bluuebunny/crossref_metadata_2025 huggingface dataset:
Flare Irradiance Spectral Model (FISM): Daily component algorithms and results
Authors: Chamberlin Phillip C. , Woods Thomas N. , Eparvier Francis G.
Published: July 2007

The solar photon output from the Sun was once thought to be constant but is now known to vary considerably over timescales from seconds during solar flares to years due to the solar cycle. This is especially true in the wavelengths shorter than 208 nm (Al ionization edge), where measurements and models of the solar irradiance at these short wavelengths are needed. The time‐variant solar irradiance drives physical changes in the Earth's atmosphere that can affect many things including GPS accuracy and satellite drag rates. The Flare Irradiance Spectral Model (FISM) is an empirical model that estimates the solar irradiance at wavelengths from 0.1 to 190 nm at 1 nm resolution with a time cadence of 60 s. This is a high enough temporal resolution to model variations due to solar flares, where few accurate measurements at these wavelengths exist, as well as the solar cycle and solar rotation variations. The modeling of the FISM daily component, including the solar irradiance variations due to the solar cycle and the solar rotation, is the topic of this paper. The daily component algorithms are given, and results and comparisons of the daily component demonstrate that the FISM estimations agree within the stated uncertainties to the various measurements of the solar VUV irradiance. A third type of event that causes sudden changes in the solar irradiance is solar flares. The FISM modeling of the variations in the solar irradiance due to solar flares is the topic of the second paper.
Coaccumulation of Calcium and β-Amyloid in the Thalamus after Transient Middle Cerebral Artery Occlusion in Rats
Authors: Mäkinen Susanna , van Groen Thomas , Clarke Jared , Thornell Anders , Corbett Dale , Hiltunen Mikko , Soininen Hilkka , Jolkkonen Jukka
Published: July 2007

Transient occlusion of the middle cerebral artery (MCAO) in rats leads to abnormal accumulation of β-amyloid (Aβ) peptides in the thalamus. This study investigated the chemical composition of these deposits. Adult male human β-amyloid precursor protein (APP) overexpressing ( hAPP695) rats and their wild-type littermates were subjected to transient MCAO for 2 h or sham operation. After 26-week survival time, histological examination revealed an overlapping distribution pattern for rodent and human Aβ in the thalamus of hAPP695 rats subjected to MCAO. X-ray microanalysis showed that the deposits did not contain significant amount of iron, zinc, or copper typical to senile plaques. In contrast, the deposit both in hAPP695 and non-transgenic rats contained calcium and phosphorus in a ratio (1.28 ± 0.15) characteristic to hydroxyapatites. Alizarin red staining confirmed that calcium coaccumulated in these Aβ deposits. It is suggested that APP expression is induced by ischemic insult in cortical neurons adjacent to infarct, which in turn is reflected as increased release of Aβ peptides by their corticothalamic axon endings. This together with insufficient clearance or atypical degradation of Aβ peptides lead to dysregulation of calcium homeostatis and coaccumulation in the thalamus.
Noninvasive Estimation of Normalized Distribution Volume: Application to the Muscarinic-2 Ligand [18F]FP-TZTP
Authors: Ichise Masanori , Cohen Robert M , Carson Richard E
Published: July 2007

Reference tissue methods to estimate neuroreceptor binding are not applicable to [18F]FP-TZTP (a muscarinic-2 cholinergic receptor ligand), because there is no suitable receptor-free reference region. We evaluated a new method to estimate, without using arterial data or a receptor-free reference region, a receptor parameter called the normalized distribution volume, V*T, using a region containing receptors as the input tissue. V*T is defined as VT /K′1 (distribution volume ( VT) normalized by K′1 of the input region). We used a two-parameter multilinear reference tissue model (MRTM2) to generate parametric images of V*T and R1 ( R1 = K1/ K′1) from [18F]FP-TZTP PET data of healthy aged subjects (10 with apolipoprotein E-ε4 alleles (APOE-ε4(+)) and nine without (APOE-ε4(–)). VT and VT were normalized by plasma-free fraction, fP. By one-tissue kinetic analysis (1TKA) with metabolite-corrected plasma data, VT was previously reported as higher in the APOE-ε4(+) group. The noise magnitude of MRTM2 V*T and R1 images were nearly identical to those of 1TKA VT and K1 images. K′1 or fP was not different between the two groups. V*T (mins) (1,659 ± 497) and VT (mL/cm3) (701 ± 99) in APOE-ε4(+) were higher by 38 and 22% than those (1,209 ± 233 and 577 ± 112) in APOE-ε4(–), respectively. The statistical significance for V*T (0.041) was lower than that for VT (0.025), due to the higher intersubject variability of V*T (25%) than that of VT (17%). We conclude that MRTM2 V*T allows detection of group differences in receptor binding without arterial blood or a receptor-free reference region.
Cystic fibrosis transmembrane conductance regulator (CFTR) functionality is dependent on coatomer protein I (COPI)
Authors: Yu Ying , Platoshyn Oleksandr , Safrina Olga , Tsigelny Igor , Yuan Jason X.‐J. , Keller Steven H.
Published: July 2007

