bluuebunny/crossref_metadata_2025 huggingface dataset:
Different sensitivity of rabbit heart and skeletal muscle to endotoxin‐induced impairment of mitochondrial function
Authors: Trumbeckaite Sonata , Opalka Jens R , Neuhof Christiane , Zierz Stephan , Gellerich Frank N
Published: March 2003

The involvement of mitochondrial dysfunction in septic disturbances of tissues is controversial. The aim of this study was to investigate the effects of endotoxin‐induced sepsis on the function of heart and skeletal muscle mitochondria. Rabbits were made septic by subcutaneous injection of endotoxin (lipopolysaccharide, LPS) from Escherichia coli at concentrations of 100 or 150 µg LPS·kg−1 24 h prior to the experiments. Mitochondrial respiration was measured in saponin‐skinned muscle fibers and compared with photometrically detected activities of respiratory chain enzymes as well as with function of perfused hearts. In heart fibers a dosage of 100 µg LPS·kg−1 caused a significant decrease of state 3‐respiration for the substrates pyruvate (−38%), octanoyl‐carnitine (−38%) and succinate (−30%) with correspondingly decreased respiratory control indexes (RCI). In addition, endotoxin caused a decreased temporal stability of the rate of state 3‐respiration. At least in part these changes can be attributed to a reduced activity of complex I + III (−50%) of the respiratory chain. State 4‐respiration rates were not significantly altered. The lowered state 3‐respiration in heart mitochondria seems to contribute to the impairment of heart muscle function as detected by an increase of coronary vascular resistance (CVR) in endotoxin‐treated hearts. Functional properties of mitochondria from M. Vastus lasteralis were not affected by 100 µg LPS·kg−1 but a higher dosage of 150 µg LPS·kg−1 caused decreased RCI for the substrates pyruvate (−29%) and octanoyl‐carnitine (−32%). Also the activity of complex I + III was not significantly affected at lower dose of endotoxin but decreased (−42%) after treatment with 150 µg LPS·kg−1. Results demonstrate the involvement of impaired mitochondria in the pathophysiology of septic organ failure and a tissue specifity of endotoxaemia.
Expression of coproporphyrinogen oxidase and synthesis of hemoglobin in human erythroleukemia K562 cells
Authors: Taketani Shigeru , Furukawa Takako , Furuyama Kazumichi
Published: March 2003

Coproporphyrinogen oxidase (CPOX), the sixth enzyme in the heme‐biosynthetic pathway, catalyzes oxidative decarboxylation of coproporphyrinogen to protoporphyrinogen and is located in the intermembrane space of mitochondria. To clarify the importance of CPOX in the regulation of heme biosynthesis in erythroid cells, we established human erythroleukemia K562 cells stably expressing mouse CPOX. The CPOX cDNA‐transfected cells had sevenfold higher CPOX activity than cells transfected with vector only. Expression of ferrochelatase and heme content in the transfected cells increased slightly compared with the control. When K562 cells overexpressing CPOX were treated with δ‐aminolevulinic acid (ALA), most became benzidine‐positive without induction of the expression of CPOX or ferrochelatase, and the heme content was about twofold higher than that in ALA‐treated control cells. Increases in cellular heme concomitant with a marked induction of the expression of heme‐biosynthetic enzymes, including CPOX, ferrochelatase and erythroid‐specific δ‐aminolevulinic acid synthase, as well as of α‐globin synthesis, were observed when cells were treated with transforming growth factor (TGF)β1. These increases in the transfected cells were twice those in control cells, indicating that overexpression of CPOX enhanced induction of the differentiation of K562 cells mediated by TGFβ1 or ALA. Conversely, the transfection of antisense oligonucleotide to human CPOX mRNA into untreated and TGFβ1‐treated K562 cells led to a decrease in heme production compared with sense oligonucleotide‐transfected cells. These results suggest that CPOX plays an important role in the regulation of heme biosynthesis during erythroid differentiation.
Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by 13C‐NMR
Authors: Yamaguchi Satoru , Yonebayashi Koka , Konishi Hidemasa , Tuzi Satoru , Naito Akira , Lanyi Janos K. , Needleman Richard , Saitô Hazime
Published: March 2003

