bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli
Authors: Bizouarn Tania , Fjellström Ola , Axelsson Magnus , Korneenko Tatyana V. , Pestov Nikolay B. , Ivanova Maria V. , Egorov Maxim V. , Shakhparonov Michail , Rydström Jan 
Published: March 2003

Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the α and the β subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease‐treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His‐tagged intact and trypsin‐treated E. coli transhydrogenase.Despite harsh treatments with, e.g. detergents and denaturing agents, the α and β subunits remained tightly associated. A monoclonal antibody directed towards the α subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin‐digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II.Replacement of domain I of intact, as well as trypsin‐digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3‐acetyl‐pyridine‐NAD+ by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions.
A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley
Authors: Ferré Henrik , Broberg Anders , Duus Jens Ø. , Thomsen Karl K. 
Published: March 2003

An arabinoxylan arabinofuranohydrolase was isolated from barley malt. The enzyme preparation, Ara 1, contained two polypeptides with apparent molecular masses of ≈ 60 and ≈ 66 kDa, a pI of 4.55 and almost identical N‐terminal amino‐acid sequences. With p‐nitrophenyl α‐l‐arabinofuranoside (pNPA) as substrate, Ara 1 exhibited a Km of 0.5 mm and a Vmax of 6.7 µmol·min−1·(mg of protein)−1. Maximum activity was displayed at pH 4.2 and 60 °C, and, under these conditions, the half‐life of the enzyme was 8 min. The Ara 1 preparation showed no activity against p‐nitrophenyl α‐l‐arabinopyranoside or p‐nitrophenyl β‐d‐xylopyranoside. Substrate preference and specificity were investigated using pure oligosaccharides and analysis by TLC and nano‐probe NMR. Ara 1 released arabinose from high‐molecular‐mass arabinoxylan and arabinoxylan‐derived oligosaccharides but was inactive against linear or branched‐chain arabinan. Arabinose was readily released from both singly and doubly substituted xylo‐oligosaccharides. Whereas single 2‐O‐linked and 3‐O‐linked arabinose substituents on non‐reducing terminal xylose were released at similar rates, there was a clear preference for 2‐O‐linked arabinose on internal xylose residues. When Ara 1 acted on oligosaccharides with doubly substituted, non‐reducing terminal xylose, the 3‐O‐linked arabinose group was preferred as the initial point of attack. Oligosaccharides with doubly substituted internal xylose were poor substrates and no preference could be determined. The enzyme described here is the first reported arabinoxylan arabinofuranohydrolase which is able to release arabinose from both singly and doubly substituted xylose, and it hydrolyses p‐nitrophenyl α‐l‐arabinofuranoside at a rate similar to that observed for oligosaccharide substrates.
Structure–function relationships in UCP1, UCP2 and chimeras
Authors: Chomiki Nathalie , Voss John C. , Warden Craig H. 
Published: March 2003

Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C‐terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1–198) and hUCP2 (amino acids 211–309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1–210) and mUCP1 (amino acids 199–307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide‐binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide‐binding site is present in the C‐terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N‐terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C‐terminal domain on nucleotide binding, they also demonstrate that interactions with the N‐terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold‐induced thermogenesis.
Stimulation of the ICAM‐1 gene transcription by the peroxovanadium compound [bpV(Pic)] involves STAT‐1 but not NF‐κB activation in 293 cells
Authors: Audette Marie , Larouche Lucie , Lussier Isabelle , Fugère Nathalie 
Published: March 2003