Background information. Cystic fibrosis results from mutations in the ABC transporter CFTR (cystic fibrosis transmembrane conductance regulator), which functions as a cAMP‐regulated anion channel. The most prevalent mutation in CFTR, the Phe508 deletion, results in the generation of a trafficking and functionally deficient channel. The cellular machineries involved in modulating CFTR trafficking and function have not been fully characterized. In the present study, we identified a role for the COPI (coatomer protein I) cellular trafficking machinery in the development of the CFTR polypeptide into a functional chloride channel. To examine the role of COPI in CFTR biosynthesis, we employed the cell line ldlF, which harbours a temperature‐sensitive mutation in ε‐COP, a COPI subunit, to inhibit COPI function and then determined whether the CFTR polypeptide produced from the transfected gene developed into a cAMP‐regulated chloride channel.

Results. When COPI was inactivated in the ldlF cells by an elevated temperature pulse (39°C), the CFTR polypeptide was detected on the cell surface by immunofluorescence microscopy and cell‐surface biotinylation. Therefore, CFTR proceeded upstream in the secretory pathway in the absence of COPI function, a result demonstrated previously by others. In contrast, electrophysiological measurements indicated an absence of cAMP‐stimulated chloride efflux, suggesting that channel function was impaired. In comparison, expression of CFTR at the same elevated temperature (39°C) in an ε‐COP‐rescued cell line [ldlF(ldlF)], which has an introduced wild‐type ε‐COP gene in addition to the mutant ε‐COP gene, showed restoration of cAMP‐stimulated channel activity, confirming the requirement of COPI for channel function.

Conclusions. These results therefore suggest that generation of the folded‐functional conformation of CFTR requires COPI.
Morphological effects of transcardially perfused SDS on the rat brain
Authors: Kovács Betti , Bukovics Péter , Gallyas Ferenc
Published: July 2007

Background information. For explanation of the formation of ‘dark’ neurons, an enigmatic phenomenon in neuropathology, we hypothesized recently that all spaces between the ultrastructural elements visible in the traditional transmission electron microscope are filled with a gel structure that stores free energy in the form of non‐covalent interactions, is continuous in the whole soma—dendrite domains of neurons, and is capable of whole‐cell phase transition. This hypothesis was deduced from the fact that ‘dark’ neurons can be formed, even under conditions extremely unfavourable for enzyme‐mediated biochemical processes, if initiated by a physical damage. In order to gain further information on this gel structure, we perfused transcardially rats for 5 min with physiological saline containing 1 mM SDS before the perfusion of a fixative for electron microscopy.

Results. Dramatic compaction of visibly intact ultrastructural elements was caused in the whole soma—dendrite domains of thinly scattered neurons (‘dark’ neurons), whereas substantial cytoplasmic swelling and patchy ultrastructural disintegration occurred in numerous other neurons (‘light’ neurons). Similar morphological changes were observed in scattered astrocytes, oligodendrocytes, pericytes and endothelial cells.

Conclusions. These observations: (i) support the existence of the above intracellular gel structure in neurons; (ii) allow the conclusion that this gel structure is present in the form of an ubiquitous trabecular network surrounded by a confluent system of fluid cytoplasm; (iii) draw attention to the possibility that the previous two statements also apply to other cell types of the brain tissue; and (iv) suggest that pressure‐induced direct channels exist between neurons and astrocytes.
Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells
Authors: Yu Jinhua , Wang Yijing , Deng Zhihong , Tang Liang , Li Yuanfei , Shi Junnan , Jin Yan
Published: July 2007

Background information. Although adult bone‐marrow‐derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non‐dental stem‐cell‐mediated odontogenesis remain an open question.

Results. STRO‐1+ (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic‐activated cell‐sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2‐day‐old rat incisors. Co‐cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up‐regulated alkaline phosphatase activity, cell‐cycle modification, and the expression of tooth‐specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth‐shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin—pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin‐like structures and fibrous connective tissues respectively.

Conclusions. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non‐dental stem cell populations.
Regulation of matrix turnover: fibroblasts, forces, factors and fibrosis
Authors: Laurent G.J. , Chambers R.C. , Hill M.R. , McAnulty R.J.
Published: July 2007

Fibroblasts are multifunctional cells that are responsible for matrix homoeostasis, continuously synthesizing and degrading a diverse group of extracellular molecules and their receptors. Rates of turnover of matrix molecules and the proteases that degrade them are normally under the control of diverse chemical and mechanical cues, with cytokines, growth factors, proteases, lipid mediators and mechanical forces playing roles. The maintenance of this homoeostasis is vital to the preservation of normal tissue function and is clearly lost in chronic diseases of the joints, skin and internal organs where destruction and excessive deposition is seen. Current research is focusing on defining the key pathways of activation either in resident fibroblasts, matrix-producing cells derived from circulating fibrocytes, or from transdifferentiation of resident cells. The common downstream signalling pathways are also being defined, as well as the gene interactions leading to altered cell phenotype. The present article reviews these findings and our current concepts of the key molecular events leading to tissue damage and excessive matrix deposition in tissue fibrosis. These studies are leading to an appreciation of the complexity of events with multiple pathways involved, but, as the facts emerge, we are finding promising new ways to treat fibrosis and halt the inexorable progression that is a feature of so many fibrotic and remodelling disorders.
Matrix synthesis and degradation in human intervertebral disc degeneration
Authors: Le Maitre C.L. , Pockert A. , Buttle D.J. , Freemont A.J. , Hoyland J.A.
Published: July 2007

