bluuebunny/crossref_metadata_2025 huggingface dataset:
Adrenergic activation modulates the signal from the Reissner fiber to cerebrospinal fluid-contacting neurons during development
Authors: Cantaut-Belarif Yasmine , Orts Del'Immagine Adeline , Penru Margot , Pézeron Guillaume , Wyart Claire , Bardet Pierre-Luc
Published: October 2020

The cerebrospinal fluid (CSF) contains an extracellular thread conserved in vertebrates, the Reissner fiber, which controls body axis morphogenesis in the zebrafish embryo. Yet, the signaling cascade originating from this fiber to ensure body axis straightening is not understood. Here, we explore the functional link between the Reissner fiber and undifferentiated spinal neurons contacting the CSF (CSF-cNs). First, we show that the Reissner fiber is required in vivo for the expression of urp2, a neuropeptide expressed in CSF-cNs. We show that the Reissner fiber is also required for embryonic calcium transients in these spinal neurons. Finally, we study how local adrenergic activation can substitute for the Reissner fiber-signaling pathway to CSF-cNs and rescue body axis morphogenesis. Our results show that the Reissner fiber acts on CSF-cNs and thereby contributes to establish body axis morphogenesis, and suggest it does so by controlling the availability of a chemical signal in the CSF.
Exploring chromosomal structural heterogeneity across multiple cell lines
Authors: Cheng Ryan R , Contessoto Vinicius G , Lieberman Aiden Erez , Wolynes Peter G , Di Pierro Michele , Onuchic Jose N
Published: October 2020

Using computer simulations, we generate cell-specific 3D chromosomal structures and compare them to recently published chromatin structures obtained through microscopy. We demonstrate using machine learning and polymer physics simulations that epigenetic information can be used to predict the structural ensembles of multiple human cell lines. Theory predicts that chromosome structures are fluid and can only be described by an ensemble, which is consistent with the observation that chromosomes exhibit no unique fold. Nevertheless, our analysis of both structures from simulation and microscopy reveals that short segments of chromatin make two-state transitions between closed conformations and open dumbbell conformations. Finally, we study the conformational changes associated with the switching of genomic compartments observed in human cell lines. The formation of genomic compartments resembles hydrophobic collapse in protein folding, with the aggregation of denser and predominantly inactive chromatin driving the positioning of active chromatin toward the surface of individual chromosomal territories.
Full-length Plasmodium falciparum myosin A and essential light chain PfELC structures provide new anti-malarial targets
Authors: Moussaoui Dihia , Robblee James P , Auguin Daniel , Krementsova Elena B , Haase Silvia , Blake Thomas CA , Baum Jake , Robert-Paganin Julien , Trybus Kathleen M , Houdusse Anne
Published: October 2020

Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity, and ultimately pathogenesis ofPlasmodium falciparumrely on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here, we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor’s mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future anti-malarials targeting both the glideosome motor and its regulatory elements.
Icelandic V3 orders with temporal adjuncts
Authors: Sigurðardóttir Sigríður Sæunn
Published: October 2020

Although Icelandic is a verb second language (V2), it sometimes allows for V3 orders. In this paper, I focus on a type of Icelandic V3 which consists of an adverbial adjunct occurring in front of wh-questions and present the results of a pilot study that investigated the effects of the length of prosodic break and clause type in relation to V3 structures. Participants were presented with an adverbial clause sandwiched between two sentences (A and B), of which the latter varied between a wh-question and a subject-initial sentence. The breaks between the adverbial and the sentences varied in length. The task was to judge which sentence (A or B) was longer, with the response reflecting which sentence the adverbial clause was parsed with. The results indicate that both the clause type of the B sentence and the length of the prosodic break between the adverbial and the B sentence had a significant effect on how the sentences were parsed.
Humour and intertextuality in Steve Bell's political cartoons
Authors: Pinar Maria Jesús
Published: October 2020

The aim of this paper is to analyse 12 political cartoons published by Steve Bell in the left-wing oriented newspaper The Guardian to show how visual metaphors and metonymies and intertextual references are powerful strategies to present potent rhetorical depictions of political candidates and political issues. These devices are used to establish intertextual links across political cartoons and historical events, contemporary culture, paintings, literary works and illustrations. The themes that appear regularly in political cartoons have been identified, as well as a number of categories of source domains in visual metaphors. The analysis of the cartoons reveals that the interpretation of the cartoon and the appreciation of humour depend on the audience’s access to background knowledge, both of the political situation described in the cartoon and the intertextual references presented, on the audience’s ideology and on the decoding of the characteristics mapped onto the target of the metaphor.
Utilising one minute and four minute recovery when employing the resistance training contrast method does not negatively affect subsequent jump performance in the presence of concurrent training
Authors: Ritchie Dean , Keogh Justin W.L. , Reaburn Peter , Bartlett Jonathan D.
Published: October 2020

Background

Little is known about contrast training and post-activation performance enhancement (PAPE) in a same day concurrent training model. The aim of the current study was to examine the use of two short duration (1-min and 4-min) recovery periods on drop jump performance in same day concurrently trained athletes.