We have examined the 13C‐NMR spectra of [3‐13C] Ala‐labeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors such as ionic strength, temperature, pH, truncation of the C‐terminal α helix, and site‐directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C‐terminal α helix and loops. It is found that the surface structure can be characterized as a complex stabilized by salt bridges or metal‐mediated linkages among charged side chains. The surface complex in bacteriorhodopsin is most pronounced under the conditions of 10 mm NaCl at neutral pH but is destabilized to yield relaxed states when environmental factors are changed to high ionic strength, low pH and higher temperature. These two states were readily distinguished by associated spectral changes, including suppressed (cross polarization‐magic angle spinning NMR) or displaced (upfield) 13C signals from the C‐terminal α helix, or modified spectral features in the loop region. It is also noteworthy that such spectral changes, when going from the complexed to relaxed states, occur either when the C‐terminal α helix is deleted or site‐directed mutations were introduced at a cytoplasmic loop. These observations clearly emphasize that organization of the cytoplasmic surface complex is important in the stabilization of the three‐dimensional structure at ambient temperature, and subsequently plays an essential role in biological functions.
Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity
Authors: Stathopoulos Constantinos , Tekos Apostolos , Zarkadis Ioannis K. , Drainas Denis
Published: March 2003

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5′‐hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.
Varying competence for protein import into chloroplasts during the cell cycle in Chlamydomonas
Authors: Su Qingxiang , Schild Christof , Schumann Philipp , Boschetti Arminio
Published: March 2003

By studying the import of radioactively labelled small subunit of ribulose‐1,5‐bisphosphate carboxylase (pSS) into chloroplasts of the green alga C. reinhardtii cw‐15 protein delivery to chloroplasts was found to vary during the cell cycle. Chloroplasts were isolated from highly synchronous cultures at different time points during the cell cycle. When pSS was imported into ‘young’ chloroplasts isolated early in the light period about three times less pSS was processed to small subunit SS than in ‘mature’ chloroplasts from the middle of the light period. In ‘young’ chloroplasts also, less pSS was bound to the envelope surface. During the second half of the light period the import competence of isolated chloroplasts decreased again when based on chlorophyll content or cell volume, but did not change significantly when related to chloroplast number. Measurements of pSS binding to the surface of chloroplasts of different age indicated that the adaptation of protein import competence during the cell cycle is due to a variation of the number of binding sites per chloroplast surface area, rather than to modulation of the binding constant.
Specific inhibition of transforming growth factor‐β2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides
Authors: Shen Zhong‐Jian , Kim Sang Kook , Kwon Oh‐Shin , Lee Young Sup , Moon Byung Jo
Published: March 2003

To elucidate the role of endogenous transforming growth factor (TGF)‐β2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S‐ODNs) complementary to regions in mRNA of TGF‐β2 were synthesized and examined their effects on TGF‐β2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S‐ODNs were designated for three different target regions in the mRNA of TGF‐β2. Among several antisense S‐ODN analyzed, an oligonucleotide (AS‐11) complementary to the translation initiation site of mRNA of TGF‐β2 demonstrated a selective and strong inhibitory effect on TGF‐β2 production in osteoblast cells. Other antisense S‐ODNs which were designated for other regions in mRNA of TGF‐β2 and one‐ or three‐base mismatched analogs of AS‐11 showed little or much less antisense activities than AS‐11. Therefore, the most effective target site in mRNA of TGF‐β2 is at the initiation codon region. The antisense effects of AS‐11 were observed without reduction of levels of mRNA of TGF‐β2. Furthermore, the inhibition of TGF‐β2 expression by antisense S‐ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF‐β2 in osteoblast cells.
A novel RNA polymerase binding site upstream of the galactose promoter in Escherichia coli exhibits promoter‐like activity
Authors: Sur Runa , Debnath Dipanwita , Mukhopadhyay Jayanta , Parrack Pradeep
Published: March 2003

RNA polymerase is known to bind and utilize the overlapping promoters P1 and P2 in Escherichia coli galactose operon. We have identified an additional specific site upstream of P2, where RNA polymerase binds in a heparin‐resistant manner. Binding of polymerase to this site, termed P3, occurs simultaneous to its binding at P1/P2. We have located this P3 site by DNase I footprinting. A 63 base pair region centered around position − 100 with respect to galP1 is protected by polymerase. Interestingly, a Pribnow box TATAAT is present within this protected region (−103 to −108). We have shown that transcription occurs from P3 in vitro. Primer extension analysis provides direct evidence that P3 is transcribed in vivo. The start site of transcription has been mapped at −96 position relative to galP1. β‐galactosidase assays with different gal promoter constructs reveal that while P3 alone functions as a weak in vivo promoter, it has a synergistic effect on transcription from the gal operon, since deletion of P3 or specifically mutating its −10 region result in a substantial reduction in the gal promoter activity.
Multisite control of the Crabtree effect in ascites hepatoma cells
Authors: Rodríguez‐Enríquez Sara , Juárez Oscar , Rodríguez‐Zavala José S. , Moreno‐Sánchez Rafael
Published: March 2003

