bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of nuclear ferritin and mechanism of translocation
Authors: SURGULADZE Nodar , PATTON Stephanie , COZZI Anna , FRIED Michael G. , CONNOR James R.
Published: June 2005

Ferritin, normally considered a cytoplasmic iron-storage protein, is also found in cell nuclei. It is an established fact that H-ferritin is the major form of nuclear ferritin, but little is known about the roles of ferritin in nuclei or about the mechanisms that control its appearance within the nuclear volume. In the present study, we show that, for human SW1088 astrocytoma cells, the nuclear and cytoplasmic forms of H-ferritin are products of the same mRNA. Histochemical and biochemical evidence is presented showing that ferritin is distributed non-randomly within the nuclear volume and that it preferentially associates with heterochromatin. Both cytoplasmic and nuclear populations of H-ferritin contain mixtures of non- and O-glycosylated forms, but the nuclear population is enriched in O-glycosylated forms. Cells treated with alloxan, a potent inhibitor of O-glycosylation, contained significantly less nuclear ferritin compared with cells grown in control media. Alloxan inhibited the reappearance of H-ferritin in nuclei of cells released from conditions of iron depletion, but did not prevent its disappearance from nuclei of cells undergoing iron depletion. These results suggest that O-glycosylation accompanies the transfer of ferritin from the cytoplasm to the nucleus, but does not influence the reverse process. The picture that emerges is one in which ferritin translocation between the cytoplasm and the nucleus is post-translationally regulated and responds to environmental and nutritional cues.
Steady-state analysis of glucose repression reveals hierarchical expression of proteins under Mig1p control in Saccharomyces cerevisiae
Authors: VERMA Malkhey , BHAT Paike J. , VENKATESH K. V.
Published: June 2005

Glucose repression is a global transcriptional regulatory mechanism commonly observed in micro-organisms for the repression of enzymes that are not essential for glucose metabolism. In Saccharomyces cerevisiae, Mig1p, a homologue of Wilms' tumour protein, is a global repressor protein dedicated to glucose repression. Mig1p represses genes either by binding directly to the upstream repression sequence of structural genes or by indirectly repressing a transcriptional activator, such as Gal4p. In addition, some genes are repressed by both of the above mechanisms. This raises a fundamental question regarding the physiological relevance of the varied mechanisms of repression that exist involving Mig1p. We address this issue by comparing two well-known glucose-repression systems, that is, SUC2 and GAL gene expression systems, which encompass all the above three mechanisms. We demonstrate using steady-state analysis that these mechanisms lead to a hierarchical glucose repression profile of different family of genes. This switch over from one carbon source to another is well-calibrated as a function of glucose concentration through this hierarchical transcriptional response. The mechanisms prevailing in this repression system can achieve amplification and sensitivity, as observed in the well-characterized MAPK (mitogen-activated protein kinase) cascade system, albeit through a different structure. A critical feature of repression predicted by our steady-state model for the mutant strain of S. cerevisiae lacking Gal80p agrees well with the data reported here as well as that available in the literature.
Activation of protein synthesis in cardiomyocytes by the hypertrophic agent phenylephrine requires the activation of ERK and involves phosphorylation of tuberous sclerosis complex 2 (TSC2)
Authors: ROLFE Mark , McLEOD Laura E. , PRATT Phillip F. , PROUD Christopher G.
Published: June 2005

The hypertrophic Gq-protein-coupled receptor agonist PE (phenylephrine) activates protein synthesis. We showed previously that activation of protein synthesis by PE requires MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and mTOR (mammalian target of rapamycin). However, it remained unclear whether ERK activation was required and which downstream components were involved in activating mTOR and protein synthesis. Using an adenovirus encoding the MKP3 (MAPK phosphatase 3) to inhibit ERK activity, we demonstrate that ERK is essential for the activation of protein synthesis by PE. Activation and phosphorylation of S6K1 (ribosomal protein S6 kinase 1) and phosphorylation of eIF4E (eukaryotic initiation factor 4E)-binding protein (both are mTOR targets) were also inhibited by MKP3, suggesting that ERK is also required for the activation of mTOR signalling. PE stimulation of cardiomyocytes induced the phosphorylation of TSC2 (tuberous sclerosis complex 2), a negative regulator of mTOR activity. TSC2 was phosphorylated only weakly at Thr1462, but phosphorylated at additional sites within the sequence RXRXX(S/T). This differs from the phosphorylation induced by insulin, indicating that MEK/ERK signalling targets distinct sites in TSC2. This phosphorylation may be mediated by p90RSK (90 kDa ribosomal protein S6K), which is activated by ERK, and appears to involve phosphorylation at Ser1798. Activation of protein synthesis by PE is partially insensitive to the mTOR inhibitor rapamycin. Inhibition of the MAPK-interacting kinases by CGP57380 decreases the phosphorylation of eIF4E and PE-induced protein synthesis. Moreover, CGP57380+rapamycin inhibited protein synthesis to the same extent as blocking ERK activation, suggesting that MAPK-interacting kinases and regulation of mTOR each contribute to the activation of protein synthesis by PE in cardiomyocytes.
Multiple interactions of the ‘transducer’ govern its function in calpain activation by Ca2+
Authors: BOZÓKY Zoltán , ALEXA Anita , TOMPA Peter , FRIEDRICH Peter
Published: June 2005