Degeneration of the intervertebral disc has been implicated in chronic low back pain. Type II collagen and proteoglycan (predominantly aggrecan) content is crucial to proper disc function, particularly in the nucleus pulposus. In degeneration, synthesis of matrix molecules changes, leading to an increase in the synthesis of collagens type I and III and a decreased production of aggrecan. Linked to this is an increased expression of matrix-degrading molecules including MMPs (matrix metalloproteinases) and the aggrecanases, ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) 1, 4, 5, 9 and 15, all of which are produced by native disc cells. Importantly, we have found that there is a net increase in these molecules, over their natural inhibitors [TIMP-1 (tissue inhibitor of metalloproteinases-1), 2 and 3], suggesting a deregulation of the normal homoeostatic mechanism. Growth factors and cytokines [particularly TNFα (tumour necrosis factor α) and IL-1 (interleukin 1)] have been implicated in the regulation of this catabolic process. Our work has shown that in degenerate discs there is an increase in IL-1, but no corresponding increase in the inhibitor IL-1 receptor antagonist. Furthermore, treatment of human disc cells with IL-1 leads to a decrease in matrix gene expression and increased MMP and ADAMTS expression. Inhibition of IL-1 would therefore be an important therapeutic target for preventing/reversing disc degeneration.
Brain matrix: structure, turnover and necessity
Authors: Rauch U.
Published: July 2007

Aided by mice with multiple deleted brain matrix protein genes, the biochemical analysis of mouse brain matrix molecules indicates a constitutive production of more proteoglycans than can be integrated in multimolecular matrix structures. Possible functions of non-matrix integrated proteoglycans, and aspects of incomplete compensatory mechanisms in knockout mice are discussed.
TGF-β, Smad3 and the process of progressive fibrosis
Authors: Gauldie J. , Bonniaud P. , Sime P. , Ask K. , Kolb M.
Published: July 2007

Transient adenovirus-mediated gene transfer of active TGF-β1 (transforming growth factor-β1) induces severe and progressive fibrosis in rodent lung without apparent inflammation. Alternatively, transfer of IL-1β (interleukin 1β) induces marked tissue injury and inflammation, which develops into progressive fibrosis, associated with an increase in TGF-β1 concentrations in lung fluid and tissue. Both vector treatments induce a fibrotic response involving myofibroblasts and progressive matrix deposition starting at the peri-bronchial site of expression and extending over days to involve the entire lung and pleural surface. Administration of the TGF-β1 vector to the pleural space induces progressive pleural fibrosis, which minimally extends into the lung parenchyma. The mechanisms involved in progressive fibrosis need to account for the limitation of fibrosis to specific organs (lung fibrosis and not liver fibrosis or vice versa) and the lack of effect of anti-inflammatory treatments in regulating progressive fibrosis. TGF-β1 is a key cytokine in the process of fibrogenesis, using intracellular signalling pathways involving the ALK5 receptor and signalling molecules Smad2 and Smad3. Transient gene transfer of either TGF-β1 or IL-1β to Smad3-null mouse lung provides little evidence of progressive fibrosis and no fibrogenesis-associated genes are induced. These results suggest that mechanisms of progressive fibrosis involve factors presented within the context of the matrix that define the microenvironment for progressive matrix deposition.
HtrA1: a novel regulator of physiological and pathological matrix mineralization?
Authors: Canfield A.E. , Hadfield K.D. , Rock C.F. , Wylie E.C. , Wilkinson F.L.
Published: July 2007

HtrA1 (high-temperature requirement protein A1) is a secreted multidomain protein with proven serine protease activity and the ability to regulate TGF-β (transforming growth factor-β)/BMP (bone morphogenetic protein) signalling. There is increasing evidence that HtrA1 regulates several pathological processes, including tumour development, Alzheimer's disease, age-related macular degeneration and osteoarthritis, although the mechanism(s) by which it regulates these processes have not been fully elucidated. Using overexpression and knock-down strategies, we have evidence demonstrating that HtrA1 is also a key regulator of physiological and pathological matrix mineralization in vitro. We propose that HtrA1 regulates mineralization by inhibiting TGF-β/BMP signalling and/or by cleaving specific matrix proteins, including decorin and MGP (matrix Gla protein). Taken together, these studies suggest that HtrA1 may be a novel therapeutic target for several diseases.
F-actin cytoskeletal organization in intervertebral disc health and disease
Authors: Li S. , Duance V.C. , Blain E.J.
Published: July 2007