AS‐30D hepatoma cells, a highly oxidative and fast‐growing tumor line, showed glucose‐induced and fructose‐induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mm glucose or 10 mm fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6‐phosphate, fructose 6‐phosphate and fructose 1,6‐bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS‐30D cells, incubated in media with low Pi (0.6 mm) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.
Molecular cloning and functional expression of rat liver cytosolic acetyl‐CoA hydrolase
Authors: Suematsu Naoya , Okamoto Kazuki , Shibata Kiyotaka , Nakanishi Yoko , Isohashi Fumihide
Published: March 2003

A cytosolic acetyl‐CoA hydrolase (CACH) was purified from rat liver to homogeneity by a new method using Triton X‐100 as a stabilizer. We digested the purified enzyme with an endopeptidase and determined the N‐terminal amino‐acid sequences of the two proteolytic fragments. From the sequence data, we designed probes for RT‐PCR, and amplified CACH cDNA from rat liver mRNA. The CACH cDNA contains a 1668‐bp ORF encoding a protein of 556 amino‐acid residues (62 017 Da). Recombinant expression of the cDNA in insect cells resulted in overproduction of functional acetyl‐CoA hydrolase with comparable acyl‐CoA chain‐length specificity and Michaelis constant for acetyl‐CoA to those of the native CACH. Database searching shows no homology to other known proteins, but reveals high similarities to two mouse expressed sequence tags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.
Spectroscopic investigation and determination of reactivity and structure of the tetraheme cytochrome c3 from Desulfovibrio desulfuricans Essex 6
Authors: Einsle Oliver , Foerster Stefanie , Mann Karlheinz , Fritz Günter , Messerschmidt Albrecht , Kroneck Peter M. H.
Published: March 2003

Cytochrome c3, a small (14‐kDa) soluble tetraheme protein was isolated from the periplasmic fraction of Desulfovibrio desulfuricans strain Essex 6. Its major physiological function appears to be that of an electron carrier for the periplasmic hydrogenase. It has been also shown to interact with the high‐molecular‐mass cytochrome complex in the cytoplasmic membrane, which eventually feeds electrons into the membraneous quinone pool, as well as with the membrane‐associated dissimilatory sulfite reductase. The EPR spectra show features of four different low‐spin Fe(III) hemes. Orthorhombic crystals of cytochrome c3 were obtained and X‐ray diffraction data were collected to below 2 Å resolution. The structure was solved by molecular replacement using cytochrome c3 from D. desulfuricans ATCC 27774 as a search model.
Mechanism of nitrite‐stimulated catalysis by lactoperoxidase
Authors: Brück T. B. , Fielding R. J. , Symons M. C. R. , Harvey P. J.
Published: March 2003

The reactions of lactoperoxidase (LPO) intermediates compound I, compound II and compound III, with nitrite ( ) were investigated. Reduction of compound I by

was rapid (k2 = 2.3 × 107 m−1·s−1; pH = 7.2) and compound II was not an intermediate, indicating that

radicals are not produced when

reacts with compound I. The second‐order rate constant for the reaction of compound II with

at pH = 7.2 was 3.5 × 105 m−1·s−1. The reaction of compound III with

exhibited saturation behaviour when the observed pseudo first‐order rate constants were plotted against

concentrations and could be quantitatively explained by the formation of a 1 : 1 ratio compound III/NO2− complex. The Km of compound III for

was 1.7 × 10−4 m and the first‐order decay constant of the compound III/ complex was 12.5 ± 0.6 s−1. The second‐order rate constant for the reaction of the complex with

was 3.3 × 103 m−1·s−1. Rate enhancement by

does not require

as a redox intermediate.

accelerates the overall rate of catalysis by reducing compound II to the ferric state. With increasing levels of H2O2, there is an increased tendency for the catalytically dead‐end intermediate compound III to form. Under these conditions, the ‘rescue’ reaction of

with compound III to form compound II will maintain the peroxidatic cycle of the enzyme.
Synthesis of novel anti‐inflammatory peptides derived from the amino‐acid sequence of the bioactive protein SV‐IV
Authors: Ialenti Armando , Santagada Vincenzo , Caliendo Giuseppe , Severino Beatrice , Fiorino Ferdinando , Maffia Pasquale , Ianaro Angela , Morelli Francesco , Di Micco Biagio , Cartenì Maria , Stiuso Paola , Metafora Vittoria , Metafora Salvatore
Published: March 2003

SV‐IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies‐specific immunomodulatory, anti‐inflammatory, and pro‐coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV‐IV, we became interested in the study of its structure–function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti‐inflammatory properties of the protein not associated with other biological activities (pro‐coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti‐inflammatory activity is located in the N‐terminal region of the molecule, the 8–16 peptide fragment being the most active; (c) the identified anti‐inflammatory peptide derivatives do not seem to possess pro‐coagulant activity, even though this particular function has been located in the 1–70 segment of the molecule.
Changes in the levels of low‐abundance brain proteins induced by kainic acid
Authors: Krapfenbauer Kurt , Berger Michael , Friedlein Arno , Lubec Gert , Fountoulakis Michael
Published: March 2003