Typical calpains in mammals become activated on binding of 8–12 Ca2+ ions per enzyme molecule, giving an example of integrated, manifold regulation by calcium. Besides two identified Ca2+ sites in catalytic domain II and several EF-hand motifs in domains IV and VI, an acidic loop in the centrally positioned domain III seems to harbour Ca2+. The mediator of distant Ca2+-induced structural transitions is an elongated structural element, the ‘transducer’. By site-directed mutagenesis along the transducer, we have generated various forms of rat m-calpain in which critical intramolecular interactions, as judged from the X-ray structure, would be abolished or modified. The kinetic parameters of these mutant enzymes support a model featuring shrinkage of transducer as a contributor to structural changes involved in calpain activation.
Molecular characterization of human xanthine oxidoreductase: the enzyme is grossly deficient in molybdenum and substantially deficient in iron-sulphur centres
Authors: GODBER Benjamin L. J. , SCHWARZ Guenter , MENDEL Ralf R. , LOWE David J. , BRAY Robert C. , EISENTHAL Robert , HARRISON Roger
Published: June 2005

XOR (xanthine oxidoreductase) purified from human milk was shown to contain 0.04 atom of Mo and 0.09 molecule of molybdopterin/subunit. On the basis of UV/visible and CD spectra, the human enzyme was approx. 30% deficient in iron-sulphur centres. Mo(V) EPR showed the presence of a weak rapid signal corresponding to the enzyme of low xanthine oxidase activity and a slow signal indicating a significant content of desulpho-form. Resulphuration experiments, together with calculations based on enzymic activity and Mo content, led to an estimate of 50–60% desulpho-form. Fe/S EPR showed, in addition to the well-known Fe/S I and Fe/S II species, the presence of a third Fe/S signal, named Fe/S III, which appears to replace partially Fe/S I. Comparison is made with similarly prepared bovine milk XOR, which has approx. 15-fold higher enzymic activity and Mo content. Taken along with evidence of low Mo content in the milk of other mammals, these findings add further support to the idea that XOR protein plays a physiological role in milk (e.g. in secretion) equal in importance to its catalytic function as an enzyme.
Multidrug-resistant cancer cells contain two populations of P-glycoprotein with differently stimulated P-gp ATPase activities: evidence from atomic force microscopy and biochemical analysis
Authors: BARAKAT Stéphane , GAYET Landry , DAYAN Guila , LABIALLE Stéphane , LAZAR Adina , OLEINIKOV Vladimir , COLEMAN Anthony W. , BAGGETTO Loris G.
Published: June 2005

Considerable interest exists about the localization of P-gp (P-glycoprotein) in DRMs (detergent-resistant membranes) of multidrug resistant cancer cells, in particular concerning the potential modulating role of the closely related lipids and proteins on P-gp activity. Our observation of the opposite effect of verapamil on P-gp ATPase activity from DRM and solubilized-membrane fractions of CEM-resistant leukaemia cells, and results from Langmuir experiments on membrane monolayers from resistant CEM cells, strongly suggest that two functional populations of P-gp exist. The first is located in DRM regions: it displays its optimal P-gp ATPase activity, which is almost completely inhibited by orthovanadate and activated by verapamil. The second is located elsewhere in the membrane; it displays a lower P-gp ATPase activity that is less sensitive to orthovanadate and is inhibited by verapamil. A 40% cholesterol depletion of DRM caused the loss of 52% of the P-gp ATPase activity. Cholesterol repletion allowed recovery of the initial P-gp ATPase activity. In contrast, in the solubilized-membrane-containing fractions, cholesterol depletion and repletion had no effect on the P-gp ATPase activity whereas up to 100% saturation with cholesterol induced a 58% increased P-gp ATPase activity, while no significant modification was observed for the DRM-enriched fraction. DRMs were analysed by atomic force microscopy: 40–60% cholesterol depletion was necessary to remove P-gp from DRMs. In conclusion, P-gp in DRMs appears to contain closely surrounding cholesterol that can stimulate P-gp ATPase activity to its optimal value, whereas cholesterol in the second population seems deprived of this function.
Identification, characterization and structure of a new Delta class glutathione transferase isoenzyme
Authors: UDOMSINPRASERT Rungrutai , PONGJAROENKIT Saengtong , WONGSANTICHON Jantana , OAKLEY Aaron J. , PRAPANTHADARA La-aied , WILCE Matthew C. J. , KETTERMAN Albert J.
Published: June 2005

