bluuebunny/crossref_metadata_2025 huggingface dataset:
Imaging phosphoinositide dynamics using GFP‐tagged protein domains
Authors: Halet Guillaume
Published: May 2008

Phosphoinositides are important regulators of cellular homoeostasis and numerous signal‐transduction pathways. One of their major features is their ability to recruit signalling proteins to membranes by direct interaction with phosphoinositide‐binding modules. The distribution and dynamics of membrane phosphoinositides are therefore major determinants in the spatiotemporal control of cell signalling and membrane trafficking. However, standard biochemical approaches cannot reveal the dynamics of phosphoinositides at the single‐cell level. A major technical advance has been the development of genetically encoded fluorescent phosphoinositide probes on the basis of the phosphoinositide‐binding domains found in signalling proteins, such as the PH (pleckstrin homology) domain. This review describes the diverse fluorescent phosphoinositide probes available for imaging specific phosphoinositide species and how their use has improved the understanding of phosphoinositide signalling at the single‐cell level.
Collagen reorganization in leech wound healing
Authors: Tettamanti Gianluca , Grimaldi Annalisa , Congiu Terenzio , Perletti Gianpaolo , Raspanti Mario , Valvassori Roberto , de Eguileor Magda
Published: May 2008

Background Information. Leeches respond to surgical lesions with the same sequence of events as that described for wound healing in vertebrates, where collagen is important for the development of tensions in healing wounds, functioning as an extracellular scaffold for accurate regeneration of the structures disrupted by surgical or traumatic actions.

Results. In surgically lesioned leeches, newly synthesized collagen is arranged in hierarchical structures. Fibrils can be packed and shaped to form cords or tubular structures, thus acting as an extracellular scaffold that directs and organizes the outgrowth of new vessels and the migration of immune cells towards lesioned tissues. In these animals, the general architecture of collagen fibrils, generated during tissue regeneration, shows similarities to both the structural pattern of collagen bundles and assembly processes observed in several vertebrate systems (fish scales, amphibian skin and human cornea).

Conclusions. The production of extracellular matrix during wound healing in leeches is a surprising example of conservation of an extremely close relationship between the structure and function of molecular structures. It could be hypothesized that collagen structures, characterized not only by a striking structural complexity, but also by multifunctional purposes, are anatomical systems highly conserved throughout evolution.
Sulphated glycoconjugates are powerful inhibitors of spermatozoa binding to the vitelline envelope in amphibian eggs
Authors: Caputo Mariangela , Riccio Stefania , Paglierucci Paolo , Tretola Loredana , Diglio Clementina , Carotenuto Rosa , De Marco Nadia , Campanella Chiara
Published: May 2008

Background information. In amphibians, the role of sulphated glycans has not been determined in spermatozoa—egg interaction, although they are known to be involved in other systems. In previous studies, it was found that, in Discoglossus pictus, a VE (vitelline envelope) glycoprotein of 63 kDa exhibits high homology to Xenopus laevis gp69/gp64 and to ZP2 of mammals. gp63 and a glycoprotein of 75 kDa are both capable of binding the spermatozoa in in vitro assays and, having similar peptide maps and different glycosylation, are probably two glycoforms of the same protein.

Results. In the present study, binding assays performed by treating dejellied eggs with metaperiodate suggest that hydroxy groups of sugars are not directly involved in spermatozoa—vitelline envelope binding. Competition assays between dejellied eggs and spermatozoa preincubated with dextran, dextran sulphate or fucoidan indicated that sulphated oligosaccharides have an inhibitory effect on spermatozoa binding. In similar competition assays, Lex (Lewisx) trisaccharide 3′‐sulphate inhibited spermatozoa binding to VE in contrast with 3′‐sialyl‐Lex tetrasaccharide. Assays performed with gp75‐ or gp63‐coated beads and spermatozoa treated with fucoidan or dextran sulphate indicated that sulphated oligosaccharides competitively inhibit spermatozoa binding to gp75‐coated beads, yet not to gp63‐coated beads. Finally, solubilized VE digested with N‐glycosidase F retains the inhibitory activity in spermatozoa—VE binding assays in contrast with VE treated with α‐N‐acetylgalactosaminidase.

Conclusion. It was concluded that VE sulphate groups are involved in spermatozoa binding. These groups are present in gp75 glycoconjugates and are probably located in O‐linked glycoconjugates.
The role of Munc18‐1 in docking and exocytosis of peptide hormone vesicles in the anterior pituitary
Authors: Korteweg Niki , Maia Ascanio S. , Thompson Brenda , Roubos Eric W. , Burbach J. Peter H. , Verhage Matthijs
Published: May 2008

Background information. Many neurons secrete classical transmitters from synaptic vesicles as well as peptide transmitters from LDCVs (large dense‐core vesicles). Little is known about the mechanistic differences between these two secretory pathways. The soluble protein Munc18‐1 is essential for synaptic vesicle secretion [Verhage, Maia, Plomp, Brussaard, Heeroma, Vermeer, Toonen, Hammer, van den Berg, Missler, et al. (2000) Science 287, 864–869.].