Low‐abundance gene products are of interest in proteomic studies, because they are probably involved in disease‐related changes and their altered levels or modifications may carry significant biological information. Detection of low‐abundance proteins of a proteome is one of the major limitations of proteomics and a scientific challenge. We investigated the changes in the levels of low‐abundance rat brain cytosolic proteins after administration of kainic acid, a potent neurotoxin and excitatory amino acid. The cytosolic proteins from controls and animals treated with kainic acid were fractionated on an ion‐exchange column. The fractions collected were analyzed by 2D electrophoresis, and the proteins with altered levels were identified by matrix‐assisted laser desorption ionization or ion‐spray MS. We found a manifold decrease in annexin VII, heat‐shock cofactor HOP/p60 and SP‐22 and a manifold increase in heparin‐binding protein p30. The results suggest, respectively, the involvement of an apoptotic pathway, recruitment of the heat‐shock protein machinery, generation of an antioxidant response, and, probably, induction of repair mechanisms. Three of the four proteins with altered levels had not been previously detected in the cytosolic fraction, and detection of the altered levels was possible only after the protein‐enriching step.
SP3 and AP‐1 mediate transcriptional activation of the lamin A proximal promoter
Authors: Muralikrishna Bh , Parnaik Veena K.
Published: March 2003

Lamin A is a major component of the nuclear lamina that is expressed in various types of differentiated cells. We have analysed previously the putative promoter sequences of the gene and shown that the rat lamin A proximal promoter contains two essential motifs, a GC box that can bind to Sp1 and Sp3, and an AP‐1 motif that can bind to c‐Jun and c‐Fos. In this study we have investigated the role of Sp1 and Sp3 in transactivation of the promoter. Functional analysis of the promoter in Drosophila SL2 cells has demonstrated that it is inactive in the absence of Sp proteins. Activation by expression of Sp3 is more pronounced than that by Sp1 although both proteins can bind to the GC box in vitro; activation clearly depends on an intact GC box as deduced from mutant analysis. Promoter activity in SL2 cells also requires an intact AP‐1 motif, which can bind to endogenous Drosophila Jun and Fos proteins. Furthermore, overexpression of c‐Jun and c‐Fos results in fourfold activation of the promoter in PCC‐4 embryonal carcinoma cells. Our demonstration that activation of the lamin A proximal promoter is mediated by Sp3 and AP‐1 transcription factors affords a basis for further studies on the regulation of this important gene during development and disease.
Regulation of laminin β2 chain gene expression in human cancer cell lines
Authors: Durkin Marian E. , Nielsen Finn C. , Loechel Frosty , Albrechtsen Reidar , Wewer Ulla M.
Published: March 2003

The laminin β2 chain is a basement membrane component expressed in a tissue‐ and developmental stage‐specific manner. In this report we have examined the transcriptional and post‐transcriptional regulation of the human laminin β2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma and clone A colon carcinoma cells express the laminin β2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the β2 chain. Segments of the β2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested by transient transfection. Sequences downstream of the transcription start site between nucleotides +91 and +120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides −164 to −667 and between nucleotides −667 to −1724. Genomic DNA at the 3′ end of the gene also appeared to have enhancer activity, as a 1.1‐kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides −667/+297 promoter segment ≈ threefold. Alternative splicing of the first intron of the human laminin β2 chain gene generates two isoforms of the 5′ untranslated region of the β2 chain mRNA. The translational efficiencies of the two laminin β2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell β2 chain mRNA, transient transfection of chimeric β2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates.
Transcriptionally and post‐transcriptionally regulated response of 13 calmodulin genes to tobacco mosaic virus‐induced cell death and wounding in tobacco plant
Authors: Yamakawa Hiromoto , Mitsuhara Ichiro , Ito Naoko , Seo Shigemi , Kamada Hiroshi , Ohashi Yuko
Published: March 2003