The insect GST (glutathione transferase) supergene family encodes a varied group of proteins belonging to at least six individual classes. Interest in insect GSTs has focused on their role in conferring insecticide resistance. Previously from the mosquito malaria vector Anopheles dirus, two genes encoding five Delta class GSTs have been characterized for structural as well as enzyme activities. We have obtained a new Delta class GST gene and isoenzyme from A. dirus, which we name adGSTD5-5. The adGSTD5-5 isoenzyme was identified and was only detectably expressed in A. dirus adult females. A putative promoter analysis suggests that this GST has an involvement in oogenesis. The enzyme displayed little activity for classical GST substrates, although it possessed the greatest activity for DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] observed for Delta GSTs. However, GST activity was inhibited or enhanced in the presence of various fatty acids, suggesting that the enzyme may be modulated by fatty acids. We obtained a crystal structure for adGSTD5-5 and compared it with other Delta GSTs, which showed that adGSTD5-5 possesses an elongated and more polar active-site topology.
Human transthyretin in complex with iododiflunisal: structural features associated with a potent amyloid inhibitor
Authors: GALES Luís , MACEDO-RIBEIRO Sandra , ARSEQUELL Gemma , VALENCIA Gregorio , SARAIVA Maria João , DAMAS Ana Margarida
Published: June 2005

Ex vivo and in vitro studies have revealed the remarkable amyloid inhibitory potency and specificity of iododiflunisal in relation to transthyretin [Almeida, Macedo, Cardoso, Alves, Valencia, Arsequell, Planas and Saraiva (2004) Biochem. J. 381, 351–356], a protein implicated in familial amyloidotic polyneuropathy. In the present paper, the crystal structure of transthyretin complexed with this diflunisal derivative is reported, which enables a detailed analysis of the protein–ligand interactions. Iododiflunisal binds very deep in the hormone-binding channel. The iodine substituent is tightly anchored into a pocket of the binding site and the fluorine atoms provide extra hydrophobic contacts with the protein. The carboxylate substituent is involved in an electrostatic interaction with the Nζ of a lysine residue. Moreover, ligand-induced conformational alterations in the side chain of some residues result in the formation of new intersubunit hydrogen bonds. All these new interactions, induced by iododiflunisal, increase the stability of the tetramer impairing the formation of amyloid fibrils. The crystal structure of this complex opens perspectives for the design of more specific and effective drugs for familial amyloidotic polyneuropathy patients.
Emergent mechanistic diversity of enzyme-catalysed β-diketone cleavage
Authors: GROGAN Gideon
Published: June 2005

The enzymatic cleavage of C–C bonds in β-diketones is, comparatively, a little studied biochemical process, but one that has important relevance to human metabolism, bioremediation and preparative biocatalysis. In recent studies, four types of enzymes have come to light that cleave C–C bonds in the β-diketone functionality using different chemical mechanisms. OPH [oxidized poly(vinyl alcohol) hydrolase from Pseudomonas sp. strain VM15C], which cleaves nonane-4,6-dione to butyrate and pentan-2-one is a serine-triad hydrolase. Dke1 (diketone-cleaving enzyme from Acinetobacter johnsonii) is a dioxygenase, cleaving acetylacetone to methylglyoxal and acetate. Fumarylacetoacetate hydrolase cleaves fumarylacetoacetate to fumarate and acetoacetate using a water molecule, activated by a catalytic His/Asp dyad, aided by a calcium ion that both chelates the enol acid form of the substrate and indirectly positions the water for nucleophilic attack at a carbonyl group. 6-Oxocamphor hydrolase cleaves nonenolizable cyclic β-diketones and is a homologue of the crotonase superfamily, employing a catalytic His/Asp dyad to activate a water molecule for nucleophilic attack at a carbonyl group on one prochiral face of the diketone substrate, effecting desymmetrizations of symmetrical substrates.
FRAP analysis of nucleocytoplasmic dynamics of the vitamin D receptor splice variant VDRB1: preferential targeting to nuclear speckles
Authors: SUNN Kathryn L. , EISMAN John A. , GARDINER Edith M. , JANS David A.
Published: June 2005

Although the key components of the cellular nuclear transport machinery have largely been characterized through extensive efforts in recent years, in vivo measurements of the kinetics of nuclear protein import/export are patently few. The present study applies the approach of FRAP (fluorescence recovery after photobleaching) to examine the nucleocytoplasmic flux of a novel human VDRB1 (vitamin D receptor B1) isoform in living cells. Through an N-terminal extension containing a consensus nuclear targeting sequence, VDRB1 is capable of localizing in nuclear speckles adjacent to SC-35 (35 kDa splicing component)-containing speckles as well as in the nucleoplasm, dependent on ligand. Investigation of VDRB1 nucleocytoplasmic transport using FRAP indicates for the first time that the VDRB1 has a serum-modulated, active nuclear import mechanism. There is no evidence of an efficient, active export mechanism for VDRB1, probably as a result of nuclear retention. VDRB1 nuclear import in the absence of serum occurred more rapidly and to a greater extent to nuclear speckles compared with import to other nuclear sites. This preferential transport from the cytoplasm to and accumulation within nuclear speckles is consistent with the idea that the latter represent dynamic centres of VDRB1 interaction with other nuclear proteins. The results are consistent with the existence of specialized pathways to target proteins to nuclear subdomains.
Pseudomonas aeruginosa MutL protein functions in Escherichia coli
Authors: JACQUELÍN Daniela K. , FILIBERTI Adrián , ARGARAÑA Carlos E. , BARRA José L.
Published: June 2005