Results. In the present study, we tested if Munc18 genes are also involved in peptidergic secretion from LDCVs using the anterior pituitary as a model system. We show that Munc18‐1 is the dominant isoform expressed in the anterior pituitary. In Munc18‐1 null mutant mice, the anterior pituitary developed normally and the five major endocrine cell types had a normal distribution. However, circulating peptide hormone levels were decreased by up to 50‐fold in the null mutant, whereas the intracellular levels were significantly higher than that in controls. Ultrastructural analysis using the tannic acid method revealed striking differences in the distribution of secretory vesicles: (i) the number of exocytotic figures was mostly decreased in the null mutants and (ii) the LDCVs accumulated near but not at their target membrane. This is in contrast with the apparently normal distribution of synaptic vesicles in developing synapses in the null mutant (Verhage et al., 2000).

Conclusions. We conclude that Munc18‐1 is involved in the secretion of peptide hormones and in the docking of LDCVs. These results unmask an apparent mechanistic difference between LDCVs and synaptic vesicles.
Aquaporin‐3 functions as a glycerol transporter in mammalian skin
Authors: Hara‐Chikuma Mariko , Verkman A.S.
Published: May 2008

The AQPs (aquaporins) are a family of homologous water transporting proteins expressed in many mammalian epithelial, endothelial and other cell types. Phenotype analysis of mice lacking individual AQPs has been informative in elucidating their role in mammalian physiology. For example, phenotype analysis has indicated an important role of AQPs in the renal urinary concentrating mechanism (AQP1–AQP4), brain water balance and neural signal transduction (AQP4), exocrine gland secretion (AQP5) and ocular fluid balance (AQP1, AQP5). In skin, the aquaglyceroporin AQP3 is expressed in the basal layer of epidermal keratinocytes. Mice deficient in AQP3 have dry skin with reduced SC (stratum corneum) hydration, decreased elasticity and impaired biosynthesis. Mechanistic analysis of the altered skin phenotype in AQP3 deficiency suggested that the glycerol rather than the water transporting function of AQP3 is important in skin physiology. The glycerol content of SC and epidermis of AQP3 deficient mice is reduced, whereas that of dermis and serum is normal. The dry, relatively inelastic skin in AQP3 null mice is probably related to the humectant properties of glycerol, and the impaired SC repair to impaired epidermal biosynthetic function. The key role of AQP3 in epidermal physiology might be exploited in the development of improved cosmetics and new therapies for skin diseases associated with altered skin water content.
Centrioles resist forces applied on centrosomes during G2/M transition
Authors: Abal Miguel , Keryer Guy , Bornens Michel
Published: May 2008

Background information. Centrosome movements at the onset of mitosis result from a balance between the pulling and pushing forces mediated by microtubules. The structural stability of the centrosome core structure, the centriole pair, is correlated with a heavy polyglutamylation of centriole tubulin.

Results. Using HeLa cells stably expressing centrin—green fluorescent protein as a centriole marker, we monitored the effect of microinjecting an anti‐(polyglutamylated tubulin) monoclonal antibody, GT335, in G1/S or G2 cells. In contrast with the slow effect of the monoclonal antibody GT335 during interphase, a dramatic and rapid centrosome fragmentation occurred in cells microinjected in G2 that was both Eg5‐ and dynein‐dependent. Inhibition of either one of these two motors significantly decreased the scattering of centrosome fragments, and inhibition of centrosome segregation by impairing microtubule dynamics abolished centrosome fragmentation.

Conclusions. Our results demonstrate that the compact structure of the mitotic centrosome is capable of absorbing most of the pulling and pushing forces during G2/M transition and suggest that centrosomes could act as mechanosensors integrating tensions during cell division.
MIPDB: a relational database dedicated to MIP family proteins
Authors: El Karkouri Khalid , Gueuné Hervé , Delamarche Christian
Published: May 2008

Background information. The MIPs (major intrinsic proteins) constitute a large family of membrane proteins that facilitate the passive transport of water and small neutral solutes across cell membranes. Since water is the most abundant molecule in all living organisms, the discovery of selective water‐transporting channels called AQPs (aquaporins) has led to new knowledge on both the physiological and molecular mechanisms of membrane permeability. The MIPs are identified in Archaea, Bacteria and Eukaryota, and the rapid accumulation of new sequences in the database provides an opportunity for large‐scale analysis, to identify functional and/or structural signatures or to infer evolutionary relationships. To help perform such an analysis, we have developed MIPDB (database for MIP proteins), a relational database dedicated to members of the MIP family.

Results. MIPDB is a motif‐oriented database that integrates data on 785 MIP proteins from more than 200 organisms and contains 230 distinct sequence motifs. MIPDB proposes the classification of MIP proteins into three functional subgroups: AQPs, glycerol‐uptake facilitators and aquaglyceroporins. Plant MIPs are classified into three specific subgroups according to their subcellular distribution in the plasma membrane, tonoplast or the symbiosome membrane. Some motifs of the database are highly selective and can be used to predict the transport function or subcellular localization of unknown MIP proteins.