We isolated 13 tobacco calmodulin (CaM) genes, NtCaM1–13, and analyzed their expression profile in response to pathogen infection and wounding using specific DNA probes for individual CaM genes and specific antibodies for CaM proteins in groups I (NtCaM1/2), II (NtCaM3/4/5/6/7/8/11/12 and 9/10) and III (NtCaM13), respectively. Synchronous cell death in tobacco mosaic virus (TMV)‐infected N‐gene‐containing tobacco leaves accompanied a predominant accumulation of NtCaM1, 2 and 13 transcripts and NtCaM13‐type protein, which is a possible ortholog of soybean defense‐involved CaM (SCaM‐4), preceding induction of PR‐1 and PR‐3 defense genes. Accumulation of NtCaM1, 2, 3 and 4 transcripts was induced within 30 min after wounding and NtCaM1‐type protein accumulated transiently after wounding. NtCaM13‐type protein, which was found at a low level in healthy leaves, decreased instantly after wounding. The treatment with a proteasome inhibitor, lactacystin, enhanced wound‐induced accumulation of NtCaM1‐type protein and inhibited wound‐induced decrease of NtCaM13‐type protein, suggesting that proteasome activity is involved in the degradation of these CaMs. Thus, our results indicate that levels of individual CaM proteins are differentially regulated both transcriptionally and post‐transcriptionally in tobacco plants that are exposed to stresses such as pathogen‐induced hypersensitive cell death and wounding.
The contribution of metal ions to the conformational stability of ribonuclease T1
Authors: Deswarte Joeri , De Vos Stefan , Langhorst Ulrike , Steyaert Jan , Loris Remy
Published: March 2003

In the crystalline state, ribonuclease T1 binds calcium ions at different lattice‐dependent positions. In solution, its conformational stability is also remarkably increased in the presence of divalent metal ions. Combining urea unfolding studies and X‐ray crystallography, we compared the presence of several metal ions at specific sites in the protein to their contribution to the overall stabilizing effect in solution. We constructed thermodynamic cycles involving particular metal ions and specific carboxylate functions. The resulting coupling energies indicate that some (but not all) metal ions found at lattice contacts in crystal structures may indeed significantly contribute to stability enhancement in the presence of metal ions in solution.
Galactofuranoic‐oligomannose N‐linked glycans of α‐galactosidase A fromAspergillus niger
Authors: Wallis Gregg L. F. , Easton Richard L. , Jolly Karen , Hemming Frank W. , Peberdy John F.
Published: March 2003

Extracellular α‐galactosidase A was purified from the culture filtrate of an over‐producing strain ofAspergillus nigercontaining multiple copies of the encodingaglAgene under the control of the glucoamylase (glaA) promoter. Endoglycosidase digestion followed by SDS/PAGE, lectin and immunoblotting suggested that glycosylation accounted for ≈ 25% of the molecular size of the purified protein. Monosaccharide analysis showed that this was composed ofN‐acetyl glucosamine, mannose and galactose. Mild acid hydrolysis, mild methanolysis, immunoblotting and exoglycosidase digestion indicated that the majority of the galactosyl component was in the furanoic conformation (β‐d‐galactofuranose, Galf). At least 20 differentN‐linked oligosaccharides were fractionated by high‐pH anion‐exchange chromatography following release from the polypeptide by peptide‐N‐glycosidase F. The structures of these were subsequently determined by fast atom bombardment mass spectrometry to be a linear series of Hex7−26HexHAc2. Indicating that oligosaccharides from GlcNAc2Man7, increasing in molecular size up to GlcNAc2Man24were present. Each of these were additionally substituted with up to three β‐Galfresidues. Linkage analysis confirmed the presence of mild acid labile terminal hexofuranose residues. These results show that filamentous fungi are capable of producing a heterogeneous mixture of high molecular‐sizeN‐linked glycans substituted with galactofuranoic residues, on a secreted glycoprotein.
The hemoglobin system of the brown moray Gymnothorax unicolor
Authors: Tamburrini Maurizio , Verde Cinzia , Olianas Alessandra , Giardina Bruno , Corda Marcella , Sanna Maria T. , Fais Antonella , Deiana Anna M. , di Prisco Guido , Pellegrini Mariagiuseppina
Published: March 2003

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion‐exchange chromatography. The oxygen‐binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5–8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co‐operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co‐operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co‐operativity, and displayed a normal Bohr effect; the addition of chloride increased co‐operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino‐acid sequences of the α and β chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.
Chicken interleukin‐6
Authors: Schneider Kirsten , Klaas Reinhard , Kaspers Bernd , Staeheli Peter
Published: March 2003

Suppression subtractive hybridization technology was used to identify differentially expressed genes in spleens of chickens that had been treated with the synthetic immune modifier S‐28463. One induced chicken gene encoded a protein with about 35% sequence identity to human interleukin‐6 (IL‐6). It consists of 241 amino acids including a putative N‐terminal signal peptide of 47 residues. Bacterially expressed chicken IL‐6 (ChIL‐6) carrying a histidine tag in place of the signal peptide was biologically active: it induced proliferation of the IL‐6‐dependent murine hybridoma cell line 7TD1. The concentration of ChIL‐6 required for half‐maximal proliferative response was ≈ 60 pg·mL−1. When injected intravenously into adult chickens, purified recombinant ChIL‐6 induced an increase in serum corticosterone levels. Supernatants of chicken LMH and monkey COS‐7 cells transiently transfected with a ChIL‐6 expression construct induced proliferation of 7TD1 cells, demonstrating that recombinant ChIL‐6 from eukaryotic cells is also active.
Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1
Authors: Vetter Stefan W. , Leclerc Estelle
Published: March 2003