Escherichia coli MutS, MutL and MutH proteins act sequentially in the MMRS (mismatch repair system). MutH directs the repair system to the newly synthesized strand due to its transient lack of Dam (DNA-adenine methylase) methylation. Although Pseudomonas aeruginosa does not have the corresponding E. coli MutH and Dam homologues, and consequently the MMRS seems to work differently, we show that the mutL gene from P. aeruginosa is capable of complementing a MutL-deficient strain of E. coli. MutL from P. aeruginosa has conserved 21 out of the 22 amino acids known to affect functioning of E. coli MutL. We showed, using protein affinity chromatography, that the C-terminal regions of P. aeruginosa and E. coli MutL are capable of specifically interacting with E. coli MutH and retaining the E. coli MutH. Although, the amino acid sequences of the C-terminal regions of these two proteins are only 18% identical, they are 88% identical in the predicted secondary structure. Finally, by analysing (E. coli–P. aeruginosa) chimaeric MutL proteins, we show that the N-terminal regions of E. coli and P. aeruginosa MutL proteins function similarly, in vivo and in vitro. These new findings support the hypothesis that a large surface, rather than a single amino acid, constitutes the MutL surface for interaction with MutH, and that the N- and C-terminal regions of MutL are involved in such interactions.
Arabidopsis thaliana β1,2-xylosyltransferase: an unusual glycosyltransferase with the potential to act at multiple stages of the plant N-glycosylation pathway
Authors: BENCÚR Peter , STEINKELLNER Herta , SVOBODA Barbara , MUCHA Jan , STRASSER Richard , KOLARICH Daniel , HANN Stephan , KÖLLENSPERGER Gunda , GLÖSSL Josef , ALTMANN Friedrich , MACH Lukas
Published: June 2005

XylT (β1,2-xylosyltransferase) is a unique Golgi-bound glycosyltransferase that is involved in the biosynthesis of glycoprotein-bound N-glycans in plants. To delineate the catalytic domain of XylT, a series of N-terminal deletion mutants was heterologously expressed in insect cells. Whereas the first 54 residues could be deleted without affecting the catalytic activity of the enzyme, removal of an additional five amino acids led to the formation of an inactive protein. Characterization of the N-glycosylation status of recombinant XylT revealed that all three potential N-glycosylation sites of the protein are occupied by N-linked oligosaccharides. However, an unglycosylated version of the enzyme displayed substantial catalytic activity, demonstrating that N-glycosylation is not essential for proper folding of XylT. In contrast with most other glycosyltransferases, XylT is enzymatically active in the absence of added metal ions. This feature is not due to any metal ion directly associated with the enzyme. The precise acceptor substrate specificity of XylT was assessed with several physiologically relevant compounds and the xylosylated reaction products were subsequently tested as substrates of other Golgi-resident glycosyltransferases. These experiments revealed that the substrate specificity of XylT permits the enzyme to act at multiple stages of the plant N-glycosylation pathway.
Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane
Authors: den HERTOG Alice L. , van MARLE Jan , van VEEN Henk A. , van't HOF Wim , BOLSCHER Jan G. M. , VEERMAN Enno C. I. , NIEUW AMERONGEN Arie V.
Published: June 2005

The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process.
Mechanistic and functional changes in Ca2+ entry after retinoic acid-induced differentiation of neuroblastoma cells
Authors: BROWN Anna M. , RIDDOCH Fiona C. , ROBSON Andrew , REDFERN Christopher P. F. , CHEEK Timothy R.
Published: June 2005

We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal.
Heterodimeric amino acid transporter glycoprotein domains determining functional subunit association
Authors: FRANCA Raffaella , VELJKOVIC Emilija , WALTER Stefan , WAGNER Carsten A. , VERREY François
Published: June 2005

The heteromeric amino acid transporter glycoprotein subunits rBAT and 4F2hc (heavy chains) form, with different catalytic subunits (light chains), functional heterodimers that are covalently stabilized by a disulphide bridge. Whereas rBAT associates with b0,+AT to form the cystine and cationic amino acid transporter defective in cystinuria, 4F2hc associates with other homologous light chains, for instance with LAT1 to form a system L neutral amino acid transporter. To identify within the heavy chains the domain(s) involved in recognition of and functional interaction with partner light chains, chimaeric and truncated forms of rBAT and 4F2hc were co-expressed in Xenopus laevis oocytes with b0,+AT or LAT1. Heavy chain–light chain association was analysed by co-immunoprecipitation, and transport function was tested by tracer uptake experiments. The results indicate that the cytoplasmic tail and transmembrane domain of rBAT together play a dominant role in selective functional interaction with b0,+AT, whereas the extracellular domain of rBAT appears to facilitate specifically L-cystine uptake. For 4F2hc, functional interaction with LAT1 was mediated by the N-terminal part, comprising cytoplasmic tail, transmembrane segment and neck, even in the absence of the extracellular domain. Alternatively, functional association with LAT1 was also supported by the extracellular part of 4F2hc comprising neck and glycosidase-like domain linked to the complementary part of rBAT. In conclusion, the cytoplasmic tail and the transmembrane segment together play a determinant role for the functional interaction of rBAT with b0,+AT, whereas either cytoplasmic or extracellular glycosidase-like domains are dispensable for the functional interaction of 4F2hc with LAT1.
Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are differentially expressed and regulated in rat tissue
Authors: RICHTER Wito , JIN S.-L. Catherine , CONTI Marco
Published: June 2005