Conclusions. MIPDB offers a user‐friendly and intuitive interface for a rapid and easy access to MIP resources and to sequence analysis tools. MIPDB is a web application, publicly accessible at http:idefix.univ‐rennes1.fr:8080Protindex.html.
My Favorite Sites: Linkage of genetics and ethics: more crossing over is needed
Authors: Lissemore James L.
Published: May 2008

Since the development of recombinant DNA technology in the mid 1970s, there has been increasing interest in the ethical, legal, and social implications of genetics and related fields. The web sites of five different organizations (government, academic, and independent not‐for‐profit) that deal explicitly with genetics and ethics are reviewed here. Some of the sites cover genetics and other issues in bioethics while others cover human genetics exclusively. The target audiences for the sites include medical and scientific professionals, students, and the general public. Among the issues examined are genetic testing, genetic discrimination in employment and health insurance, genetically modified foods, stem cells and DNA patenting. Resources for those interested in legal issues are particularly well‐represented on these sites.
Phylogeny and evolution of the major intrinsic protein family
Authors: Zardoya Rafael
Published: May 2008

Background information. MIPs (major intrinsic proteins) form channels across biological membranes that control recruitment of water and small solutes such as glycerol and urea in all living organisms. Because of their widespread occurrence and large number, MIPs are a sound model system to understand evolutionary mechanisms underlying the generation of protein structural and functional diversity. With the recent increase in genomic projects, there is a considerable increase in the quantity and taxonomic range of MIPs in molecular databases.

Results. In the present study, I compiled more than 450 non‐redundant amino acid sequences of MIPs from NCBI databases. Phylogenetic analyses using Bayesian inference reconstructed a statistically robust tree that allowed the classification of members of the family into two main evolutionary groups, the GLPs (glycerol‐uptake facilitators or aquaglyceroporins) and the water transport channels or AQPs (aquaporins). Separate phylogenetic analyses of each of the MIP subfamilies were performed to determine the main groups of orthology. In addition, comparative sequence analyses were conducted to identify conserved signatures in the MIP molecule.

Conclusions. The earliest and major gene duplication event in the history of the MIP family led to its main functional split into GLPs and AQPs. GLPs show typically one single copy in microbes (eubacteria, archaea and fungi), up to four paralogues in vertebrates and they are absent from plants. AQPs are usually single in microbes and show their greatest numbers and diversity in angiosperms and vertebrates. Functional recruitment of NOD26‐like intrinsic proteins to glycerol transport due to the absence of GLPs in plants was highly supported. Acquisition of other MIP functions such as permeability to ammonia, arsenite or CO2is restricted to particular MIP paralogues. Up to eight fairly conserved boxes were inferred in the primary sequence of the MIP molecule. All of them mapped on to one side of the channel except the conserved glycine residues from helices 2 and 5 that were found in the opposite side.
Expression and subcellular localization of the AQP8 and AQP1 water channels in the mouse gall‐bladder epithelium
Authors: Calamita Giuseppe , Ferri Domenico , Bazzini Claudia , Mazzone Amelia , Bottà Guido , Liquori Giuseppa E. , Paulmichl Markus , Portincasa Piero , Meyer Giuliano , Svelto M.
Published: May 2008

Background information. Transepithelial transport of water is one of the most distinctive functions by which the gall‐bladder rearranges its bile content. Water is reabsorbed from the gall‐bladder lumen during fasting, whereas it is secreted into the lumen following meal ingestion. Nevertheless, the molecular mechanism by which water is transported across the gall‐bladder epithelium remains mostly unclear.

Results. In the present study, we investigate the presence and subcellular localization of AQP (aquaporin) water channels in the mouse gall‐bladder epithelium. Considerable AQP8 mRNA was detected in the gall‐bladder epithelium of mouse, calf, rabbit, guinea pig and man. Studies of subcellular localization were then addressed to the mouse gall‐bladder where the transcript of a second AQP, AQP1, was also detected. Immunoblotting experiments confirmed the presence of AQP8 and AQP1 at a protein level. Immunohistochemistry showed intense expression of AQP8 and AQP1 in the gall‐bladder epithelial cells where AQP8 was localized in the apical membrane, whereas AQP1 was seen both in the apical and basolateral membranes, and in vesicles located in the subapical cytoplasm.

Conclusions. The pattern of subcellular distribution of AQP8 and AQP1 strongly corroborates the hypothesis of a transcellular route for the movement of water across the gall‐bladder epithelium. Osmotic water would cross the apical membrane through AQP8 and AQP1, although AQP1 would be the facilitated pathway for the movement of water across the basolateral membrane. The presence of two distinct AQPs in the apical membrane is an unusual finding and may relate to the membrane's ability both to absorb and secrete fluid. It is tempting to hypothesize that AQP1 is hormonally translocated to the gall‐bladder apical membrane to secrete water as in the bile duct epithelium, a functional homologue of the gall‐bladder epithelium, whereas apical AQP8 may account for the absorption of water from gall‐bladder bile.
Aquaporins in yeasts and filamentous fungi
Authors: Pettersson Nina , Filipsson Caroline , Becit Evren , Brive Lars , Hohmann Stefan
Published: May 2008