We have previously characterized the calcium‐dependent calmodulin (CaM)‐binding domain (Ser76–Ser92) of the 135‐kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem.258, 567–671]. Here we demonstrate that phosphorylation of two serine residues within the 17‐residue peptide alters their ability to adopt α helical conformation in a position‐dependent manner. The helical content of the peptides was determined by CD‐spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di‐phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82–Phe86. The ability of the peptides to adopt α helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM‐binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide‐binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides.
Mutational analysis of the J recombination signal sequence binding protein (RBP‐J)/Epstein–Barr virus nuclear antigen 2 (EBNA2) and RBP‐J/Notch interaction
Authors: Fuchs Klaus Peter , Bommer Guido , Dumont Elisabeth , Christoph Barbara , Vidal Marc , Kremmer Elisabeth , Kempkes Bettina
Published: March 2003

Epstein–Barr virus nuclear antigen 2 (EBNA2) and the Notch protein both function within the nucleus as transcriptional adaptor proteins. EBNA2 plays a key role during the immortalization of primary B‐cells by Epstein–Barr virus (EBV). Notch proteins are involved in lymphomagenesis as well as in multiple cell fate decisions during tissue differentiation and development. Both, EBNA2 and Notch interact with the DNA binding protein RBP‐J and thereby gain access to the promoter of their target genes. In order to identify regions within the J recombination signal sequence binding protein (RBP‐J), that are relevant for either the Notch or the EBNA2 interaction, we have performed a mutational analysis of RBP‐J. A library of RBP‐J mutants was screened by a reverse two‐hybrid system for alleles that fail to bind to either EBNA2 or Notch. The sequence analysis of these alleles reveals that a limited and particularly distinct number of amino‐acid positions are relevant for either interaction only. Given the important role of RBP‐J in B‐cell immortalization, the EBNA2/RBP‐J protein–protein interaction could be a candidate target for therapeutic intervention in EBV related diseases.
Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803
Authors: Lamparter Tilman , Esteban Berta , Hughes Jon
Published: March 2003

The phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 forms holoprotein adducts with close spectral similarity to plant phytochromes when autoassembled in vitro with bilin chromophores. Cph1 is a 85‐kDa protein that acts as a light‐regulated histidine kinase seemingly involved in ‘two‐component’ signalling. This paper describes the improvement of Cph1 purification, estimation of the extinction coefficient of holo‐Cph1, spectral analyses of the assembly procedure and studies on quaternary structure. During assembly with the natural chromophore phycocyanobilin (PCB), a red‐shifted intermediate is observed. A similar result was obtained when phycoerythrobilin was used as chromophore. As shown by SDS/PAGE and Zn2+ fluorescence, the covalent attachment of PCB is blocked by 1 mm iodoacetamide, a cysteine‐derivatizing agent. When PCB was incubated with blocked apo‐Cph1, again a shoulder at longer wavelengths appeared. It is therefore proposed that the long‐wavelength‐absorbing form represents the protonated, noncovalently bound bilin. Biliverdin, which is neither protonated nor covalently attached, undergoes spectral changes in its blue‐absorbing band upon incubation with apo‐Cph1. On the basis of these data we therefore propose a three‐step model for phytochrome autoassembly. Size‐exclusion chromatography revealed different mobilities for the apoprotein, red‐absorbing Cph1‐PCB and far‐red‐absorbing Cph1‐PCB. The major peaks of both holoprotein adducts had apparent molecular masses ≈ 200 kDa, a result in agreement with the notion that autophosphorylation in sensory histidine kinases requires dimerization. When Cph1‐PCB was further purified by preparative native electrophoresis, the mobility on size‐exclusion chromatography was ≈ 100 kDa, and it was found to have lost its kinase activity, results implying that the material had lost its capacity to dimerize.
Reproductive Decisions in Female European Ground Squirrels: Factors Affecting Reproductive Output and Maternal Investment
Authors: Millesi Eva , Huber Susanne , Everts Lammina G. , Dittami John P.
Published: March 2003