Cyclic nucleotide PDE4 (phosphodiesterase 4) inhibitors are being developed as potent anti-inflammatory drugs for use in chronic lung diseases, but the complexity of the PDE4 family has hampered this process. The four genes comprising the PDE4 family, PDE4A, PDE4B, PDE4C and PDE4D, are all expressed as multiple splice variants. The most widely used criterion to identify PDE4 variants expressed endogenously is their migration on SDS/PAGE. However, when a PDE4D3-selective antibody was used for immunoprecipitation, the pattern of expression obtained did not confirm the expression predicted by SDS/PAGE. This observation, together with the recent discovery of additional PDE4D transcripts, prompted us to re-evaluate the pattern of expression of these variants. The nine rat PDE4D splice variants, PDE4D1 to PDE4D9, were cloned, their electrophoretic properties compared, and their in vivo mRNA and protein levels determined. Using this approach, we found that the pattern of distribution of the PDE4D splicing variants is more complex than previously reported. Multiple variants co-migrate in single immunoreactive bands, and variant-selective antibodies were necessary to discriminate between splice variants. Tissues that were thought to express only PDE4D3, express three closely related proteins, with PDE4D8 and PDE4D9 as the predominantly expressed forms. In addition, activation of cAMP signalling produces phosphorylation and activation of variants other than PDE4D3, and expression of PDE4D mRNA does not always correlate with the pattern of protein expression. As PDE4 inhibitors have different affinities for distinct PDE4D splicing variants, our results indicate that a better definition of the pattern of PDE4 expression is required for target validation.
Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine
Authors: TANG Dale D. , BAI Ying , GUNST Susan J.
Published: June 2005

Vimentin intermediate filaments undergo spatial reorganization in endothelial cells and fibroblasts in response to stimulation with platelet-derived growth factor and epidermal growth factor. In the present study, the vimentin network exhibited a curved filamentous structure in unstimulated smooth muscle cells. Vimentin filaments became straight and were arranged along the long axis of cells upon stimulation with 5-hydroxytryptamine (5-HT; serotonin). Stimulation of smooth muscle cells with 5-HT also induced phosphorylation of vimentin on Ser-56. Treatment of cells with small interfering RNA selectively down-regulated the expression of PAK1 (p21-activated kinase 1) without affecting the content of smooth muscle α-actin. The silencing of PAK1 inhibited the site-specific phosphorylation and spatial rearrangement of the vimentin network in response to stimulation with 5-HT. Neither the disruption of stress fibres by cytochalasin D nor the inhibition of protein tyrosine phosphorylation affects the spatial reorganization of vimentin intermediate filaments in response to stimulation with 5-HT. In addition, stimulation of smooth muscle cells with 5-HT increased the ratio of soluble to insoluble vimentin. PAK1 silencing attenuated increases in the ratio of soluble to insoluble vimentin upon stimulation with 5-HT. These results suggest that the PAK-mediated site-specific phosphorylation of vimentin may play a role in regulating the reorganization of vimentin intermediate filaments during stimulation of smooth muscle cells with 5-HT.
Molecular directionality in crystalline β-chitin: hydrolysis by chitinases A and B from Serratia marcescens 2170
Authors: HULT Eva-Lena , KATOUNO Fuminori , UCHIYAMA Taku , WATANABE Takeshi , SUGIYAMA Junji
Published: June 2005

β-Chitin microfibrils were treated with ChiA and ChiB (chitinases A and B respectively) from Serratia marcescens 2170. The β-chitin microfibrils were shortened, and the tips appeared narrowed and sharpened at both ends, after either consecutive or simultaneous degradation by ChiA and ChiB. Increased production of reducing sugars by simultaneous degradation (by ChiA and ChiB) of β-chitin, but not of glycol chitin, suggests synergistic interactions between the two enzymes. A combined analysis using the tilt microdiffraction method to determine the crystallographic axes, together with the biotin–streptavidin–gold-labelling method specific to the reducing ends, was used to investigate the polarity of the degraded β-chitin microcrystals. The digestion of the β-chitin fibrils by ChiA occurred from the reducing end to the nonreducing end, whereas digestion by ChiB occurred from the non-reducing end to the reducing end. The results are in agreement with the previously determined three-dimensional structures of these enzymes.
Rgt1, a glucose sensing transcription factor, is required for transcriptional repression of the HXK2 gene in Saccharomyces cerevisiae
Authors: PALOMINO Aaron , HERRERO Pilar , MORENO Fernando
Published: June 2005