The cytoskeleton, which in most cell types, including the intervertebral disc described here, comprises microfilaments, microtubules and intermediate filaments, plays important functions in many fundamental cellular events, including cell division, motility, protein trafficking and secretion. The cytoskeleton is also critical for communication; for example, alterations to the architecture of the F-actin (filamentous actin) cytoskeletal networks can affect communication between the cells and the extracellular matrix, potentially compromising tissue homoeostasis. Although there are limited studies to date, this paper aims to review current knowledge on F-actin cytoskeletal element organization in intervertebral disc cells, how F-actin differs with pathology and its implications for mechanotransduction.
Astrocyte–leucocyte interactions and the mechanisms regulating matrix degradation in CNS tuberculosis
Authors: Green J.A. , Friedland J.S.
Published: July 2007

The CNS (central nervous system) has a unique pattern of immune response to infection. TB (tuberculosis) of the CNS is devastating with widespread tissue destruction. In TB, astrocyte–leucocyte interactions are key in regulating MMP (matrix metalloproteinase) activity and are regulated by complex signalling pathways. A synergistic interaction between interferon γ and monocyte-derived mediators drives high-level astrocyte MMP-9 secretion; this and other networking effects are inhibited by steroids. Better understanding of regulatory mechanisms may identify potential switch points that could be future therapeutic targets.
Bone marrow stromal cells promote growth and survival of prostate cancer cells
Authors: Cross N.A. , Papageorgiou M. , Eaton C.L.
Published: July 2007

Prostate cancers frequently metastasize to the skeleton, and it has been hypothesized that this environment selectively supports the growth of these tumours. Specifically there is strong evidence that interactions between tumour cells and BMSCs (bone marrow stromal cells) play a major role in supporting prostate cancer growth and survival in bone. Here, we examine factors shown to be secreted by BMSCs, such as IGFs (insulin-like growth factors) and IL-6 (interleukin 6), shown to promote prostate cancer cell proliferation and to potentially replace the requirement for androgens. In addition we discuss another factor produced by BMSCs, osteoprotegerin, which may promote tumour cell survival by suppressing the biological activity of the pro-apoptotic ligand TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand).
Structural basis of natural ligand binding and activation of the Class II G-protein-coupled secretin receptor
Authors: Miller L.J. , Dong M. , Harikumar K.G. , Gao F.
Published: July 2007

The secretin receptor is prototypic of Class II GPCRs (G-protein-coupled receptors), based on its structural and functional characteristics and those of its natural agonist ligand. Secretin represents a linear 27-residue peptide with diffuse pharmacophoric domain. The secretin receptor includes the typical signature sequences for this receptor family within its predicted transmembrane segments and the highly conserved six cysteine residues contributing to three intradomain disulfide bonds within its long N-terminus. This domain is critical for secretin binding based on receptor mutagenesis and photoaffinity labelling studies. Full agonist analogues of secretin incorporating a photolabile moiety at various positions throughout the pharmacophore covalently label residues within this region, while only N-terminal probes have labelled the core helical bundle domain. Combining insights coming from receptor structural studies, peptide structure–activity relationship considerations, photoaffinity labelling, and application of fluorescence techniques has resulted in the development of a working model of the secretin–receptor complex. This supports the initial docking of the peptide agonist within a cleft in the receptor N-terminus, providing the opportunity for an endogenous sequence within that domain to interact with the core of the receptor. This interaction is believed to be key in the molecular basis of conformational change associated with activation of this receptor. The site of action of this endogenous agonist could also provide a possible target for small molecule agonists to act.
Structural characterization of the parathyroid hormone receptor domains determinant for ligand binding
Authors: Mierke D.F. , Mao L. , Pellegrini M. , Piserchio A. , Plati J. , Tsomaia N.
Published: July 2007

Over the years, the association of peptide ligands to Family B GPCRs (G-protein coupled receptors) has been characterized by a number of experimental and theoretical techniques. For the PTH (parathyroid hormone) ligand–receptor system, important insight has been provided by photoaffinity labelling experiments and the elucidation of direct contact points between ligand and receptor. Our research has focused on the structural elucidation of the receptor domains shown to be involved in the binding of PTH. Employing a combination of carefully designed receptor domains, solution-state NMR carried out in the presence of membrane mimetics and extensive computer simulations, we have obtained a well-resolved model of the ligand–receptor complex for PTH. Here, we review the development of this model and highlight some inherent limitations of the methods employed and their consequences on interpretation of the ligand–receptor model.
Agonist selectivity in the oxytocin/vasopressin receptor family: new insights and challenges
Authors: Chini B. , Manning M.
Published: July 2007