Recently, genome sequences from different fungi have become available. This information reveals that yeasts and filamentous fungi possess up to five aquaporins. Functional analyses have mainly been performed in budding yeast, Saccharomyces cerevisiae, which has two orthodox aquaporins and two aquaglyceroporins. Whereas Aqy1 is a spore‐specific water channel, Aqy2 is only expressed in proliferating cells and controlled by osmotic signals. Fungal aquaglyceroporins often have long, poorly conserved terminal extensions and differ in the otherwise highly conserved NPA motifs, being NPX and NXA respectively. Three subgroups can be distinguished. Fps1‐like proteins seem to be restricted to yeasts. Fps1, the osmogated glycerol export channel in S. cerevisiae, plays a central role in osmoregulation and determination of intracellular glycerol levels. Sequences important for gating have been identified within its termini. Another type of aquaglyceroporin, resembling S. cerevisiae Yfl054, has a long N‐terminal extension and its physiological role is currently unknown. The third group of aquaglyceroporins, only found in filamentous fungi, have extensions of variable size. Taken together, yeasts and filamentous fungi are a fruitful resource to study the function, evolution, role and regulation of aquaporins, and the possibility to compare orthologous sequences from a large number of different organisms facilitates functional and structural studies.
In situ localization of sulphur in the thioautotrophic symbiotic model Lucina pectinata (Gmelin, 1791) by cryo‐EFTEM microanalysis
Authors: Lechaire Jean‐Pierre , Frébourg Ghislaine , Gaill Françoise , Gros Olivier
Published: May 2008

Background information. Lucina pectinata is a large tropical lucinid known to harbour sulphide‐oxidizing bacteria in specialized gill cells. Conventional TEM (transmission electron microscopy) has shown that bacteriocytes also harbour visibly ‘empty’ vesicles whose chemical content remains, to date, only roughly determined.

Results. In the present study, L. pectinata gill tissues were cryo‐fixed as fast as possible by performing high‐pressure freezing before a freeze‐substitution process and finally performing a cryo‐embedding in Lowicryl. Ultrathin sections were then used for a cryo‐EFTEM (where EFTEM stands for energy‐filtered TEM) microanalysis. Results show that bacteriocytes within the gill tissues contain elemental sulphur in small vesicles produced by the host itself. In instances of sporadic depletion of sulphur in the environment, such structures may act as energy sources for bacterial endosymbionts.

Conclusions. The cryo‐EFTEM techniques represent (i) the only method used to date to locate and preserve sulphur at the cellular level and (ii) a powerful tool for sulphur metabolism analysis in thioautotrophic symbiont relationships.
Identification of novel neuronal isoforms of the Rho‐GEF Trio
Authors: Portales‐Casamar Elodie , Briançon‐Marjollet Anne , Fromont Sylvie , Triboulet Robinson , Debant Anne
Published: May 2008

Background information. The large family of GEFs (guanine nucleotide‐exchange factors) for Rho GTPases activate the GTPases by accelerating their GDP/GTP exchange. The multidomain protein Trio is the founding member of an intriguing subfamily of Rho‐GEFs exhibiting two Rho‐GEF and numerous additional domains. The members of the Trio family play an important role in neuronal physiology, and their structural organization is very well conserved through evolution. It has previously been shown that all the members, except mammalian Trio, display several isoforms, the functions of which have been well established.

Results. In this study, we have identified, by a combination of different approaches, novel Trio isoforms that have been generated by alternative splicing, giving rise to proteins that exhibit one or two Rho‐GEF domains (GEFDs). These isoforms are specifically expressed in the nervous system, at a higher level than the full‐length Trio, which is ubiquitously expressed. In addition, we show that all the GEFD1‐containing isoforms induce neurite outgrowth in neuroblastoma cells.

Conclusions. We have identified neuronal specific isoforms of Trio which could be essential for Trio function in neuronal morphology.
Linking exocytosis and endocytosis during phagocytosis
Authors: Braun Virginie , Niedergang Florence
Published: May 2008

Phagocytosis is used by macrophages, dendritic cells and neutrophils to capture and destroy pathogens and particulate antigens. Although localized assembly of actin filaments is the driving force for particle internalization, exocytosis of intracellular compartments, and in particular endocytic compartments, has been shown recently to be required for the early steps of phagosome formation. Here we report on the different compartments undergoing exocytosis during phagocytosis, with a special focus on late endosomes. We then compare this process with secretion from lysosomes or lysosome‐related organelles in specialized cells. Finally, we discuss how some of the molecular mechanisms responsible for lysosome‐related organelle secretion could also be implicated in phagosome formation.
Modelling the effect of specific inositol 1,4,5‐trisphosphate receptor isoforms on cellular Ca2+ signals
Authors: Dupont Geneviève , Combettes Laurent
Published: May 2008

Background information. Oscillations of cytosolic Ca2+ are well‐known to rely on the regulatory properties of the InsP3R (inositol 1,4,5‐trisphosphate receptor). Three isoforms of this channel have been identified. They differ in their regulatory properties by Ca2+ and InsP3. Experiments in different cell types clearly indicate that the relative amounts of each isoform affect the time course of Ca2+ changes after agonist stimulation. In the present study, we investigate whether different steady‐state curves for the open probability of the InsP3Rs as a function of Ca2+ imply different dynamical behaviours when these receptors are present in a cellular environment. We therefore describe by a specific phenomenological model the three main types of curves that have been reported: (i) the classical bell‐shaped curve, (ii) the bell‐shaped curve that is shifted towards higher Ca2+ concentrations when InsP3 is increased, and (iii) a monotonous increasing function of cytosolic Ca2+.