Expression of HXK2, a gene encoding a Saccharomyces cerevisiae bifunctional protein with catalytic and regulatory functions, is controlled by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are low. In the present study, we identified Rgt1 as a transcription factor that, together with the Med8 protein, is essential for repression of the HXK2 gene in the absence of glucose. Rgt1 represses HXK2 expression by binding specifically to the motif (CGGAAAA) located at −395 bp relative to the ATG translation start codon in the HXK2 promoter. Disruption of the RGT1 gene causes an 18-fold increase in the level of HXK2 transcript in the absence of glucose. Rgt1 binds to the RGT1 element of HXK2 promoter in a glucose-dependent manner, and the repression of target gene depends on binding of Rgt1 to DNA. The physiological significance of the connection between two glucose-signalling pathways, the Snf3/Rgt2 that causes glucose induction and the Mig1/Hxk2 that causes glucose repression, was also analysed.
Guanine-specific DNA damage induced by γ-irradiated histone
Authors: FURUKAWA Ayako , HIRAKU Yusuke , OIKAWA Shinji , LUXFORD Catherine , DAVIES Michael J. , KAWANISHI Shosuke
Published: June 2005

In γ-irradiation, •OH is directly generated from water and causes DNA damage leading to carcinogenesis. Exposure of proteins to γ-irradiation, in the presence of oxygen, gives high yields of hydroperoxides. To clarify whether these hydroperoxides, particularly those formed on DNA-binding histone proteins, participate in γ-irradiation-induced carcinogenesis, experiments using 32P-labelled DNA fragments obtained from human cancer-related genes were undertaken. Histone protein-hydroperoxides induced significant DNA damage in the presence of Cu(I). Histone H1- and H3-hydroperoxides showed stronger DNA damage compared with histone H2A- and H4-hydroperoxides at 0.7 μM. Histone H1-hydroperoxides caused Cu(I)-dependent DNA damage predominantly at guanine residues, especially at 5′-GGC-3′, 5′-GGA-3′, 5′-GGT-3′ and single G bases. In contrast, histone H3-hydroperoxides/Cu(I) induced DNA damage at 5′-G in GG sequences; this sequence specificity is identical with that generated by 2,2′-azobis (2-amidinopropane) dihydrochloride, which is known to produce peroxyl radicals (RO2•). The difference in site specificity of DNA damage induced by histone H1- and H3-hydroperoxides may arise from their amino acid composition or their mode of binding to DNA. The histone H1-hydroperoxides/Cu(I) system also induced 8-oxo-7,8-dihydro-2′-deoxyguanosine formation in calf thymus DNA. It is concluded that histone protein-hydroperoxides can induce guanine-specific DNA damage, which may contribute to γ-irradiation-induced carcinogenesis.
Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4
Authors: KIYOHARA Masashi , SAKAGUCHI Keishi , YAMAGUCHI Kuniko , ARAKI Toshiyoshi , NAKAMURA Takashi , ITO Makoto
Published: June 2005

We cloned a novel β-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the β-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed β-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse β-1,4-xylan, β-1,4-mannan, β-1,4-glucan, β-1,3-xylobiose or p-nitrophenyl-β-xyloside. When β-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of β-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble β-1,3-xylan, but not that of soluble glycol-β-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.
Simultaneous Dual Fast and Slow Pathway Conduction upon Induction of Typical Atrioventricular Nodal Reentrant Tachycardia: Electrophysiologic Characteristics in a Series of Patients
Authors: TOMASI CORRADO , DE PONTI ROBERTO , TRITTO MASSIMO , BARILLI ANGELA LUCIANA , BOTTONI NICOLA , ZARDINI MARCO , MENOZZI CARLO , SPADACINI GIAMMARIO , SALERNO‐URIARTE JORGE ANTONIO
Published: June 2005

Introduction: Simultaneous dual atrioventricular nodal conduction (SDNC) through slow (SP) and fast pathway (FP) is a rare phenomenon observed upon the induction of atrioventricular nodal reciprocating tachycardia (AVNRT). The aim of this study is to report the electrophysiological features of patients showing typical AVNRT induced through SDNC.

Methods and Results: Among 461 consecutive patients with typical AVNRT submitted to radiofrequency catheter ablation (RFCA), seven patients (1.5%) with SDNC at tachycardia onset (group I: 6 female; age 60–72 years, mean 65.2 ± 3.8 years) and 118 age‐matched controls (group II: 60 female; age 60–88 years, mean 68.4 ± 6.8 years) were considered. Controls were further subdivided into two subgroups according to age: subgroup A (94 patients, age 60–75 years) and subgroup B (24 patients, age >75 years). The value of the following parameters was significantly higher in group I than in group II and in subgroup A: A–H interval [113 ± 26 vs. 89 ± 27 (P < 0.01) vs. 84 ± 19 (P < 0.001)], ventriculoatrial conduction effective refractory period [355 ± 85 vs. 293 ± 87 (P < 0.05) vs. 281 ± 82 (P < 0.05)], SP conduction time upon AVNRT induction [444 ± 104 vs. 350 ± 72 (P < 0.01); vs. 345 ± 67 (P < 0.001)], AVNRT cycle length [484 ± 103 vs. 396 ± 71 ms (P < 0.05); vs. 384 ± 69 (P < 0.05)], and rate of AVNRT induction from ventricle [71% vs. 10% (P = 0.001); vs. 6% (P = 0.001)]. Differences were mostly not significant between group I and subgroup B. SP location and RFCA success rate were similar in all groups.