The design and development of selective agonists acting at the OT (oxytocin)/AVP (vasopressin) receptors has been and continues to be a difficult task because of the great similarity among the different receptor subtypes as well as the high degree of chemical similarity between the active ligands. In recent decades, at least a thousand synthetic peptides have been synthesized and examined for their ability to bind to and activate the different OT/AVP receptors; an effort that has led to the identification of several receptor subtype-selective agonists in the rat. However, owing to species differences between rat and human AVP/OT receptors, these peptides do not exhibit the same selectivities in human receptor assays. Furthermore, the discovery of receptor promiscuity, which is the ability of a single receptor subtype to couple to several different G-proteins, has led to the definition of a completely new class of compounds, referred to here as coupling-selective ligands, which may activate, within a single receptor subtype, only a specific signalling pathway. Finally, the accumulating evidence that GPCRs (G-protein-coupled receptors) do not function as monomers, but as dimers/oligomers, opens up the design of another class of specific ligands, bivalent ligands, in which two agonist and/or antagonist moieties are joined by a spacer of the appropriate length to allow the simultaneous binding at the two subunits within the dimer. The pharmacological properties and selectivity profiles of these bivalent ligands, which remain to be investigated, could lead to highly novel research tools and potential therapeutic agents.
Phenotypic classification of mutants: a tool for understanding ligand binding and activation of muscarinic acetylcholine receptors
Authors: Hulme E.C. , Bee M.S. , Goodwin J.A.
Published: July 2007

GPCRs (G-protein-coupled receptors) such as the M1 muscarinic receptor have so far proved recalcitrant to direct structure determination. Nevertheless systematic mutagenesis, particularly alanine scanning, has advanced our understanding of their structure–function relationships. GPCRs exhibit multiple conformational states with different affinities for and abilities to activate their cognate G-proteins. Ligand binding alters these conformational equilibria, thus promoting or inhibiting signalling. Alanine-scanning mutagenesis probes the relative contributions of a particular amino acid side chain to the stability of the ground and activated states of the receptor and its complexes. These determine the phenotype of the mutant receptor. Classification of the phenotypes suggests functional roles for particular amino acid side chains, allowing us to group them accordingly. From a rhodopsin-based homology model of the M1 mAChR, a coherent view emerges of how these clusters of residues function in ligand anchoring, transduction of binding energy, global structural stabilization and selective stabilization of the ground state or the activated state of the receptor. We can identify differences in ligand-binding modes, and suggest inter- and intra-molecular interactions that are weakened or broken, or formed or intensified during acetylcholine-induced activation. In due course, we may be able to extend these insights to activation by unconventional agonists.
Assessing GPCR activation using protein complementation: a novel technique for HTS
Authors: Eglen R.M.
Published: July 2007

GPCRs (G-protein-coupled receptors) are critical targets in drug discovery. Although most HTS (high-throughput screening) assays are routinely used to identify functional agonism or antagonism, they are suboptimal as methods to screen for modulators of other, novel, aspects of GPCR function. Indeed, it is now evident that GPCRs are highly complex proteins that interact with RAMPs (receptor-activity-modifying partners), β-arrestins, G-proteins, as well as functioning in potential homo- or hetero-meric complexes. Consequently, novel HTS technologies are now required that would facilitate interrogation of GPCRs in terms of their cellular protein–protein interactions. One approach is oligomerization-assisted complementation of monomeric protein fragments and detection of fragment reassembly. Notably, the use of enzymes has advantages in this regard, since complementation results in catalytically competent protein. The assay signal generated in this fashion results in assays of high sensitivity, thereby enabling protocols to be developed in HTS systems that require extremely low fluid volumes. The use of complementing proteins that generate a luminescent signal also provides assays that are markedly free from artefactual interferences.
Unravelling the mechanisms underpinning chemokine receptor activation and blockade by small molecules: a fine line between agonism and antagonism?
Authors: Wise E. , Pease J.E.
Published: July 2007

Chemokines are a family of small basic proteins which induce the directed migration of cells, notably leucocytes, by binding to specific GPCRs (G-protein-coupled receptors). Both chemokines and their receptors have been implicated in a host of clinically important diseases, leading to the notion that antagonism of the chemokine–chemokine receptor network may be therapeutically advantageous. Consequently, considerable effort has been put into the development of small-molecule antagonists of chemokine receptors and several such compounds have been described in the literature. One curious by-product of this activity has been the description of several small-molecule agonists of the receptors, which are typically discovered following the optimization of lead antagonists. In this review we discuss these findings and conclude that these small-molecule agonists might be exploited to further our understanding of the molecular mechanisms by which chemokine receptors are activated.
Neurotensin receptor type 1: Escherichia coli expression, purification, characterization and biophysical study
Authors: Harding P.J. , Attrill H. , Ross S. , Koeppe J.R. , Kapanidis A.N. , Watts A.
Published: July 2007

NT (neurotensin) is an endogenous tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. One receptor for NT, NTS1, belongs to the GPCR (G-protein-coupled receptor) superfamily, has seven putative transmembrane domains, and is being studied by a range of single-molecule, functional and structural approaches. To enable biophysical characterization, sufficient quantities of the receptor need to be expressed and purified in an active form. To this end, rat NTS1 has been expressed in Escherichia coli in an active ligand-binding form at the cell membrane and purified in sufficient amounts for structural biology studies either with or without fluorescent protein [YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein)] fusions. Ligand binding has been demonstrated in a novel SPR (surface plasmon resonance) approach, as well as by conventional radioligand binding measurements. These improvements in production of NTS1 now open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.
Applications of cell-penetrating peptides in regulation of gene expression
Authors: Järver P. , Langel K. , El-Andaloussi S. , Langel Ü.
Published: July 2007

