog pyridoxal-5'-diphospho-5'-guanosine. This compound specifically modifies a single lysine residue in elongation factor 1 alpha from Artemia, indicating that this residue is in close contact with the reactive part of the guanine nucleotide analog. This result is discussed in terms of the structure of the nucleotide-binding domain of the factor.

The human leucine zipper-containing guanine-nucleotide exchange protein elongation factor-1 delta.

Copy-DNA clones containing the complete coding region of the human elongation factor-1 delta (EF-1 delta) mRNA have been isolated and characterized. We present the deduced amino acid sequence and observe in it a leucine zipper motif seen recently in EF-1 delta from Artemia and Xenopus laevis. The human EF-1 delta sequence shows a strong conservation in its C-terminal domain. The homology between the N-terminal domains of EF-1 delta proteins is low and almost exclusively limited to the leucine zipper motif.

The effect of insulin, serum and dexamethasone on mRNA levels for the insulin receptor in the human lymphoblastoic cell line IM-9.

The effect of insulin, serum and dexamethasone on mRNA levels in the insulin receptor in the human lymphoblastoic cell line IM-9 was examined. To this end, mRNA levels were quantitated by Northern blot analysis using a labeled cDNA probe for the insulin receptor. The presence of 0.1 microM dexamethasone in the medium had a strong stimulatory effect on mRNA levels in insulin receptor, suggesting the presence of a glucocorticoid inducible enhancer element near the insulin receptor gene. Also, the nature of the serum had an effect on insulin receptor mRNA levels, as cells maintained in 10% fetal calf serum had insulin receptor mRNA levels that were 40-50% of those found in IM-9 cells maintained in 1% newborn serum. Variations in insulin receptor mRNA levels led in each situation to concordant variations in insulin binding. Insulin levels of up to 1 microM had no effect on hybridizable insulin receptor mRNA levels making an insulin-induced feed-back mechanism on gene expression or mRNA stability unlikely.

The leucine-zipper in elongation factor EF-1 delta, a guanine-nucleotide exchange protein, is conserved in Artemia and Xenopus.

Elongation factor 1, a complex involved in protein biosynthesis, contains two guanine-nucleotide-exchange proteins EF-1 beta and EF-1 delta. The sequence of EF-1 delta of Artemia was determined with the purified protein. When compared to EF-1 delta from Xenopus, a high degree of identify (80%) was found in the C-terminal domains of the proteins, which contain the guanine-nucleotide-exchange activity. The N-terminal domains share only 23% of the amino acids at identical positions, and therefore they were further analysed for less obvious types of homology. To this end, a published approach for sequence analysis, which can detect peculiar amino acid patterns in proteins was applied. In this way, a weak albeit unmistakable similarity between the two EF-1 delta proteins was demonstrated in the region of the leucine-zippers, apart from the leucine repeat itself. Apparently, they display a common structural pattern in their N-terminal domains, which so far has been observed mainly in transcription factors.

Elongation factor 1 beta of artemia: localization of functional sites and homology to elongation factor 1 delta.

Elongation factor (EF)-1 beta, a 26 kDa protein, is the eukaryotic equivalent of bacterial EF-Ts, the nucleotide exchange factor in protein synthesis. EF-1 beta catalyzes the exchange of guanine nucleotides bound to EF-1 alpha; the latter protein is the eukaryotic equivalent of bacterial EF-Tu. Limited proteolytic cleavage studies on EF-1 beta lead to the following picture: the protein is composed of two domains, an aminoterminal and a carboxyterminal domain, connected to each other by a stretch of hydrophilic, charged amino acids situated in the middle of the molecule. The carboxyterminal domain supplies the catalytic site for the nucleotide exchange reaction, whereas the aminoterminal domain interacts with EF-1 gamma, the third component of elongation factor 1. The regulatory, serine phosphate residue, Ser-89, localized in the hydrophilic stretch of EF-1 beta, does not appear to be necessary for the basic exchange reaction. The fourth component of the high molecular weight elongation factor complex (EF-1H), named EF-1 delta or 28 K protein, is homologous to EF-1 beta and contains regions very similar to the carboxyterminal part. EF-1 delta was found to be active in the nucleotide exchange reaction.

Ribosomal proteins L7/L12 of Escherichia coli. Localization and possible molecular mechanism in translation.