Vanadate and peroxovanadium derivatives are potent inhibitors of protein tyrosine phosphatases (PTPs) and exhibit insulinomimetic activities in several cell systems. We have found that in 293 and 293T cells, intercellular adhesion molecule‐1 (ICAM‐1) gene transcription is activated by bpV(Pic), a picolinic acid‐stabilized peroxovanadium derivative. To identify the bpV(Pic)‐responsive element(s), several deletion and site‐specific mutants of the ICAM‐1 gene promoter cloned upstream from the firefly luciferase reporter gene were transiently transfected into both cell lines. Deletion or site‐specific mutation of the NF‐κB site did not affect bpV(Pic) responsiveness, whereas deletion or mutation of the palindromic interferon‐γ‐responsive element (pIγRE)/γ‐interferon activated sequence site greatly decreased bpV(Pic) responsiveness in both cell types. bpV(Pic) synergistically co‐operated with interferon‐γ to increase the transcriptional activity of the ICAM‐1 promoter. Electrophoretic mobility‐shift assays showed that bpV(Pic) induces signal transducers and activators of transcription (STAT)‐1 binding to the ICAM‐1 pIγRE/GAS in 293T cells, suggesting that the peroxovanadium compound specifically inhibits the phosphatase(s) required to regulate the JAK/STAT signal‐transduction pathway.
Formation and properties of hybrid photosynthetic F1‐ATPases
Authors: Tucker Ward C. , Du Ziyun , Gromet‐Elhanan Zippora , Richter Mark L. 
Published: March 2003

A hybrid ATPase composed of cloned chloroplast ATP synthase β and γ subunits (βC and γC) and the cloned α subunit from the Rhodospirillum rubrum ATP synthase (αR) was assembled using solubilized inclusion bodies and a simple single‐step folding procedure. The catalytic properties of the assembled

were compared to those of the core

complex of the native chloroplast coupling factor 1 (CF1) and to another recently described hybrid enzyme containing R. rubrumα and β subunits and the CF1γ subunit (

). All three enzymes were similarly stimulated by dithiothreitol and inhibited by copper chloride in response to reduction and oxidation, respectively, of the disulfide bond in the chloroplast γ subunit. In addition, all three enzymes exhibited the same concentration dependence for inhibition by the CF1ε subunit. Thus the CF1γ subunit conferred full redox regulation and normal ε binding to the two hybrid enzymes. Only the native CF1 complex was inhibited by tentoxin, confirming the requirement for both CF1α and β subunits for tentoxin inhibition. However, the

complex, like the

complex, was stimulated by tentoxin at concentrations in excess of 10 µm. In addition, replacement of the aspartate at position 83 in βC with leucine resulted in the loss of stimulation in the

hybrid. The results indicate that both inhibition and stimulation by tentoxin require a similar structural contribution from the β subunit, but differ in their requirements for α subunit structure.
Structural elucidation of a capsular polysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn’s disease
Authors: Hashimoto Masahito , Kirikae Fumiko , Dohi Taeko , Kusumoto Shoichi , Suda Yasuo , Kirikae Teruo 
Published: March 2003

The structure of a capsular polysaccharide (CPS) from a clinical isolate of Bacteroides vulgatus was elucidated. B. vulgatus IMCJ 1204 was isolated from feces of a patient with Crohn’s disease. CPS was prepared by phenol/water extraction of the bacterial cells followed by hydrophobic interaction chromatography and then gel filtration chromatography of the extract. The structure of CPS was determined by chemical analysis and NMR spectroscopy including DQF‐COSY, TOCSY, ROESY, HSQC‐TOCSY, HMQC and HMBC to be a polysaccharide composed of the following repeating unit: →3)β‐d‐Glcp(1→6)[α‐d‐GalpNAc(1→2)β‐d‐Galp(1→4)]β‐d‐GlcpNAc(1→3)α‐d‐Galp(1→4)β‐d‐Manp(1→.
The interaction of human apolipoprotein C‐I with sub‐micellar phospholipid
Authors: Atcliffe Benjamin W. , MacRaild Christopher A. , Gooley Paul R. , Howlett Geoffrey J. 
Published: March 2003