Methods

Ten professional Australian Rules footballers (age, 20.6 ± 1.9 yr; height, 184.8 ± 6.9 cm; body mass, 85.8 ± 8.4 kg) completed two resistance training sessions with different PAPE recovery durations; 1-min and 4-min, 1 h following a field-based endurance session. Baseline (pre) drop jumps were compared to post-test maximal drop jumps, performed after each set of three squats (where each participant was encouraged to lift as heavy as they could), to determine changes between 1-min and 4-min recovery periods. Data were analysed by fitting a mixed model (significance was set at P ≤ 0.05). Corrected Hedges’ g standardised effect sizes ±95% confidence limits were calculated using group means ± SDs.

Results

There were no significant differences between baseline and experimental sets 1, 2 and 3 for reactive strength index (RSI), flight time, and total and relative impulse for either recovery duration. However, for contact time, 1-min baseline was significantly different from set 2 (mean difference; 95% CI [0.029; 0.000–0.057 s], P = 0.047, ES; 95% CI [−0.27; −1.20 to 0.66]). For RSI and flight time, 1-min was significantly higher than 4-min (RSI: 0.367; 0.091 to 0.642, P = 0.010, ES; 95% CI [0.52; −0.37 to 1.42]; flight time: 0.033; 0.003 to 0.063 s, P = 0.027, ES; 95% CI [0.86; −0.06 to 1.78]).

Discussion

Short recovery periods of 1-min may be a time-efficient form of prescribing strength-power exercise in contrast loading schemes. Longer recovery periods do not appear to benefit immediate, subsequent performance.
Belowground fungal community diversity, composition and ecological functionality associated with winter wheat in conventional and organic agricultural systems
Authors: Garnica Sigisfredo , Rosenstein Ronja , Schön Max Emil
Published: October 2020

Understanding the impacts of agricultural practices on belowground fungal communities is crucial in order to preserve biological diversity in agricultural soils and enhance their role in agroecosystem functioning. Although fungal communities are widely distributed, relatively few studies have correlated agricultural production practices. We investigated the diversity, composition and ecological functionality of fungal communities in roots of winter wheat (Triticum aestivum) growing in conventional and organic farming systems. Direct and nested polymerase chain reaction (PCR) amplifications spanning the internal transcribed spacer (ITS) region of the rDNA from pooled fine root samples were performed with two different sets of fungal specific primers. Fungal identification was carried out through similarity searches against validated reference sequences (RefSeq). The R package ‘picante’ and FUNGuild were used to analyse fungal community composition and trophic mode, respectively. Either by direct or cloning sequencing, 130 complete ITS sequences were clustered into 39 operational taxonomic units (OTUs) (25 singletons), belonging to the Ascomycota (24), the Basidiomycota (14) and to the Glomeromycota (1). Fungal communities from conventional farming sites are phylogenetically more related than expected by chance. Constrained ordination analysis identified total N, total S and Pcal that had a significant effect on the OTU’s abundance and distribution, and a further correlation with the diversity of the co-occurring vegetation could be hypothesised. The functional predictions based on FUNGuild suggested that conventional farming increased the presence of plant pathogenic fungi compared with organic farming. Based on diversity, OTU distribution, nutrition mode and the significant phylogenetic clustering of fungal communities, this study shows that fungal communities differ across sampling sites, depending on agricultural practices. Although it is not fully clear which factors determine the fungal communities, our findings suggest that organic farming systems have a positive effect on fungal communities in winter wheat crops.
Biosorption of Bromo-based Dyes from Wastewater Using Low-Cost Adsorbents: A Review
Authors: Dada Ebenezer Olujimi , Ojo Ilesanmi Ademola , Alade Abass Olanrewaju , Afolabi Tinuade Jolaade , Jimoh Monsurat Omolola , Dauda Monsuru Olatunji
Published: October 2020

The dyes in the effluents discharged into water bodies, aimlessly, are displeasing aesthetically and pose hazards to aquatic communities. The use of adsorption process has been adopted for effective treatment of wastewater containing dyes. The removal of Bromophenol blue (BPB), Bromocresol green (BCG), Bromocresol purple (BCP), and Bromothymol blue (BTB) dyes (a family of triarylmethane dyes) through adsorption process using several cheaply available non-conventional agricultural-waste based adsorbents was reviewed in this report. The gaps in the treatment trend further indicate the prospect of adapting various lignocellulose and other biogenic materials for the removal of Bromo-based dyes from wastewater.
Genotypic Detection and Characterization of Adhesins in Clinical Escherichia coli Isolates
Authors: Iliyasu M. Y. , Mustapha I. , Yakubu H. , Shuaibu H. M. , Umar A. F. , Agbo E. B. , Uba A. , Deeni Y. Y.
Published: October 2020

Background of Study: Many virulence determinants contribute to the pathogenicity of Gram negative bacteria, like Escherichia coli, which is the most common cause of many infections worldwide such as urinary tract infection (UTI), profuse diarrhoea and septicaemia.

Aim: To determine the genotypic characteristics of adhesin-producing E. coli isolates from clinical specimens.

Place and Duration of Study: Conducted at the Infectious diseases hospital Bayara, Bauchi state, Nigeria, between February to March, 2019.

Methods: A total of twelve (12) Gram negative bacterial isolates were selected based on the ability to grow on Luria-Bertani (LB) agar medium containing 100 µg/ml ampicillin. The isolates were from urine, stool, and blood specimens. The isolates were screened for multidrug resistant pattern according to Kirby-Bauer disc diffusion method. Adhesion factors, Fimbrial adhesin (fimH) and Invasive plasmid adhesin (ipaH) was genotyped by conventional PCR and sequenced.