Results. We show that, although these types of curves can be ascribed to slight differences in the channel regulation by Ca2+ and InsP3, they can indicate important variations as to the receptor role in cellular Ca2+ control. Thus the receptor associated with the classical bell‐shaped curve appears to be the most robust Ca2+ oscillator. If the steady‐state curve is supposed to be a monotonous increasing function of cytosolic Ca2+, the modelled receptor cannot sustain Ca2+ oscillations in the absence of Ca2+ exchanges with the extracellular medium. When the bell‐shaped curve is shifted towards higher Ca2+ concentrations with increasing InsP3 levels, the model predicts that the receptor is less robust to changes in density; this receptor, however, provides a finer control of the steady‐state level of Ca2+ when varying the InsP3 concentration.

Conclusions. Our model allows us to propose an explanation for the experimental observations about the effect of selectively expressing or down‐regulating InsP3R isoforms, as well as to make theoretical predictions.
Control of cell migration: a tumour suppressor function for p53?
Authors: Roger Lauréline , Gadea Gilles , Roux Pierre
Published: May 2008

Much remains to be learned about how cancer cells acquire the property of migration, a prerequisite for invasiveness and metastasis. Loss of p53 functions is assumed to be a crucial step in the development of many types of cancers, leading to dysregulation of cell cycle checkpoint controls and apoptosis. However, emerging evidence shows that the contribution of the tumour suppressor p53 to the control of tumorigenesis is not restricted to its well‐known anti‐proliferative activities, but is extended to other stages of cancer development, i.e. the modulation of cell migration. This interesting alternative function has been proposed in light of the effect of p53 on specific features of migrating cells, including cell spreading, establishment of cell polarization and the production of protrusions. The effects of p53 on cell motility are largely mediated through the regulation of Rho signalling, thereby controlling actin cytoskeletal organization. These recent studies connect the regulation of proliferation to the control of cell migration and define a new concept of p53 function as a tumour suppressor gene, suggesting that p53 might be involved in tumour invasion and metastasis. This review focuses on emerging data concerning the properties of p53 that contribute to its atypical role in the regulation of cell migration.
Initial size and dynamics of viral fusion pores are a function of the fusion protein mediating membrane fusion
Authors: Plonsky Ilya , Kingsley David H. , Rashtian Afshin , Blank Paul S. , Zimmerberg Joshua
Published: May 2008

Background information. Protein‐mediated merger of biological membranes, membrane fusion, is an important process. To investigate the role of fusogenic proteins in the initial size and dynamics of the fusion pore (a narrow aqueous pathway, which widens to finalize membrane fusion), two different fusion proteins expressed in the same cell line were investigated: the major glycoprotein of baculovirus Autographa californica (GP64) and the HA (haemagglutinin) of influenza X31.

Results. The host Sf9 cells expressing these viral proteins, irrespective of protein species, fused to human RBCs (red blood cells) upon acidification of the medium. A high‐time‐resolution electrophysiological study of fusion pore conductance revealed fundamental differences in (i) the initial pore conductance; pores created by HA were smaller than those created by GP64; (ii) the ability of pores to flicker; only HA‐mediated pores flickered; and (iii) the time required for pore formation; HA‐mediated pores took much longer to form after acidification.

Conclusion. HA and GP64 have divergent electrophysiological phenotypes even when they fuse identical membranes, and fusion proteins play a crucial role in determining initial fusion pore characteristics. The structure of the initial fusion pore detected by electrical conductance measurements is sensitive to the nature of the fusion protein.
Nuclear localization, DNA binding and restricted expression in neural and germ cells of zebrafish Dmrt3
Authors: Li Qin , Zhou Xiang , Guo Yiqing , Shang Xuan , Chen Hao , Lu Heng , Cheng Hanhua , Zhou Rongjia
Published: May 2008

Background information. The DM (doublesex and male aberrant‐3) genes implicated in sexual development in diverse metazoan organisms have been proved to be involved in development of non‐gonadal tissues. The aim of the present study was to identify and characterize Dmrt3 (DM‐related transcription factor 3) of zebrafish.