CPPs (cell-penetrating peptides) can be defined as short peptides that are able to efficiently penetrate cellular lipid bilayers. Because of this remarkable feature, they are excellent candidates regarding alterations in gene expression. CPPs have been utilized in in vivo and in vitro experiments as delivery vectors for different bioactive cargoes. This review focuses on the experiments performed in recent years where CPPs have been used as vectors for multiple effectors of gene expression such as oligonucleotides for antisense, siRNA (small interfering RNA) and decoy dsDNA (double-stranded DNA) applications, and as transfection agents for plasmid delivery.
Tumour-homing peptides: tools for targeting, imaging and destruction
Authors: Enbäck J. , Laakkonen P.
Published: July 2007

Each normal organ and pathological condition contains organ- or disease-specific molecular tags on its vasculature that constitute a vascular ‘zip code’ system. Tissue-selective tumour metastasis may also depend on vascular addresses. We have used phage display peptide libraries to map disease-specific differences in the vasculature. By using this technology, we have isolated several peptides which are targeted specifically to tumour blood vessels, lymphatic vessels and/or tumour cells. Some of the tumour-homing peptides recognize common angiogenesis markers and are capable of binding to several types of tumour, whereas other peptides recognize tumour-type-specific differences. We have also shown that the vasculature of a pre-malignant lesion differs from that of a full-blown tumour and also from the vasculature of the corresponding normal organ. Our peptides have revealed molecules that act as novel biomarkers of this vascular heterogeneity. Interestingly, some of our homing peptides are able to penetrate the target cells. This internalization differs from that of the Tat, penetratins and other related peptides in that our peptides enter the cell in a cell-type-specific manner. These peptides appear to be able to concentrate in the target tissue, making them particularly efficient delivery vectors for the targeting of drugs, other therapeutic moieties and imaging agents.
Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells
Authors: Poon G.M.K. , Gariépy J.
Published: July 2007

Polycationic macromolecules and cationic peptides acting as PTDs (protein transduction domains) and CPPs (cell-penetrating peptides) represent important classes of agents used for the import and delivery of a wide range of molecular cargoes into cells. Their entry into cells is typically initiated through interaction with cell-surface HS (heparan sulfate) molecules via electrostatic interactions, followed by endocytosis of the resulting complexes. However, the endocytic mechanism employed (clathrin-mediated endocytosis, caveolar uptake or macropinocytosis), defining the migration of these peptides into cells, depends on parameters such as the nature of the cationic agent itself and complex formation with cargo, as well as the nature and distribution of proteoglycans expressed on the cell surface. Moreover, a survey of the literature suggests that endocytic pathways should not be considered as mutually exclusive, as more than one entry mechanism may be operational for a given cationic complex in a particular cell type. Specifically, the observed import may best be explained by the distribution and uptake of cell-surface HSPGs (heparan sulfate proteoglycans), such as syndecans and glypicans, which have been shown to mediate the uptake of many ligands besides cationic polymers. A brief overview of the roles of HSPGs in ligand internalization is presented, as well as mechanistic hypotheses based on the known properties of these cell-surface markers. The identification and investigation of interactions made by glycosaminoglycans and core proteins of HSPGs with PTDs and cationic polymers will be crucial in defining their uptake by cells.
Protein transduction: identification, characterization and optimization
Authors: Tilstra J. , Rehman K.K. , Hennon T. , Plevy S.E. , Clemens P. , Robbins P.D.
Published: July 2007

Protein transduction domains (PTDs), both naturally occurring and synthetic, have been increasingly employed to deliver biologically active agents to a variety of cell types in vitro and in vivo. In addition to the previously characterized arginine-rich PTDs, including Tat (transactivator of transcription), Antp (Antennapedia) and PTD-5, we have demonstrated that lysine and ornithine, as well as arginine, homopolymers are able to mediate transduction of a wide variety of agents. To screen for optimal PTDs, we have used as a therapeutic cargo a peptide derived from IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} β, able to bind to the IKK regulatory subunit [NEMO (NF-κB essential modulator)], preventing formation of an active kinase complex. This peptide, termed NBD, is able to block activation of NF-κB, but not basal activity. We demonstrate that PTD-mediated delivery of NBD using certain PTDs, in particular 8K (octalysine), is therapeutic following systemic delivery in murine models of inflammatory bowel disease, diabetes and muscular dystrophy. In addition, we have developed a peptide phage display library screening method for novel transduction peptides able to facilitate tissue-specific internalization of marker protein complexes. Using this approach, we have identified transduction peptides that are able to facilitate internalization of large protein complexes into tumours, airway epithelia, synovial fibroblasts, cardiac tissue and HEK-293 (human embryonic kidney) cells in culture and/or in vivo.
Tatp-mediated intracellular delivery of pharmaceutical nanocarriers
Authors: Torchilin V.P.
Published: July 2007