Results: All the isolates were resistant to Ampicillin, Cephalothin, Erythromycin, Fusidic acid, Novobiocin and Oxacillin, but sensitive to Augmentin, Colistin sulphate and Imipenem. Presence of fimH and ipaH genes were observed in nine isolates that expressed strong relationship with. Multidrug resistance (MDR). The fimH was the most prevalent found in urine, stool and blood isolates. Most of the adhesion genes sequence (61.8%) in this study had significant alignment (95 to 100% homology) with E.coli genome in the NCBI database.

Conclusion: This study revealed the role of adhesin as virulence markers in MDR Gram negative bacteria and FimH is one of the commonest gene in MDR E.coli pathotypes.
Monoclonal antibodies used as probes for the structural organization of the central region of fibronectin
Authors: Dziadek M. , Richter H. , Schachner M. , Timpl R.
Published: July 2002

Two monoclonal mouse antibodies against human plasma fibronectin were compared in their reactivity for proteolytic fragments of the antigen by enzyme immunoassay and immunoblotting. These antibodies were shown to react with two different structures within a short segment (about 30 kDa) located about one‐third away from the C‐terminus of the fibronectin chains.
Inhibition by the protein ceruloplasmin of lipid peroxidation stimulated by an Fe3+—ADP—adriamycin complex
Authors: Nakano Hiroko , Ogita Kouji , Gutteridge John M.C. , Nakano Minoru
Published: July 2002

Self‐reduction of an Fe3+—ADP—adriamycin complex under anaerobic conditions and reduction of ferricytochrome c by the complex under aerobic conditions were strongly inhibited by ceruloplasmin, but not by superoxide dismutase or albumin at the same protein concentration. Ceruloplasmin, a protein with ferroxidase activity, is able to catalyse oxidation of Fe2+ to the ferric state. The inhibitory activity of ceruloplasmin towards reactions stimulated by the complex suggests that Fe2+ is formed during the self‐reduction process. As expected, the Fe3+—ADP—adriamycin complex stimulated lipid peroxidation in which the Fe2+ moiety was implicated. This stimulation was again effectively prevented by ceruloplasmin but not by superoxide dismutase.
Conformationally constrained chemotactic peptide analogs of high biological activity
Authors: Iqbal M. , Balaram P. , Showell H.J. , Freer R.J. , Becker E.L.
Published: July 2002

The stereochemically constrained chemotactic peptide analogs, formylmethionyl‐α‐aminoisobutyrylphenylalanine (formyl‐Met‐Aib‐Phe‐OH) and formylmethionylcycloleucinylphenylalanine (formyl‐Met‐Cyl‐Phe‐OH) are highly effective in inducing lysosomal enzyme release from rabbit neutrophils. NMR studies of the Aib2 analog in (CD3)2SO favor a folded conformation in which the Phe NH group is inaccessible to solvent. Intramolecularly hydrogen‐bonded conformations involving a Met‐Aib‐β‐turn or a γ‐turn centered at Aib2 are considered. The results suggest that folded conformations may allow highly active interactions with the neutrophil formylpeptide receptor.
Activation of protein kinase C by an aromatic xenobiotic diacylglycerol analogue
Authors: Moorhouse Kathryn G. , Hutson David H. , Dodds Peter F.
Published: July 2002

Protein kinase C; Xenobiotic lipid; 3‐Phenoxybenzoic acid; Diacylglycerol; Phorbol ester
Inhibition of phosphatidic acid phosphohydrolase activity by sphingosine
Authors: Lavie Yaakov , Piterman Orly , Liscovitch Mordechai
Published: July 2002

Recent evidence indicates that a major fraction of diacylglycerol that is produced in hormonally stimulated cells arises by phosphatidylcholine hydrolysis via the sequential action of phospholipase D and phosphatidic acid phosphohydrolase (PAP). We have previously reported that sphingoid bases stimulate phospholipase D activity in NG108‐15 cells. The evidence presented here demonstrates that in sphingosine‐treated NG108‐15 cells, elevated phosphatidic acid levels are accompanied by a parallel, time‐ and dose‐dependent decrease in diacylglycerol levels. DL‐propranolol, a known inhibitor of PAP, exerted similar effects, suggesting that the action of sphingosine may have been due to inhibition of PAP activity. This prediction was confirmed in in vitro experiments in which it was demonstrated that sphingosine is as potent an inhibitor of both cytosolic and membrane‐associated PAP activity as propranolol. The hypothesis that sphingoid bases may exert a dual action in diacylglycerol signal termination is proposed.
A protein with homology to the C‐terminal repeat sequence of Octopus rhodopsin and synaptophysin is a member of a multigene family in Dictyostelium discoideum
Authors: Noegel Angelika A. , Gerisch Günther , Lottspeich Friedrich , Schleicher Michael
Published: July 2002

Monoclonal antibodies were raised against a protein with a molecular mass of 24 kDa that has been described as a membrane‐associated, actin binding protein from Dictyostelium discoideum [(1985) J. Cell Biol. 100, 727–735]. Using these monoclonal antibodies we isolated from a λgt11 expression library cDNA clones coding for this protein. The cDNA deduced amino acid sequence revealed the presence of an unusual carboxy‐terminus which has homologies to the C‐termini of Octopus rhodopsin and synaptophysin. This part of the protein sequence contains 5 direct repeats with the motif GYP (P)Q(P). Southern and Northern blots showed that this sequence is present in a series of Dictyostelium genes transcribed in all stages of development.
Solution structure of μ‐conotoxin GIIIA analysed by 2D‐NMR and distance geometry calculations
Authors: Ott Karl Heinz , Becker Stefan , Gordon Robert D. , Rüterjans Heinz
Published: July 2002