Results. Zebrafish Dmrt3 has a conserved DMA domain, besides a common DM domain, which clustered it into the DMRTA subfamily. During embryogenesis, Dmrt3 expression increases gradually to a high level at pharyngula stage, which is restricted to the olfactory placode and the neural tube. In the juvenile zebrafish, the gene expression is first detected in undifferentiated gonad on 17 dpf (day post‐fertilization). In adult, Dmrt3 is expressed only in the developing germ cells of both gonads, mainly in spermatogonia, spermatocytes and developing oocytes. The Dmrt3 has a functional NLS (nuclear localization signal) K41GHKR45 within the DM domain, which ensures that Dmrt3 exerts its role in the nucleus. Moreover, EMSA (electrophoretic mobility‐shift assay) indicates that the Dmrt3‐derived DM polypeptide binds to similar sites of both targets of DSX (doublesex) and MAB‐3 (male aberrant‐3).

Conclusion. These results suggest that as a DNA‐binding protein, zebrafish Dmrt3 may function in the nucleus as a potential transcription factor to exert potential roles in the development of the olfactory placode, the neural tube and germ cells.
αvβ3 and αvβ5 integrins and their role in muscle precursor cell adhesion
Authors: Sinanan Andrea C. M. , Machell Jon R. A. , Wynne‐Hughes G. Trevor , Hunt Nigel P. , Lewis Mark P.
Published: May 2008

Background information. Functional adaptation of skeletal muscle is a requirement for different muscle groups (e.g. craniofacial, ocular and limb) to undergo site‐specific changes. Such tissue remodelling depends on dynamic interactions between muscle cells and their extracellular matrix, via participation of multifunctional molecules such as integrins. In view of data suggesting a role in fundamental muscle biology and muscle development in other systems, the present study has focused on expression and function of αv integrins, in cultured adult human craniofacial muscle (masseter) precursor cells and myotubes, and the predominantly fibroblastic IC (interstitial cells) population.

Results and conclusions. Flow‐cytometric phenotyping and immunofluorescence phenotyping show that αv, αvβ3 and αvβ5 are expressed in all mononuclear cells (muscle precursors and IC) seeded on muscle extracellular molecules such as gelatin, VN (vitronectin) and FN (fibronectin). In this system, blockade of αv activity using a function‐perturbing antibody abrogates cell migration on VN and FN. αv integrins act predominantly as VN receptors as cell—substrate attachment is diminished when αv neutralizing agents are introduced into cultures seeded on VN, and this inhibition is reversible; these integrins also appear to be minor FN receptors. These results demonstrate that the αv subset of integrins present on both myogenic precursors and IC is an essential cohort of VN and, to a lesser extent, FN receptors mediating cell adhesion and, either directly or indirectly, arbiters of cell motility.
Fucoganglioside α-fucosyl(α-galactosyl)-GM1: a novel member of lipid membrane microdomain components involved in PC12 cell neuritogenesis
Authors: Yamazaki Yasuhiro , Horibata Yasuhiro , Nagatsuka Yasuko , Hirabayashi Yoshio , Hashikawa Tsutomu
Published: May 2008

In order to search for novel components of lipid membrane microdomains involved in neural signalling pathways, mAbs (monoclonal antibodies) were raised against the detergent-insoluble membrane fraction of PC12 (pheochromocytoma) cells. Among the 22 hybrid clones, mAb PR#1 specifically detected a fucoganglioside Fuc(Gal)-GM1 [α-fucosyl(α-galactosyl)-GM1], a ganglioside homologous with GM1a (II3NeuAc,GgOse4Cer), as a novel member of microdomain components with biological functions. In the presence of mAb PR#1 in the culture medium, the outgrowth of neurites was induced in PC12 cells in a dose-dependent manner, with no effects on cell proliferation, suggesting that Fuc(Gal)-GM1 is preferentially involved in PC12 cell neuritogenesis. Effects through Fuc(Gal)-GM1 were different from those through GM1a during differentiation, e.g. under PR#1 treatment on Fuc(Gal)-GM1, round cell bodies with thinner cell processes were induced, whereas treatment with CTB (cholera toxin B subunit), a specific probe for GM1a, produced flattened cell bodies with thicker pro-cesses. Molecular analysis demonstrated that the PR#1–Fuc(Gal)-GM1 pathway was associated with Fyn and Yes of the Src family of kinases, although Src itself was not involved. No association was found with TrkA (tropomyosin receptor kinase A) and ERKs (extracellular-signal-regulated kinases), which are responsible for GM1a-induced differentiation. From these findings, it is suggested that a fucoganglioside Fuc(Gal)-GM1 provides a functional platform distinct from that of GM1a for signal transduction in PC12 cell differentiation.
Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach
Authors: Russwurm Michael , Mullershausen Florian , Friebe Andreas , Jäger Ronald , Russwurm Corina , Koesling Doris
Published: May 2008

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 μM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.
Mitochondrial uncouplers with an extraordinary dynamic range
Authors: Lou Phing-How , Hansen Birgit S. , Olsen Preben H. , Tullin Søren , Murphy Michael P. , Brand Martin D.
Published: May 2008