CPPs (cell-penetrating peptides), including Tatp (transactivator of transcription peptide), have been successfully used for intracellular delivery of a wide variety of cargoes including various nanoparticulate pharmaceutical carriers such as liposomes, micelles and nanoparticles. Here, we will consider the major results obtained in this area with emphasis on Tatp-mediated delivery of liposomes and various transfection vectors. We will also address the development of ‘smart’ stimuli-sensitive nanocarriers, where the cell-penetrating function can only be activated when the nanocarrier is inside the biological target, thus minimizing the interaction with non-target cells.
Cell-penetrating peptides in drug development: enabling intracellular targets
Authors: Chen L. , Harrison S.D.
Published: July 2007

A large body of literature has shown that CPPs (cell-penetrating peptides) are capable of carrying macromolecules across the plasma membrane. CPPs can penetrate a wide variety of tissue types and enable modulation of intracellular targets with molecules that, by themselves, are incapable of penetrating cells. As a result, CPPs are recognized for their potential value in validating intracellular targets that could lead to drug discovery programmes [Dietz and Bahr (2004) Mol. Cell Neurosci. 27, 85–131]. The potential for CPP–drug conjugates to be used as human therapeutic agents has not been extensively explored and there is limited knowledge regarding the characteristics of CPPs which are necessary for drug development. A better understanding of the properties of CPPs relating to in vivo pharmacology, pharmacokinetics, pharmacodynamics and safety will continue to inform and encourage novel drug development efforts using CPPs as therapeutics. Here we will discuss areas of interest for drug development of CPP-conjugated compounds.
Cell-penetrating peptide–morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo
Authors: Moulton H.M. , Fletcher S. , Neuman B.W. , McClorey G. , Stein D.A. , Abes S. , Wilton S.D. , Buchmeier M.J. , Lebleu B. , Iversen P.L.
Published: July 2007

The cellular uptake of PMOs (phosphorodiamidate morpholino oligomers) can be enhanced by their conjugation to arginine-rich CPPs (cell-penetrating peptides). Here, we discuss our recent findings regarding (R-Ahx-R)4AhxB (Ahx is 6-aminohexanoic acid and B is β-alanine) CPP–PMO conjugates in DMD (Duchenne muscular dystrophy) and murine coronavirus research. An (R-Ahx-R)4AhxB–PMO conjugate was the most effective compound in inducing the correction of mutant dystrophin transcripts in myoblasts derived from a canine model of DMD. Similarly, normal levels of dystrophin expression were restored in the diaphragms of mdx mice, with treatment starting at the neonatal stage, and protein was still detecTable 22 weeks after the last dose of an (R-Ahx-R)4AhxB–PMO conjugate. Effects of length, linkage and carbohydrate modification of this CPP on the delivery of a PMO were investigated in a coronavirus mouse model. An (R-Ahx-R)4AhxB–PMO conjugate effectively inhibited viral replication, in comparison with other peptides conjugated to the same PMO. Shortening the CPP length, modifying it with a mannosylated serine moiety or replacing it with the R9F2 CPP significantly decreased the efficacy of the resulting PPMO (CPP–PMO conjugate). We attribute the success of this CPP to its stability in serum and its capacity to transport PMO to RNA targets in a manner superior to that of poly-arginine CPPs.
Generation, application and quantification of clamp–BioShuttle carriers for plasmid delivery into nuclei of prostate cancer cells
Authors: Pipkorn R. , Waldeck W. , Jenne J.W. , Didinger B. , Braun K.
Published: July 2007

This paper presents the BioShuttle platform as a delivery vehicle for transfer of contrast agents and genetic material into target cells, which can be followed by activation of the BioShuttle inside the target cell. Here, we present a transporter system and summarize the findings on transporter use in vivo and in vitro. The results here are limited to examples where cargoes (drugs, genetically active materials or contrast agents) are covalently associated with the transporter module. A further example, in which the cargo is non-covalently attached to the BioShuttle, is also discussed. Finally, attempts have been made to solve some of the issues surrounding the efficiency of transfer of therapeutic or diagnostic agents and their later activity in the cell.
Kilovolt radiation from a laser-produced Al plasma
Authors: Cobble J. A. , Tierney T. E. , Abdallah J.
Published: July 2007

We have characterized kilo-electron-volt (keV) emission from Al plasmas for various laser illuminations at the OMEGA laser with the goal of optimizing the ability to backlight low-atomic-number materials such as beryllium for fusion ignition studies. The plasma is diagnosed by spectral-measurement comparisons to detailed theoretical atomic physics models. It is found that a significant fraction of the radiation is due to the x-ray continuum, that the electron temperature Te depends weakly on laser energy and power, and that the conversion efficiency to Lyman α (Lα, the N=2→1 transition in H-like Al) at 1.73 keV is reduced as laser energy is increased. As the number of beams is increased, the extra laser energy goes into a larger, higher-density plasma in which He-like and H-like ions are more effectively ionized.
Laser light-stimulated exoelectron emission from solid Ar pre-irradiated by an electron beam
Authors: Gumenchuk G. B. , Belov A. G. , Savchenko E. V. , Ponomaryov A. N. , Bondybey V. E.
Published: July 2007