We have investigated the structure of μ‐conotoxin GIIIA by 2D‐NMR methods. The assignment of 1H NMR spectra and a quantitative analysis of NOE and J‐coupling data are presented. These results were used for the calculation of secondary structure elements of μ‐conotoxin GIIIA. Distance geometry calculations were carried out to define the global folding of the peptide.
Go associates with another 40 kDa brain protein
Authors: Denker Bradley M. , Neer Eva J.
Published: July 2002

Guanine nucleotide binding proteins (G proteins) mediate a variety of cellular responses to external stimuli. Pure G protein, receptor, and effector are sufficient to reconstitute hormonal activation of an effector in phospholipid vesicles, but other components may be important for specificity or localization in vivo. If another protein associates with G??? the molecular weight of Go solubilized from membranes would be larger than the molecular weight of Go after purification. We find that Go solubilized from bovine brain membranes by Triton X‐100 behaves as a single population of molecules on sucrose density gradients and gel filtration columns. Its molecular mass is about 40 kDa larger than pure Go. Association of Go with the other protein is fragile as the proteins dissociate on further purification. There was no difference in ADP‐ribosylation or tryptic cleavage of Go in larger and smaller form. These studies provide a basis for future experiments to stabilize the interaction and identify the protein.
Structure of adsorbed fibrinogen obtained by scanning force microscopy
Authors: Wigren Roger , Elwing Hans , Erlandsson Ragnar , Welin Stefan , Lundström Ingemar
Published: July 2002

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three‐dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM‐results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.
Phytochromes and bacterial sensor proteins are related by structural and functional homologies Hypothesis on phytochrome‐mediated signal‐transduction
Authors: Schneider-Poetsch Hansjörg A.W. , Braun Birgit , Marx Stefan , Schaumburg Anke
Published: July 2002

Phytochrome and bacterial sensor proteins are related by functional and structural homologies. They are both sensors of environmental stimuli and share structural homologies which comprise a domain of about 250 amino acids (about 28 kg mol−1). This domain is C‐terminal in phytochromes and in several bacterial sensor proteins. In both groups of sensors this domain undergoes conformational changes which are caused by the N‐terminal part sensing the stimulus. In the case of the bacterial sensors, the conformational alteration is, regulated by additional proteins, conferred to a corresponding regulator protein which then acts on transcription. The coincidences between the two group of sensors are striking enough to assume phytochrome to transduce signals in a way comparable to the bacterial two‐component systems.
Soluble human interleukin‐6‐receptor modulates interleukin‐6‐dependent N‐glycosylation of α1‐protease inhibitor secreted by HepG2 cells
Authors: Mackiewicz Andrzej , Rose-John Stefan , Schooltink Heidi , Laciak Maria , Górny Aleksander , Heinrich Peter C.
Published: July 2002

Interleukin‐6 (IL‐6) induces changes in gene expression and the N‐glycosylation pattern of acute‐phase proteins in hepatocytes. IL‐6 exerts its action via a cell surface receptor complex consisting of an 80 kDa IL‐6 binding protein (gp80) and a 130 kDa glycoprotein (gp130) involved in signal transduction. A genetically engineered gp80‐derived soluble human IL‐6‐receptor (shIL‐6‐R) significantly enhanced the IL‐6 effect on N‐glycosylation changes (revealed by reactivity with the lectin—concanavalin A) of a1‐protease inhibitor (PI) secreted by human hepatoma cells (HepG2). Stable transfection of IL‐6‐cDNA into HepG2 cells (HepG2‐IL‐6) resulting in constitutive secretion of 2 μg of IL‐6 per 106 cells in 24 h led to a down‐regulation of surface‐bound gp80 and subsequent homologous desensitization or HepG2‐IL‐6 cells towards IL‐6. Soluble human IL‐6‐R functionally substituted membrane‐bound gp80 resulting in a reconstitution of responsiveness of HepG2‐IL‐6 cells.
Molecular cloning and characterization of a novel calcium channel from rabbit brain
Authors: Niidome Tetsuhiro , Kim Man-Suk , Friedrich Thomas , Mori Yasuo
Published: July 2002

The complete amino acid sequence of a novel calcium channel (designated BII) from rabbit brain has been deduced by cloning and sequencing the cDNA. The BII calcium channel is structurally more closely related to the BI calcium channel than to the cardiac and skeletal muscle L‐type calcium channels. Blot hybridization analysis of RNA from different tissues and from different regions of the brain shows that the BII calcium channel is distributed predominantly in the brain, being abundant in the cerebral cortex, hippocampus and corpus striatum.
Identification of a domain of ETA receptor required for ligand binding
Authors: Adachi Miki , Yang Yan-Yan , Trzeciak Arnold , Furuichi Yasuhiro , Miyamoto Chikara
Published: July 2002