Physiological and behavioural parameters associated with reproductive effort and success were investigated in female European ground squirrels Spermophilus citellus. The proportion of reproductive (lactating) females in the study population was over 90% and was not related to age. Timing of oestrus and ovulation was found to be affected by the female’s emergence date and condition. Females with low emergence mass showed delayed oestrus. Differences in ovulation dates were shown to affect reproductive output in terms of litter size and sex ratio. Early litters were larger and male biased. X‐ray techniques were used to determine intrauterine litter size in individual females. The results indicated that litter size and sex ratio were fixed prenatally. Lactation costs were reflected in the intensity of mass loss and duration of lactation. Mass loss varied with litter size, in that females with large litters showed a more rapid loss than others. The second parental investment parameter, lactation duration, varied among individual females and was dependent on the timing of reproduction and litter size (except yearlings). Early born litters, which were, in most cases, larger than later ones, were nursed longer. Prolonged lactation periods affected female condition in that they started prehibernation fattening later and entered hibernation with a lower mass than individuals that had shorter lactation periods. Yearling females probably could not afford the energetic costs of long lactation, independent of their offspring number. These results indicated that females with higher reproductive output and higher investment were unable to compensate these costs before hibernation. Consequences for these individuals could therefore be lower over‐winter survival or a delayed oestrus in the following season.
Oviposition Site Choice in the Yellow Dung Fly Scathophaga stercoraria
Authors: Ward Paul I. , Foglia Massimiliano , Blanckenhorn Wolf U.
Published: March 2003

Female yellow dung flies Scathophaga stercoraria oviposit on the dung surface, which is extremely uneven and other eggs are often already present. We examined the effects of these features on egg placement. We found that: 1. females preferred to lay on small hills on the dung surface and avoided depressions and points; and 2. they ignored the presence of other eggs. These results were confirmed in a field experiment, where the pattern of egg distribution on artificially formed dung pats was consistent with the predictions from the laboratory results. We further showed that survival to adult emergence was best when laying sites were experimentally manipulated so that the eggs were apparently laid on small hills, avoiding the drying effects of small points in the dung and possible drowning by (simulated) rain when laid in depressions in the dung surface. A female dung fly is clearly capable of making subtle decisions about the placement of her eggs, thus increasing her reproductive success.
Female Choice and the Consistency of Courtship Feeding in Black‐horned Tree Crickets Oecanthus nigricornis Walker (Orthoptera: Gryllidae: Oecanthinae)
Authors: Brown William D. , Kuns Michelle M.
Published: March 2003

Courtship feeding in insects is often strongly correlated with insemination duration and therefore provides a potential postcopulatory episode of sexual selection. We tested whether courtship feeding and other courtship traits in the black‐horned tree cricket Oecanthus nigricornis showed sufficient consistency potentially to respond to sexual selection by testing whether they differed significantly among males. Duration of courtship feeding differed among males when measured repeatedly and this caused significant differences in the duration of spermatophore attachment, a trait that determines the maximum duration of insemination and thus has important fitness consequences in crickets. We also partitioned variance in courtship behaviour between the sexes to test whether differences in courtship behaviour were attributable primarily to males, females or both sexes. Duration of spermatophore attachment was controlled by females and therefore represents a mechanism of female mate choice. Significant variation in duration of spermatophore attachment was associated with differences between individuals of both sexes. Differences among males indicate that females agree in their preference of certain males whereas differences among females indicate that females differ in their receptivity to postcopulatory courtship and insemination. The fact that differences among males in duration of spermatophore attachment were due to significant differences solely in the period of courtship feeding indicates that postcopulatory female choice was mediated through courtship feeding. Whether males manipulate female choices by allocating more or fewer resources requires further testing, but we found that males court some females more vigorously than others after females dismount. The number of previous mates had opposite effects on the duration of courtship feeding for the sexes, decreasing it for males but increasing it for females, and we discuss the possible causes of these results.
Quality, Quantity, Distribution and Audience Effects on Food Calling in Cotton—Top Tamarins
Authors: Roush Rebecca S. , Snowdon Charles T.
Published: March 2003

Despite extensive documentation in many taxa of vocalizations specific to feeding contexts, little is known of the variables leading to production of food‐associated calls. We thus experimentally investigated food‐associated calls in cotton‐top tamarins (Saguinus oedipus). In one experiment, we examined the roles of food quantity, quality and distribution and of social condition of individuals on food‐associated calling. Paired adults without offspring gave more vocalizations than immature individuals and adults with offspring. We observed no effects of food quantity or distribution on calling rate. Tamarins called more often to peaches, a food of intermediate preference that was also consumed the fastest. Tamarins gave more food‐associated calls when in closer proximity to other group members. Low rates of moderate aggression were observed in single, but not multiple, feeder conditions. We saw no consistent evidence of dominance among adults, and all aggression in family groups was directed toward immature animals. In another experiment, we presented a tamarin with food in a separate cage, either in view of or visually isolated from its mate. Tamarins presented with food called at an equal rate whether or not their mates were visible, suggesting no effect of audience on calling. Individuals not presented with food called in response to the food calls of their mates equally often, irrespective of whether or not the mate or the food was visible. Since food calling occurs at extremely low rates in the absence of food, the food calling by tamarins that cannot see the food suggests that the calls of their mates provide representational information about food.
Developmental Instability and Sexual Selection in Stag Beetles from Chernobyl and a Control Area
Authors: Møller Anders Pape
Published: March 2003