We have discovered that some weak uncouplers (typified by butylated hydroxytoluene) have a dynamic range of more than 106in vitro: the concentration giving measurable uncoupling is less than one millionth of the concentration causing full uncoupling. They achieve this through a high-affinity interaction with the mitochondrial adenine nucleotide translocase that causes significant but limited uncoupling at extremely low uncoupler concentrations, together with more conventional uncoupling at much higher concentrations. Uncoupling at the translocase is not by a conventional weak acid/anion cycling mechanism since it is also caused by substituted triphenylphosphonium molecules, which are not anionic and cannot protonate. Covalent attachment of the uncoupler to a mitochondrially targeted hydrophobic cation sensitizes it to membrane potential, giving a small additional effect. The wide dynamic range of these uncouplers in isolated mitochondria and intact cells reveals a novel allosteric activation of proton transport through the adenine nucleotide translocase and provides a promising starting point for designing safer uncouplers for obesity therapy.
Analysis of the contribution of the globin and reductase domains to the ligand-binding properties of bacterial haemoglobins
Authors: Farrés Judith , Burckhardt-Herold Susanna , Scherrer Jan , Frey Alexander D. , Kallio Pauli T.
Published: May 2008

Bacterial Hbs (haemoglobins), like VHb (Vitreoscilla sp. Hb), and flavoHbs (flavohaemoglobins), such as FHP (Ralstonia eutropha flavoHb), have different autoxidation and ligand-binding rates. To determine the influence of each domain of flavoHbs on ligand binding, we have studied the kinetic ligand-binding properties of oxygen, carbon monoxide and nitric oxide to the chimaeric proteins, FHPg (truncated form of FHP comprising the globin domain alone) and VHb-Red (fusion protein between VHb and the C-terminal reductase domain of FHP) and compared them with those of their natural counterparts, FHP and VHb. Moreover, we also analysed polarity and solvent accessibility to the haem pocket of these proteins. The rate constants for the engineered proteins, VHb-Red and FHPg, do not differ significantly from those of their natural counterparts, VHb and FHP respectively. Our results suggest that the globin domain structure controls the reactivity towards oxygen, carbon monoxide and nitric oxide. The presence or absence of a reductase domain does not affect the affinity to these ligands.
Human α2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3
Authors: Doan Ninh , Gettins Peter G. W.
Published: May 2008

Human α2M (α2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human α2M to be made. We describe here the expression and characterization of three α2M domains predicted to be involved in the stabilization of the thiol ester in native α2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the α2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of α2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1–MG8 of C3. TED is, as predicted, an α-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these α2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of α2M, and the consequent thiol ester-stabilizing domain–domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.
Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus
Authors: Ohradanova Anna , Vullo Daniela , Kopacek Juraj , Temperini Claudia , Betakova Tatiana , Pastorekova Silvia , Pastorek Jaromir , Supuran Claudiu T.
Published: May 2008

The N-terminal region of a 32 kDa cell-surface-binding protein, encoded by the D8L gene of vaccinia virus, shows sequence homology to CAs (carbonic anhydrases; EC 4.2.1.1). The active CAs catalyse the reversible hydration of CO2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein [vaccCA (vaccinia virus CA-like protein)] contains one of the three conserved histidine residues required for co-ordination to the catalytic zinc ion and for enzyme activity. In the present study, we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidine residues into the wild-type protein. The wild-type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other hCAs (human CAs) shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII. Moreover, its affinity for acetazolamide was high, comparable with that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was approx. 2-fold more efficient than in wild-type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.
Differential gene expression analysis of maize leaf at heading stage in response to water-deficit stress
Authors: Yue Guidong , Zhuang Yunlong , Li Zhaoxia , Sun Li , Zhang Juren
Published: May 2008

The whole-genomic gene-expression changes of maize (Zea mays L.) plants in response to water-deficit stress at the heading stage have not been previously studied. The present work utilized a maize oligonucleotide array (‘57K’, ~57000 sequences; http://www.maizearray.org/) representing more than 30000 unique genes, to profile transcriptome changes in maize leaves subjected to 1d (day) and 7d water-deficit stress. After 1d and 7d water-stress treatment, 195 and 1008 differential genes were identified respectively. One-third of 1d-water-stress-induced genes had known or putative functions in various cellular signalling pathways, indicating that signal-transduction-related genes play important roles in the early responses of maize leaves to water stress. The 7d-stress-regulated genes were involved in a broad range of cellular and biochemical activities. The most notable genes may function in compatible osmolyte metabolism, particularly in proline, sucrose, trehalose and raffinose metabolism in the leaves. The present study provided a valuable starting point for further elucidation of molecular mechanisms in the drought tolerance of maize plants.
Use of GafChromic film to diagnose laser generated proton beams
Authors: Hey D. S. , Key M. H. , Mackinnon A. J. , MacPhee A. G. , Patel P. K. , Freeman R. R. , Van Woerkom L. D. , Castaneda C. M.
Published: May 2008