Various chimeric ETA and ETB receptors were produced in CHO cells for the elucidation of a specific domain which influences the affinity of the receptor toward BQ‐123, a selective ETA antagonist. Replacement of the first extracellular loop domain (B‐loop) of the ETA receptor with the corresponding domain of the ETB receptor, reduced the inhibition by BQ‐123 drastically, while the replacements of other extracellular domains of ETA did not. By contrast, the introduction of the B‐loop of ETA in place of the corresponding domain of the ETB receptor endowed the ETB‐based chimeric receptor with a sensitivity to BQ‐123. These observations suggest that the B‐loop domain of the ETA receptor is involved in ligand binding.
Dephosphorylation of human p34cdc2 kinase on both Thr‐14 and Tyr‐15 by human cdc25B phosphatase
Authors: Honda Reiko , Ohba Yoshiki , Nagata Akihisa , Okayama Hiroto , Yasuda Hideyo
Published: July 2002

In mammalian cells, p34
cdc2
kinase undergoes phosphorylation at threonine‐14, tyrosine‐15 and threonine‐161 in the S and G2 phases of the cell cycle. At the onset of mitosis, the kinase becomes dephosphorylated at threonine‐14 and tyrosine‐15, resulting in activation. Cdc25 phosphatase has been shown to dephosphorylate tyrosine‐15 in vitro, but whether it also does at threonine‐14 remains unclear. In this study, we have found that human cdc25B phosphatase dephosphorylates both threonine‐14 and tyrosine‐15 but not threonine‐161.
Demonstration of (Ca2+‐Mg2+‐ATPase activity of the neural cell adhesion molecule
Authors: Dzhandzhugazyan Karine , Bock Elisabeth
Published: July 2002

In this study a possible association between (Ca2+‐Mg2+‐ATPase activity and the neural cell adhesion molecule, NCAM, was investigated. The effects of various detergents on ATPase activity were evaluated, and it was found that solubilization of rat brain microsomes with 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate, CHAPS, released a major fraction of the (Ca2+‐Mg2+‐ATPase activity together with NCAM. Using different types of solid phase immunoadsorption it was shown that NCAM antibodies selectively isolated ATPase activity. Furthermore, agarose gel immunoeleetrophoresis of solubilized brain microsomes followed by ATPase assay directly in the gel revealed ATPase activity associated with the NCAM immunoprecipitate. The NCAM‐associated enzyme activity had a broad nucleoside triphosphate specificity and no strict selectivity for divalent cations, indicating that the enzyme probably is an ecto‐ATPase. This raises a series of intriguing questions in relation to NCAM adhesive functions.
Cyclin A‐mediated inhibition of intra‐Golgi transport requires p34cdc2
Authors: Mackay Deborah , Kieckbusch Regina , Adamczewski Jörg , Warren Graham
Published: July 2002

An in vitro assay was used to study the role of p34cdc2 in cyclin A‐mediated vesicular transport inhibition. It was shown that the S‐phase kinase p33cdk2 reduced the effect of cyclin A on transport assays performed with sHeLa cytosol, even though histone kinase was strongly activated. Also, transport with FT210 cytosol (which is temperature‐sensitive for p34cdc2) was inhibited by cyclin A only at the permissive temperature. However, the phosphatase inhibitor microcystin inhibited transport without any requirement for p34cdc2 activity. These results show that transport is inhibited by cyclin A via p34cdc2, and also by another kinase, possibly downstream of p34cdc2.
The C‐terminal tripeptide of glycosomal phosphoglycerate kinase is both necessary and sufficient for import into the glycosomes of Trypanosoma brucei
Authors: Sommer Jürg M. , Peterson Gregory , Keller Gilbert-A. , Parsons Marilyn , Wang C.C.
Published: July 2002

Glycosomal phosphoglycerate kinase (gPGK) of Trypanosoma brucei differs from the cytoplasmic isozyme (cPGK) in its higher isoelectric point characterized by clusters of positive charges along the polypeptide chain, and a 20 amino acid C‐terminal extension ending in serine‐serine‐leucine (SSL). While a C‐terminal SSL tripeptide is apparently not capable of directing luciferase to the peroxisomes in mammalian cells [J. Cell Biol. 108 (1989), 1657‐1664], we show here that it is sufficient for the import of luciferase as well as an unrelated protein, β‐glucuronidase, into the glycosomes of T. brucei, as determined by immunoelectron microscopy. The analysis of luciferase‐gPGK fusion proteins indicates that the only targeting signal for import of gPGK into the glycosome resides in this C‐terminal SSL sequence.
Biosynthetic lipid‐tagging of antibodies
Authors: Keinänen Kari , Laukkanen Marja-Leena
Published: July 2002

Chemical conjugation of fatty acids to antibodies generates lipid‐modified molecules which have found use in the targeting of liposome‐mediated drug delivery and in liposome‐based immunoassays. Alternatively, bacterial expression of antibodies as single‐chain Fv fragments fused to lipoprotein signal peptide and N‐terminal sequence leads to in vivo enzymatic addition of a single glycerolipid group at the N‐terminus of the molecule. This lipid‐modification converts the antibody from a soluble protein into a functional membrane‐bound molecule. These biosynthetically lipid‐tagged antibodies may prove useful for immobilization of antibodies to membranes in various biotechnological applications.
Inducible nitric oxide synthase in human lymphomononuclear cells activated by synthetic peptides derived from extracellular matrix proteins
Authors: Pérez-Mediavilla L.A. , López-Zabalza M.J. , Calonge M. , Montuenga L. , López-Moratalla N. , Santiago E.
Published: July 2002