Conclusion: In a population of AVNRT patients, SDNC at AVNRT induction is infrequent and it prevails beyond the fifth decade of life and in females. SDNC is associated with peculiar AVN conduction features, which resemble the age‐related modifications of AVN conduction.
Ventricular Arrhythmias Following Exposure of Failing Hearts to Oxidative Stress in Vitro
Authors: BRIGADEAU FRANÇOIS , GELÉ PATRICK , MARQUIÉ CHRISTELLE , SOUDAN BENOÎT , LACROIX DOMINIQUE
Published: June 2005

Introduction: There is experimental evidence that heart failure (HF) is an oxidative stress and that HF myocytes may be damaged by oxygen‐derived free radicals. However, the arrhythmogenicity of these radicals has not been studied in HF.

Methods and Results: Isolated perfused hearts were obtained from sham‐operated (SHAM, n = 6), and fast pacing (250 ms, 2 weeks)‐induced heart failure porcines (HF, n = 8). Epicardial conduction was mapped in the longitudinal and transverse directions and ventricular arrhythmias were closely monitored after perfusion of 100, 300, and 1000 μmol/L H2O2. Left ventricular epicardium was sampled for action potentials recordings in the same conditions. Myocardial levels of thiobarbituric acid reactive substances and antioxidant enzymatic capacity were also assessed. Epicardial conduction velocities were unaffected by H2O2 in both groups. Isolated ventricular premature beats and runs of slow ventricular rhythm with H2O2 more frequently occurred in HF compared to SHAM despite an increased antioxidant capacity including Cu/Zn and Mn superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase. Sustained arrhythmias were not observed. Higher thiobarbituric acid reactive substances levels were found in HF confirming endogenous oxidative stress. Action potential duration at plateau level was increased following H2O2 in SHAM but not in HF epicardial fibers where a toxic effect developed at 1000 μmol/L.

Conclusion: Oxidative stress with concomitant increase in antioxidant capacity develops in this HF model. There is a greater proclivity to oxidative stress‐mediated arrhythmias in HF. These arrhythmias are mainly extrasystoles or slow ventricular rhythms and not dependent on abnormal myocardial conduction.
Changes of Pulmonary Vein Orifice Size and Location throughout the Cardiac Cycle: Dynamic Analysis Using Magnetic Resonance Cine Imaging
Authors: LICKFETT LARS , DICKFELD TIMM , KATO RITSUSHI , TANDRI HARIKRISHNA , VASAMREDDY CHANDRASEKHAR R. , BERGER RONALD , BLUEMKE DAVID , LÜDERITZ BERNDT , HALPERIN HENRY , CALKINS HUGH
Published: June 2005

Introduction: Anatomically guided left atrial ablation is used increasingly for treatment of atrial fibrillation (AF). Three‐dimensional mapping systems used for pulmonary veins (PV) encircling ablation procedures anticipate a stable size and position of the PV orifice. The aim of the current study was therefore to analyze changes of PV orifice size and location throughout the cardiac cycle using cine magnetic resonance imaging (MRI).

Methods and Results: Twenty‐five healthy volunteers were studied using a 1.5 Tesla MRI system. MR angiograms were acquired with a breath‐hold three‐dimensional fast‐spoiled gradient‐echo imaging (3D FSPGR) sequence in the coronal plane before and after gadolinium injection. Maximum intensity projections and multiplanar reformations were performed to reconstruct images of the PV. Bright blood cine imaging in the axial view was acquired by a steady‐state free precession pulse sequence. Twenty bright blood images were obtained per cardiac cycle. The axial (anterior‐posterior) PV orifice diameter was measured in all 20 images. For analysis of PV movement the location of the orifice posterior edge was plotted on scale paper.

PV orifice size depends on the stage of the cardiac cycle with the largest diameter in late atrial diastole and a mean decrease of 32.5% during atrial systole. Location changes of the PV orifice are in the range of up to 7.2 mm and larger in the coronal (lateral‐medial) than in the sagittal (anterior‐posterior) direction.

Conclusion: PV orifice size and location is not as stable as anticipated by three‐dimensional mapping systems used for PV encircling left atrial ablation procedures. RF application close to the presumed orifice location should therefore be avoided to minimize the risk of PV stenosis.
Lag and anticipating synchronization without time-delay coupling
Authors: Corron Ned J. , Blakely Jonathan N. , Pethel Shawn D.
Published: June 2005

We describe a new method for achieving approximate lag and anticipating synchronization in unidirectionally coupled chaotic oscillators. The method uses a specific parameter mismatch between the drive and response that is a first-order approximation to true time–delay coupling. As a result, an adjustable lag or anticipation effect can be achieved without the need for a variable delay line, making the method simpler and more economical to implement in many physical systems. We present a stability analysis, demonstrate the method numerically, and report experimental observation of the effect in radio-frequency electronic oscillators. In the circuit experiments, both lag and anticipation are controlled by tuning a single capacitor in the response oscillator.
Pretransitional behavior of a water in liquid crystal microemulsion close to the demixing transition: Evidence for intermicellar attraction mediated by paranematic fluctuations
Authors: Caggioni M. , Giacometti A. , Bellini T. , Clark N. A. , Mantegazza F. , Maritan A.
Published: June 2005