Radioactivity may have both phenotypic and genetic effects through its disruption of physiological processes and mutations. I studied the size and asymmetry of secondary sexual and ordinary morphological characters of stag beetles (Lucanus cervus) in two areas in the Ukraine: near Chernobyl, where levels of radiation are high, and in a control area with low background radiation. Developmental instability of morphological characters was estimated from the degree of fluctuating asymmetry using the restricted maximum likelihood parameter estimation (REML) method to partition measurement error from asymmetry. The degree of asymmetry estimated from unsigned differences in size of right and left secondary sexual character was similar to estimates based on the REML method. Beetles from the contaminated area had a significantly elevated level of fluctuating asymmetry in the secondary sexual character compared with animals from the control area. Male stag beetles found with a female had significantly lower asymmetry than males found alone. While mated males did not differ in asymmetry between areas, unmated males from Chernobyl were much more asymmetric than unmated males from the control area. These findings provide evidence for radiation disrupting developmental homeostasis, and thereby affecting the mating status of free‐living beetles.
The Relationship Between the Presence of Satellite Males and Nest‐Holders' Mating Success in the Azorean Rock‐Pool BlennyParablennius sanguinolentus parvicornis
Authors: Oliveira Rui F. , Carvalho Natacha , Miranda Jason , Gonçalves Emanuel J. , Grober Matthew , Santos Ricardo Serrão
Published: March 2003

In the Azorean rock‐pool blenny, sexually active males may adopt alternative reproductive tactics. In the present paper the relationship between the presence of satellite males and the reproductive success of nest‐holders was investigated by comparing nests with and without an associated satellite male. Males with an associated satellite male suffered more conspecific intrusions but they did not display a higher frequency of attacks towards conspecifics. Nest‐holder males were more aggressive towards other conspecifics than towards satellites and the tolerance of nest‐holders towards satellites was inversely correlated with the time spent by the satellites in the breeding territory, which suggests control by the nest‐holder male of the satellite investment in shared territorial defence. Nest‐holders with an associated satellite male had higher condition factors and received more female visits and more spawnings. These results bear two possible interpretations. (1) Nest‐holders benefit from the presence of a satellite male by increased attractiveness of their nests to females; satellite males are mutualists helping to defend the nest‐owner's territory and to attract females, which is why they are tolerated. (2) Satellite males associate preferentially with more successful nest‐holder males which have higher condition factors, and by doing so have more opportunities to achieve parasitic fertilizations. Only experiments will allow these two hypotheses to be distinguished.
Testosterone Manipulation of Male Attractiveness has no Detectable Effect on Female Home‐Range Size and Behavior During the Fertile Period
Authors: Neudorf Diane L. , Ziolkowski Jr David J. , Jr Val Nolan , Ketterson Ellen D.
Published: March 2003

Female dark‐eyed juncos (Junco hyemalis) are socially monogamous, but they engage in extra‐pair copulations (EPCs). We examined spatial activity and behavior of female juncos during their fertile period to determine whether they engaged in tactics likely to facilitate EPCs and whether any such tactics varied with the attractiveness of their social mates. We manipulated the attractiveness of social mates by implanting experimental males with tubes containing testosterone (T‐males) and control males with empty tubes (C‐males). Previous findings in free‐living juncos showed that females mated to C‐males were more likely to produce extra‐pair young than females mated to T‐males. We radio‐tracked 13 females (eight C‐mated, five T‐mated) for an average of 15 h each over 3 d during their fertile periods. We predicted that C‐mated females, to compensate for the induced relative unattractiveness of their social mates, would foray from their territories to seek EPCs and as a result would have larger home ranges than T‐mated females. Females of both treatment groups made extra‐territorial forays, some of considerable distances, but we observed no EPCs during forays. Further, neighboring T‐ and C‐males frequently made incursions into the home ranges of T‐ and C‐mated females but we saw no EPCs during these incursions. Our ability to detect statistical differences was limited by sample size, but given that constraint, we found no detectable difference in female home‐range size in relation to the treatment of their mates, nor did other female behavior differ according to male treatment. Male behavior was significantly affected by testosterone treatment. C‐males guarded their mates more closely than did T‐males. We conclude that female juncos make extra‐territorial movements during their fertile period without regard to male attractiveness (testosterone treatment), but we found no evidence that these function as a special tactic to gain EPCs.