Synthetic peptides with sequences present in extracellular matrix proteins are capable of causing the expression of the inducible form of nitric oxide synthase (iNOS), detected by immunocytochemistry, and the release of NO by human lymphomononuclear cells incubated in their presence. Active peptides are 15‐mers containing a characteristic 2‐6‐11 motif in which the amino acid residue at position 2 is Leu, Ile, Val, Gly, Ala or Lys; the residue at position 6 is always Pro; and residue 11 is Glu or Asp. The induction of iNOS in human monocytes and macrophages could be involved in the cytotoxicity against tumor cell lines also elicited by these peptides.
N‐(2‐Ferrocene‐ethyl)maleimide : a new electroactive sulphydryl‐specific reagent for cysteine‐containing peptides and proteins
Authors: Di Gleria K. , Hill H.A.O. , Wong L.L.
Published: July 2002

We report the synthesis and application of a specific electroactive label, N‐(2‐ferrocene‐ethyl)maleimide, which provides new redox properties to organic compounds and proteins possessing sulphydryl groups. Its reaction conditions with the cysteine‐containing peptide, glutathione, and a terminal monooxygenase enzyme, cytochrome P450cam are presented. The labelled peptide and enzyme acquired reversible electrochemical properties due to the attached ferrocene moiety.
Contraceptive use and the risk of HIV infection in Nairobi, Kenya
Authors: Mali J.K.G. , Hunter D.J. , Maggwa B.N. , Tukei P.M.
Published: July 2002

Objectives: To determine: (a) the prevalence of human immunodeficiency virus‐1 (HIV‐1) infection among women attending family planning clinics in Nairobi; and (b) the associations between contraceptive use and HIV infection. Methods: History, clinical examination and laboratory tests were used to obtain data from 4404 women attending family planning clinics in Nairobi. We conducted a case‐control study comparing HIV seropositive and seronegative women with regard to previous and current use of contraception. Results: The overall prevalence of HIV‐1 infection was 4.9% (95% C.I. 4.3–5.5). Previous and current use of oral contraceptives (OC), injectable contraceptives and the intrauterine device were not associated with a significant increase in risk, while current users of condoms had a non‐significant reduction in risk. OC use was significantly associated with cervical ectopy, but no significant association was evident between ectopy and HIV infection. Conclusion: The finding of no significant association between past or current OC use and risk of HIV infection suggests that any independent association that may exist between OC use and HIV risk is not large.
Myosin light chain phosphorylation‐dependent modulation of volume‐regulated anion channels in macrovascular endothelium
Authors: Nilius Bernd , Prenen Jean , Walsh Michael P. , Carton Iris , Bollen Mathieu , Droogmans Guy , Eggermont Jan
Published: July 2002

The Rho/Rho‐associated kinase (ROK) pathway has been shown to modulate volume‐regulated anion channels (VRAC) in cultured calf pulmonary artery endothelial (CPAE) cells. Since Rho/ROK can increase myosin light chain phosphorylation, we have now studied the effects of inhibitors of myosin light chain kinase (MLCK) or myosin light chain phosphatase (MLCP) on VRAC in CPAE. Application of ML‐9, an MLCK inhibitor, inhibited VRAC, both when applied extracellularly or when dialyzed into the cell. A similar inhibitory effect was obtained by dialyzing the cells with AV25, a specific MLCK inhibitory peptide. Conversely, NIPP1191–210, an MLCP inhibitory peptide, potentiated the activation of VRAC by a 25% hypotonic stimulus. These data indicate that activation of VRAC is modulated by MLC phosphorylation.
Opposite role of changes in mitochondrial membrane potential in different apoptotic processes
Authors: Salvioli Stefano , Barbi Cristiana , Dobrucki Jurek , Moretti Laura , Pinti Marcello , Pedrazzi Jessica , Pazienza Teresa Loredana , Bobyleva Valentina , Franceschi Claudio , Cossarizza Andrea
Published: July 2002

We have studied the role of changes in mitochondrial membrane potential (ΔΨ) in two widely‐used models of apoptosis, such as dexamethasone‐treated rat thymocytes and U937 human cells treated with tumor necrosis factor‐α and cycloheximide. To dissipate ΔΨ, we used low concentrations of valinomycin, unable per se to induce apoptosis, and demonstrated that the decline in ΔΨ exerts opposite effects in the two models. Indeed, in U937 cells, depolarization of mitochondria increased apoptosis, which decreased in rat thymocytes. This leads to the suggestion that disruption of ΔΨ plays opposite roles depending on the experimental model. In U937 cells, the drop of ΔΨ is a possible contributory cause for the apoptotic process; in rat thymocytes, it could be a limiting factor. We propose that these opposite effects could be due to the different ATP requirement of each apoptotic pathway.
Genomic Exploration of the Hemiascomycetous Yeasts: 20. Evolution of gene redundancy compared to Saccharomyces cerevisiae
Authors: Llorente Bertrand , Durrens Pascal , Malpertuy Alain , Aigle Michel , Artiguenave François , Blandin Gaëlle , Bolotin-Fukuhara Monique , Bon Elisabeth , Brottier Philippe , Casaregola Serge , Dujon Bernard , de Montigny Jacky , Lépingle Andrée , Neuvéglise Cécile , Ozier-Kalogeropoulos Odile , Potier Serge , Saurin William , Tekaia Fredj , Toffano-Nioche Claire , Wésolowski-Louvel Micheline , Wincker Patrick , Weissenbach Jean , Souciet Jean-Luc , Gaillardin Claude
Published: July 2002