Spatially separated stable charge centers, self-trapped holes and trapped electrons, were generated in Ar cryocrystals by a low-energy electron beam. A combination of the cathodoluminescence (CL) and photon-stimulated exoelectron emission (PSEE) methods was used to monitor center formation and a selected relaxation channel—exoelectron emission. It was found that photon-promoted electron current decreased exponentially under irradiation with the laser operating in the visible range. The influence of the laser parameters (power and wavelength) on the characteristic lifetime of exoelectron emission is discussed. Effective bleaching of the low-temperature peaks of thermally stimulated exoelectron emission by the laser light in a visible range is observed.
Thermoactivation spectroscopy of solid Ar doped with N2
Authors: Ponomaryov A. N. , Savchenko E. V. , Khizhniy I. V. , Gumenchuk G. B. , Frankowski M. , Bondybey V. E.
Published: July 2007

A new modification of low-temperature activation spectroscopy technique for real-time correlated study of relaxation processes in cryogenic solids is developed. This enables us to measure simultaneously the thermally stimulated exoelectron emission (TSEE) and spectrally resolved thermally stimulated luminescence (TSL) in the range from 200 to 1100nm. This paper presents the results on TSL and TSEE from solid Ar doped with N2 exposed to irradiation by a low-energy electron beam (500eV) during deposition. The TSEE and TSL yields were measured at a heating rate of 3.2K∕min. The emissions of molecular (A3Σu+→X1Σg+ transition) and atomic (D2→S4 transition) nitrogen in the TSL spectra and their temperature behavior were studied. Drastic changes in the intensity distribution of the molecular progression are observed with temperature rise. In the low-temperature range, “hot” vibrationally unrelaxed transitions are detected, in contrast to “cold” vibrationally relaxed transitions observed in “high”-temperature TSL. The mechanisms of the processes resulting in TSL in the whole temperature range of solid Ar existence are suggested.
Evolution of discrete local levels into an impurity band in solidified inert gas solution
Authors: Kosevich A. M. , Feodosyev S. B. , Gospodarev I. A. , Grishaev V. I. , Kotlyar O. V. , Kruglov V. O. , Manzhelii E. V. , Syrkin E. S.
Published: July 2007

The density of states g(ω) of disordered solutions of solidified inert gases are calculated using the Jacobian matrix method. The transformation of a discrete vibrational level into an impurity band at a growing concentration of light impurity atoms is investigated. It is shown that a 1-10% change in the impurity concentration leads to smearing the local discrete level into an impurity band. As this occurs, additional resonance levels appear which carry important information about the impurity-impurity and impurity-basic lattice force interactions in such solutions.
Exohedral silicon fullerenes: SiNPtN∕2 (20⩽N⩽60)
Authors: Pei Yong , Gao Yi , Zeng X. C.
Published: July 2007

Using density functional theory method we show that hollow silicon fullerene cages, SiN (20⩽N⩽60), can be fully stabilized by exohedrally coated platinum atoms (PtN∕2), denoted as SiNPtN∕2. The exohedral coating PtN∕2 passivates the dangling bonds of the silicon cages, thereby making the silicon cages SiN to retain the symmetry and structure of homologous carbon fullerenes CN. In particular, the Ih symmetrical, 60-atom silicon buckminsterfullerene cage (Si60) can be fully stabilized by exohedrally coated 30 Pt atoms. Properties of SiNPtN∕2, such as the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) gap and relative stability of cage isomers, are calculated and compared with their carbon counterparts. It is found that the HOMO-LUMO gaps of SiNPtN∕2 are close to their carbon fullerene counterparts (CN). The trend in relative stability for exohedral fullerene isomers SiNPtN∕2 is similar to that for the homologous carbon fullerenes (CN). The exohedral Pt coating offers a possible molecular design towards stabilizing the silicon fullerene cages.
On surface signatures generated by submerged momentum sources
Authors: Voropayev S. I. , Fernando H. J. S. , Smirnov S. A. , Morrison R.
Published: July 2007

The conditions under which submerged momentum disturbances can penetrate upward and create detectable signatures on the water surface were studied using laboratory experiments. Unsteady maneuvering motion of a self-propelled body or an impulsive turbulent jet was used as the (controlled) momentum disturbance, which was located in the upper homogeneous layer of a stable two-layer stratified fluid with a deep lower layer. The experiments were conducted at high Reynolds numbers (>104), corresponding to fully turbulent wake/jet flows. The measurement of flow characteristics was made using particle-image velocimetry, and resulting observations were explained using theoretical arguments. Criteria for the penetration of wake/jet signatures to the water surface were advanced in terms of main governing parameters, and the results are extrapolated to typical upper-ocean situations.
Multielectron coincidence spectroscopy for core-valence doubly ionized states of CO
Authors: Hikosaka Y. , Kaneyasu T. , Shigemasa E. , Lablanquie P. , Penent F. , Ito K.
Published: July 2007

Double photoionization into states which have holes in one core and one valence orbitals has been observed in CO using a state-of-the-art multielectron coincidence method. The core-valence CO2+ structures exhibited on the electron coincidence spectra are assigned by comparison with the available calculation [H. Schulte et al., J. Chem. Phys. 105, 11108 (1996)]. Features of the spectrum confirm that the properties of the CO2+ states are characterized by the interaction between the localized valence holes and the core holes.