Experiments were performed in order to determine the minimal requirement for the proteins L7/L12 in polyphenylalanine synthesis and elongation factor EF-G-dependent GTP hydrolysis. Via reconstitution, ribosomal particles were prepared containing variable amounts of L7/L12. The L7/L12 content of these particles was carefully determined by the use of 3H-labelled L7/L12 and by radioimmunoassay. The activity of the particles was determined as a function of the L7/L12 content. Our results show that only one dimer of L7/L12 is required for full activity in EF-G-dependent GTP hydrolysis. On the other hand, two L7/L12 dimers are required for polyphenylalanine synthesis. In addition, we have determined the relation between the number of L7/L12 stalks, as observed by electron microscopy, and the L7/L12 content of the 50 S particles. Our interpretation of these results is that each ribosomal particle possesses two L7/L12 binding sites, each site being involved in binding one dimer. Binding of L7/L12 dimer in one site gives rise to formation of the L7/L12 stalk, whereas binding in the other site has no effect on the number of visible stalks.

Stimulation of integrin receptors using a magnetic drag force device induces an intracellular free calcium response.

Mechanical loading of cells is of fundamental relevance in physiological processes and induces several functional responses in cells. Integrins, a family of adhesion receptors, which are responsible for the interaction with the extracellular matrix, may play a role in transmission of mechanical signals into cells. The osteogenic cell line U-2 OS expresses different integrin subunits which are uniformly distributed over the cell surface. We applied defined physical forces on individual integrin receptor subunits using paramagnetic microbeads coated with anti-integrin antibodies. Application of an inhomogeneous magnetic field consequently leads to a mechanical stress on the receptor. Intracellular Ca2+ increased when the alpha 2 or the beta 1 integrin subunits were stressed, whereas mechanical loading of the transferrin receptor had a significantly lower effect. This result indicates that forces specifically exerted to individual integrin receptors induce signal transduction pathways.

Phosphate-binding sequences in nucleotide-binding proteins.

In the three-dimensional model of adenylate kinase, the phosphate-binding site for AMP and ATP has been identified [Pai, E.F. et al. (1977) J. Mol. Biol. 114, 37--45]. In this region one can distinguish a sequence glycine XXXX glycinelysine. The same sequence is found in many other mononucleotide-binding proteins including elongation factors and oncogenic P21 proteins. Dinucleotide-binding proteins display a pyrophosphate-binding unit with a glycine pattern different from that of mononucleotide-binding proteins. It has been found that P21 ras protein possesses a strand motif typical for (pyro)phosphate binding of a mononucleotide. A single mutation at position 12 can confer oncogenic activity on the protein. Based on the assumption that amino acid residues which are critical for function are preferentially conserved, we predict from the sequence that glycine residue 15 rather than residue 12 is important for (pyro)phosphate binding.

Sequence homology between EF-1 alpha, the alpha-chain of elongation factor 1 from Artemia salina and elongation factor EF-Tu from Escherichia coli.

In the course of a structural analysis of the alpha-chain of elongation factor 1 from Artemia salina cysts, we present four amino acid sequences comprising together half of the polypeptide chain. A comparison of these sequences with the primary structure of elongation factor EF-Tu from Escherichia coli reveals a clear correspondence between the eukaryotic and prokaryotic protein throughout their polypeptide chains. The results support a basic conservation of the structure of the aminoacyl-tRNA carrying enzyme in evolution. The occurrence, in the eukaryotic factor, of several epsilon-trimethyllysine residues, is remarkable.

Primary structure of elongation factor 1 gamma from Artemia.

Complementary DNA corresponding to elongation factor 1 gamma, which forms a complex with EF-1 beta, has been cloned. A lambda gt11 cDNA library has been screened with an antiserum against EF-1 beta gamma. The derived amino acid sequence of EF-1 gamma corresponds to 429 amino acids excluding the initiator methionine, which is absent in the mature protein. About half of the protein was sequenced by direct protein sequence analysis. No clear homology with any other protein was found.

A bacterial clone carrying sequences coding for elongation factor EF-1 alpha from Artemia.

A bacterial cDNA clone was identified carrying one third of the nucleotides coding for elongation factor EF-1 alpha from the brine shrimp Artemia. The sequence of codons corresponds with the known sequence of amino acids of EF-1 alpha in the region involved.

Genes for elongation factor EF-1 alpha in the brine shrimp Artemia.

A plasmid carrying a cDNA sequence coding for elongation factor EF-1 alpha from Artemia was used to probe blots of mRNA and chromosomal DNA from Artemia. A messenger length for EF-1 alpha corresponding to 1850 nucleotides was found. Southern blots pointed to a limited number (1-4) of genes, coding for EF-1 alpha. From an Artemia gene library a recombinant phage was isolated, which contains genomic sequences of EF-1 alpha. S1-nuclease mapping indicated the presence of intervening sequences within this cloned gene.