We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others.
Dopamine β‐hydroxylase inactivation generates a protein‐bound quinone derivative
Authors: Slama Patrick , Jabre Frédéric , Tron Thierry , Réglier Marius
Published: July 2002

Bovine dopamine β‐hydroxylase (DbH) was inactivated by hydrogen peroxide and ascorbate in the presence of dioxygen. Both inactivated forms of the enzyme were investigated. We could highlight the presence of a quinone derivative bound to the protein, assumed as being dopa‐quinone, that is absent from active enzyme. Such results suggest that a tyrosinyl radical transiently forms during catalysis. Moreover we could show that addition of substrate tyramine to H2O2 incubates is responsible for a partial protection of DbH against inactivation.
Interactions of the XylS regulators with the C‐terminal domain of the RNA polymerase α subunit influence the expression level from the cognate Pm promoter
Authors: Ruiz Raquel , Ramos Juan L , Egan Susan M
Published: July 2002

The Pseudomonas putida meta‐cleavage operon encodes the enzymes for the catabolism of alkylbenzoates. Activation of meta‐operon transcription is mediated by the XylS protein which, upon activation by effectors, binds two sites between −70 and −35 with respect to the main transcription initiation point at the Pm promoter. Two naturally occurring regulators, XylS and XylS1, that differ by only five amino acids, have been analyzed with regard to potential interactions of these positive regulators with the C‐terminal domain of the α subunit of RNA polymerase (α‐CTD). For these studies we expressed a derivative of α deprived of the entire C‐terminal domain (α‐Δ235) and found that expression from Pm with XylS or XylS1 was significantly decreased. To discern whether α‐CTD activation depended on interactions with DNA and/or XylS proteins we tested a large collection of alanine substitutions within α‐CTD. Most substitutions that had an effect on XylS and XylS1‐dependent transcription were located in or adjacent to helix 1 and 4, which are known to be involved in α‐CTD interactions with DNA. Two alanine substitutions in helix 3 (residues 287 and 291) identified a putative region of α‐CTD/XylS regulator interactions.
Histone deacetylases in replicative senescence: evidence for a senescence‐specific form of HDAC‐2
Authors: Wagner M. , Brosch G. , Zwerschke W. , Seto E. , Loidl P. , Jansen-Dürr P.
Published: July 2002

To analyze mechanisms of senescence‐associated gene expression, we have investigated histone deacetylases (HDACs) in human fibroblasts undergoing replicative senescence. We found that the overall acetylation pattern of histones does not vary detectably with replicative senescence. By Northern blot and Western blot, we found a significant decrease in the abundance of HDAC‐1 in senescent cells. Biochemical analysis of deacetylase activities in extracts from old and young cells revealed a striking difference. While by anion exchange chromatography we found a single peak of activity in extracts from young cells, which coincided with the elution of both HDAC‐1 and HDAC‐2, in senescent cells a second peak of activity was found. This second peak of activity is associated with HDAC‐2 but does not contain HDAC‐1. These results suggest that HDAC‐2 is present in at least two distinct forms, one of which is specific for senescent cells. Further biochemical characterization of the enzyme activity revealed that addition of nicotinamide adenine dinucleotide (NAD) did not detectably influence the activity of any fraction, suggesting that NAD is not an essential co‐factor for the analyzed HDACs from diploid human fibroblasts.
Cloning and sequence of full‐length cDNAs encoding the human neuronal nicotinic acetylcholine receptor (nAChR) subunits β3 and β4 and expression of seven nAChR subunits in the human neuroblastoma cell line SH‐SY5Y and/or IMR‐321
Authors: Groot Kormelink Paul J , Luyten Walter H.M.L
Published: July 2002

Using PCR‐based techniques, we have isolated and sequenced the full‐length cDNAs that encode the human neuronal nAChR subunits α3,4,5,7 and β2,3,4 in the neuroblastoma cell lines SH‐SY5Y and/or IMR‐32. The predicted nAChR β3‐ and β4‐subunit proteins contain 458 and 498 amino acids, respectively. Except for the β2, all the other cloned cDNAs showed differences with the published sequences. Northern blots show expression of the nAChR subunits α3,7 and β2,4 in the human neuroblastoma cell line SH‐SY5Y, with intensity of the hybridisation signal decreasing in the order α3>β4>β2>α7.
The Na+/e− stoichiometry of the Na+‐motive NADH : quinone oxidoreductase in Vibrio alginolyticus
Authors: Bogachev Alexander V , Murtazina Rachilya A , Skulachev Vladimir P
Published: July 2002

A method is proposed to estimate the stoichiometries of primary Na+‐pumps in intact bacterial cells. It is based on technique when the H+/e− stoichiometry is measured in the presence of protophorous uncoupler and in the absence of penetrating ions other than H+. Under these conditions, the H+ influx discharges membrane potential generated by the Na+ pump so the Na+/e− and H+/e− ratios become equal. Using this approach it is shown that the Na+/e− ratio for the Na+‐motive NADH : quinone oxidoreductase of Vibrio alginolyticus is equal to 0.71±0.06. The Na+/e− stoichiometry appears to be ≈1, provided that the contribution of the non‐coupled NADH : quinone oxidoreductase, which is resistant to low HQNO concentrations, is taken into account.
Crystal structure of vipoxin at 2.0 Å: an example of regulation of a toxic function generated by molecular evolution
Authors: Perbandt M , Wilson J.C , Eschenburg S , Mancheva I , Aleksiev B , Genov N , Willingmann P , Weber W , Singh T.P , Betzel Ch
Published: July 2002