A calibration of three types of GafChromic radiochromic film (HS, MD-55, and HD-810) was carried out on the Crocker Nuclear Laboratory’s 76in. cyclotron at UC Davis over doses ranging from 0.001to15kGy. The film was digitized with a scanning microdensitometer with which it was scanned twice with two different filters to increase the film’s effective dynamic range. We demonstrate how this calibrated film can be used to measure the spectrum and total energy of a laser generated proton beam. This technique was applied to an experiment on the 10J, 100fs Callisto laser at Lawrence Livermore National Laboratory. The resulting proton spectrum was compared to that obtained by simultaneous measurement of Ti nuclear activation; the two methods give the same proton beam slope temperature and agree in number of protons to within 27%.
Abelian gauge theories on compact manifolds and the Gribov ambiguity
Authors: Kelnhofer Gerald
Published: May 2008

We study the quantization of Abelian gauge theories of principal torus bundles over compact manifolds with and without boundary. It is shown that these gauge theories suffer from a Gribov ambiguity originating in the nontriviality of the bundle of connections whose geometrical structure will be analyzed in detail. Motivated by the stochastic quantization approach, we propose a modified functional integral measure on the space of connections that takes the Gribov problem into account. This functional integral measure is used to calculate the partition function, Green’s functions, and the field strength correlating functions in any dimension by using the fact that the space of inequivalent connections itself admits the structure of a bundle over a finite dimensional torus. Green’s functions are shown to be affected by the nontrivial topology, giving rise to nonvanishing vacuum expectation values for the gauge fields.
Perturbative theory and modeling of electronic-resonance-enhanced coherent anti-Stokes Raman scattering spectroscopy of nitric oxide
Authors: Kuehner Joel P. , Naik Sameer V. , Kulatilaka Waruna D. , Chai Ning , Laurendeau Normand M. , Lucht Robert P. , Scully Marlan O. , Roy Sukesh , Patnaik Anil K. , Gord James R.
Published: May 2008

A theory is developed for three-laser electronic-resonance-enhanced (ERE) coherent anti-Stokes Raman scattering (CARS) spectroscopy of nitric oxide (NO). A vibrational Q-branch Raman polarization is excited in the NO molecule by the frequency difference between visible Raman pump and Stokes beams. An ultraviolet probe beam is scattered from the induced Raman polarization to produce an ultraviolet ERE-CARS signal. The frequency of the ultraviolet probe beam is selected to be in electronic resonance with rotational transitions in the AΣ+2←XΠ2 (1,0) band of NO. This choice results in a resonance between the frequency of the ERE-CARS signal and transitions in the (0,0) band. The theoretical model for ERE-CARS NO spectra has been developed in the perturbative limit. Comparisons to experimental spectra are presented where either the probe laser was scanned with fixed Stokes frequency or the Stokes laser was scanned with fixed probe frequency. At atmospheric pressure and an NO concentration of 100ppm, good agreement is found between theoretical and experimental spectral peak locations and relative intensities for both types of spectra. Factors relating to saturation in the experiments are discussed, including implications for the theoretical predictions.
Rotational dynamics of a soft filament: Wrapping transition and propulsive forces
Authors: Coq N. , du Roure O. , Marthelot J. , Bartolo D. , Fermigier M.
Published: May 2008

We experimentally analyze the shape of a long elastic filament rotating in a viscous liquid. We identify a continuous but sharp transition from a straight to an helical shape, resulting from the competition between viscous stresses and elastic forces. This induced helicity generates a propulsive force along the axis of rotation. In addition, we show that the shape transition is associated with an unstable branch in the force-torque relation, confirming the numerical predictions of Manghi et al. [Phys. Rev. Lett. 96, 068101 (2006)]. A linearized model of the fluid-structure interaction is proposed to account for all the features of the nonlinear filament dynamics.
Tearing instabilities driven by nonideal effects in the tail plasma sheet
Authors: Sundaram A. K.
Published: May 2008

Using an extended magnetohydrodynamic description, the excitation of tearing modes is analytically investigated in the tail plasma sheet region that includes the magnetic field components B0x(x,z) and B0z(x,z). Taking electron inertia and the Hall effect into account, a generalized technique is displayed for obtaining the tearing solutions near the singular layer, where the B0x(x,z) field reverses sign at z=0. In two-dimensional tail geometry for scale lengths of order c∕ωpe, it is shown that a localized tearing mode as well as a mode with broad spatial extent (Δ′-driven mode) is excited near the field reversal region and these modes are mainly driven by electron inertia. For appropriate current sheet parameters, it is found that the localized mode becomes unstable in a couple of minutes while the mode with broad spatial width grows faster in 10s. For three-dimensional perturbations wherein kx,ky≠0, the combined effects of the Hall term and the electron inertia are shown to excite new localized tearing modes with considerably enhanced growth rates (γ>ωci).
Spatial symmetry holes in many-electron atoms and molecules
Authors: Koga Toshikatsu , Sekiya Masahiro
Published: May 2008

When a many-electron system has a spatial symmetry, it is shown that there exist spatial symmetry holes, which imply that two or more electrons are prohibited from being at certain spatial positions simultaneously. Inversion holes, rotation holes, and reflection holes, which result from inversion, twofold rotation, and reflection symmetries, respectively, are discussed in detail. The electron-electron counterbalance hole reported in literature is a particular case of the inversion hole. The spatial symmetry holes are illustrated for simple atoms and molecules.