We present a study of a water-in-oil microemulsion in which surfactant coated water nanodroplets are dispersed in the isotropic phase of the thermotropic liquid-crystal penthyl-cyanobiphenyl (5CB). As the temperature is lowered below the isotropic to nematic phase transition of pure 5CB, the system displays a demixing transition leading to a coexistence of a droplet-rich isotropic phase with a droplet-poor nematic. The transition is anticipated, in the high T side, by increasing pretransitional fluctuations in 5CB molecular orientation and in the nanodroplet concentration. The observed phase behavior supports the notion that the nanosized droplets, while large enough for their statistical behavior to be probed via light scattering, are also small enough to act as impurities, disturbing the local orientational ordering of the liquid crystal and thus experiencing pretransitional attractive interaction mediated by paranematic fluctuations. The pretransitional behavior, together with the topology of the phase diagram, can be understood on the basis of a diluted Lebwohl–Lasher model which describes the nanodroplets simply as holes in the liquid crystal.
Efficient topological chaos embedded in the blinking vortex system
Authors: Kin Eiko , Sakajo Takashi
Published: June 2005

We consider the particle mixing in the plane by two vortex points appearing one after the other, called the blinking vortex system. Mathematical and numerical studies of the system reveal that the chaotic particle mixing, i.e., the chaotic advection, is observed due to the homoclinic chaos, but the mixing region is restricted locally in the neighborhood of the vortex points. The present article shows that it is possible to realize a global and efficient chaotic advection in the blinking vortex system with the help of the Thurston–Nielsen theory, which classifies periodic orbits for homeomorphisms in the plane into three types: periodic, reducible, and pseudo-Anosov (pA). It is mathematically shown that periodic orbits of pA type generate a complicated dynamics, which is called topological chaos. We show that the combination of the local chaotic mixing due to the topological chaos and the dipole-like return orbits realize an efficient and global particle mixing in the blinking vortex system.
X-ray and neutron reflectivity measurements of moisture transport through model multilayered barrier films for flexible displays
Authors: Vogt Bryan D. , Lee Hae-Jeong , Prabhu Vivek M. , DeLongchamp Dean M. , Lin Eric K. , Wu Wen-li , Satija Sushil K.
Published: June 2005

One encapsulation approach to extend the lifetime of flexible organic light-emitting diode (OLED) devices uses inorganic Al2O3-polymer multilayer barrier films. However, a recent theoretical examination of multilayer barriers indicated that the barriers should not be effective for OLED applications, despite empirical evidence of success. It was suggested that a long-lived transient process in the transport of water molecules through multilayer films is responsible for its practical success, but has not been directly observed experimentally. X-ray reflectivity (XR) and neutron reflectivity measurements are used to measure permeation rates and structural changes in model barrier films upon exposure to water vapor. A film consisting of a stack of an undercured organic and the typical inorganic phases was found to barely swell [(7±5)Å] after an 11-d exposure to moisture [60 °C, 100% relative humidity (RH)]. Current measurements of ultralow moisture permeation assume that 10 d is sufficient for the equilibrium measurement, but XR data show that a stack of three dyad layers may require as many as 500 d (>12000h) to reach equilibrium. Barriers with a high number of defects in the inorganic phase reached equilibrium after 6 d of exposure to moisture (60 °C, 100% RH). Over this time scale, water breakthrough at each layer can be observed from XR. Neutron reflectivity measurements with deuterated water show an accumulation of water near the aluminum oxide∕polymer interface. This interface behaves similar to a desiccant, where the permeation of water through the barrier is retarded by the strong adsorption of water to aluminum oxide. This internal desiccant effect of the multilayered structure is clearly delineated and appears to be responsible for the long-term transient behavior of these barrier materials.
Calculation of Cl2 vibrational distributions after vibrational predissociation of He2–Cl2(B3Πu)
Authors: García-Vela A.
Published: June 2005

An approximate model is tested in order to obtain the vibrational distributions of the Cl2 fragment produced after He2–Cl2(B) predissociation, when the dynamics is simulated by a full-dimensional wave-packet method. The model is intended to overcome the limitations imposed to a rigorous calculation of the vibrational distributions by the use of a finite grid size and absorbing boundary conditions. The good agreement found between the present results and the available experimental data indicates that the model is able to provide realistic estimates for the product vibrational distributions.
Wigner measures dynamics in a Coulomb potential
Authors: Keraani Sahbi
Published: June 2005

In this paper, we are concerned with the propagation of the Wigner measure of a family of solutions to the semiclassical Schrödinger evolution equation with potential having Coulomb-type singularities in three space dimensions. We prove that after collision the Wigner measure is reflected by the potential singularities under the regularized flow as in classical mechanics.