Vipoxin is the main toxic component in the venom of the Bulgarian snake Vipera ammodytes meridionalis, the most toxic snake in Europe. Vipoxin is a complex between a toxic phospholipase A2 (PLA2) and a non‐toxic protein inhibitor. The structure is of genetic interest due to the high degree of sequence homology (62%) between the two functionally different components. The structure shows that the formation of the complex in vipoxin is significantly different to that seen in many known structures of phospholipases and contradicts the assumptions made in earlier studies. The modulation of PLA2 activity is of great pharmacological interest, and the present structure will be a model for structure‐based drug design.
C‐terminal end of v‐src protein interacts with peptide coded by gadd7/adapt15‐like RNA in two‐hybrid system
Authors: Mizenina Olga , Yanushevich Yuriy , Musatkina Elena , Rodina Anna , Camonis Jacques , Tavitian Armand , Tatosyan Alexander
Published: July 2002

The significant differences in the metastatic properties of hamster fibroblasts transformed by the Rous sarcoma virus (RSV) were associated with mutations in the v‐src carboxy‐terminal region. To identify the capacity of this region for protein–protein interaction the two‐hybrid system was used. The cDNA clone (vseap1), producing the protein specifically bound with the v‐src C‐terminal part in yeast cells in vivo and in GST‐fusion system in vitro was isolated. Vseap1 shared 68% of homology with stressful agents induced RNA‐gadd7/adapt15. Two vseap1 specific messenger RNAs were identified: 0.9‐kbp RNA expressed in all transformed cells and three times less in embryo fibroblasts; 3.1‐kbp transcript was deleted in the cells with suppressed v‐src activity and H2O2resistance.
Effect of rutin and its copper complex on superoxide formation and lipid peroxidation in rat liver microsomes
Authors: Afanas'ev Igor B , Ostrachovich Elena A , Korkina Ludmila G
Published: July 2002

Two free radical scavengers, bioflavonoid rutin and the copper‐rutin complex Cu(Rut)Cl2, inhibited lucigenin‐amplified chemiluminescence and lipid peroxidation in rat liver microsomes, Cu(Rut)Cl2 being a 5–9 times more efficient inhibitor than rutin. The enhanced inhibitory activity of Cu(Rut)Cl2 was due to the presence of the additional superoxide‐dismutating center (Cu), as follows from the comparison of its effects on microsomal chemiluminescence and cytochrome c reduction by xanthine oxidase. Similar effects of both inhibitors on superoxide production and lipid peroxidation as well as the elevated activity of Cu(Rut)Cl2 indicate an important role of superoxide ion in the initiation of microsomal lipid peroxidation.
Interferon regulatory factor‐1 is a major regulator of epidermal growth factor receptor gene expression
Authors: Rubinstein Yaffa R , Proctor Kimberle N , Bergel Michael , Murphy Barbara , Johnson Alfred C
Published: July 2002

Overexpression of the epidermal growth factor receptor (EGFR) occurs in many tumors and in breast cancer correlates with poor prognosis for treatment. Here, we report that interferon regulatory factor‐1 (IRF‐1) induces EGFR promoter activity up to 200‐fold compared to 3–10‐fold induction by other regulators. The region of the promoter that is required for this induction was defined using deletion mutants. In addition, we found that IRF‐1 and tricostatin A, a deacetylase inhibitor, have a synergistic effect on EGFR promoter activity. This indicates that the increase in EGFR promoter activity by IRF‐1 may also involve changes in chromatin structure. These results identify IRF‐1 as a major regulator of EGFR gene expression.
Soluble IL‐1 receptor type I binds to human dermal fibroblasts and induces calcium flux
Authors: Spörri Bernhard , Wiesmann Ueli N. , Ochsenbein Regula M. , Loetscher Marcel , Waelti Ernst R. , Lottaz Daniel , Bickel Matthias
Published: July 2002

Soluble cytokine receptors appear to modify ligand concentrations by stabilizing ligands or by specifically inhibiting interactions of ligands with their membrane‐bound receptors. Here we describe a new function of the soluble interleukin‐1 receptor type I (IL‐1sR I). This receptor induced a transient rise of intracellular free calcium concentration in human dermal fibroblasts in a dose‐dependent fashion. Mobilization of calcium by IL‐1sR I was abolished in the presence of an equimolar concentration of IL‐1 receptor antagonist (IL‐1ra). Neutralizing antibodies against IL‐1β also abolished calcium mobilization stimulated with IL‐1sR I indicating that IL‐1β is involved. IL‐1sR I bound with high affinity (K
d 1–2 nM) to the fibroblasts. In addition, IL‐1sR I enhanced expression of IL‐6 and IL‐8 mRNA. The observation that IL‐1sR I can act as a ligand and agonist for membrane IL‐1 extends the concept of the ligand‐receptor functions of both IL‐1 and IL‐1sR I and adds a new dimension to the cytokine network.