Mature human apolipoprotein C‐I (apoC‐I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC‐I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC‐I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC‐I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC‐I with yields of approximately 4–5 mg per L of culture. The purified product has properties similar to plasma‐derived apoC‐I including self‐association in the lipid‐free state and induced α‐helical content in the presence of egg‐yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short‐chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro‐PCho), to examine the interaction of apoC‐I with submicellar phospholipid. Circular dichroism spectroscopy and cross‐linking experiments show that apoC‐I acquires helical content and remains self‐associated at submicellar concentrations of Hex2Gro‐PCho (4 mm). Sedimentation equilibrium studies of apoC‐I at submicellar levels of Hex2Gro‐PCho and analysis of the effects of apoC‐I on the 1H NMR spectrum of Hex2Gro‐PCho indicate micelle induction by apoC‐I, and establish the capacity of apoC‐I to assemble individual phospholipid molecules.
Purification and structure of the major product obtained by reaction of NADPH and NMNH with the myeloperoxidase/hydrogen peroxide/chloride system
Authors: Auchère Françoise , Bertho Gildas , Artaud Isabelle , Girault Jean Pierre , Capeillère‐Blandin Chantal 
Published: March 2003

The first spectrophotometric study of the reaction of the myeloperoxidase/H2O2/Cl− system with NADPH and NMNH showed that the reaction products were not the corresponding oxidized nucleotides and that modifications would take place on the nicotinamide part of the molecule [Auchère, F. & Capeillère‐Blandin, C. (1999) Biochem. J. 343, 603–613]. In this report, in order to obtain more precise information on the structural modifications and mechanism of the reaction, we focus on the purification and isolation of products derived from NADPH and NMNH by RP‐HPLC. Electrospray ionization mass spectra indicated that the relative height of the peaks reflected that of the natural isotopic abundance of 35Cl and 37Cl, providing evidence that the products derived from NADPH and NMNH were monochlorinated. Moreover, calculated masses revealed the 1 : 1 addition of HOCl to the molecule. Various 1D and 2D NMR experiments provided data for the assignments of the chemical shifts of protons and carbons and the coupling constants of the protons of the chlorinated nucleotides. Further NOESY experiments allowed the characterization of the spatial structure of the chlorinated product and showed that trans HOCl addition occurred at the C5=C6 carbon double bond of the nicotinamide ring, leading to a chlorohydrin.
In vitro processing of the proproteins GrdE of protein B of glycine reductase and PrdA of D‐proline reductase from Clostridium sticklandii
Authors: Bednarski Brit , Andreesen Jan R. , Pich Andreas 
Published: March 2003

GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase and d‐proline reductase, respectively, that are processed post‐translationally to form a catalytic active pyruvoyl group. The cleavage occurred on the N‐terminal side of a cysteine residue, which is thus the precursor of a pyruvoyl moiety. Both proproteins could be over‐expressed in Escherichia coli and conditions were developed for in vitro processing. GrdE could be expressed as full‐size protein, whereas PrdA had to be truncated N‐terminally to achieve successful over‐expression. Both proproteins were cleaved at the in vivo observed cleavage site after addition of 200 mm NaBH4 in Tris buffer (pH 7.6) at room temperature as analysed by SDS/PAGE and MS. Cleavage of GrdE was observed with a half‐time of ≈ 30 min. Cys242, as the precursor of the pyruvoyl group in GrdE, was changed to alanine, serine, or threonine by site‐directed mutagenesis. The Cys242→Ser and Cys242→Thr mutant proteins were also cleaved under similar conditions with extended half‐times. However, the Cys242→Ala mutant protein was not cleaved indicating a pivotal role of the thiol group of cysteine or hydroxyl group of serine and threonine during the processing of pyruvoyl group‐dependent reductases.
Co‐clustering of Fcγ and B cell receptors induces dephosphorylation of the Grb2‐associated binder 1 docking protein
Authors: Koncz Gábor , Tóth Gábor K. , Bökönyi Gyöngyi , Kéri György , Pecht Israel , Medgyesi David , Gergely János , Sármay Gabriella 
Published: March 2003

The immunoreceptor tyrosine‐based inhibitory motif (ITIM) of human type IIb Fcγ receptor (FcγRIIb) is phosphorylated on its tyrosine upon co‐clustering with the B cell receptor (BCR). The phosphorylated ITIM (p‐ITIM) binds to the SH2 domains of polyphosphoinositol 5‐phosphatase (SHIP) and the tyrosine phosphatase, SHP‐2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcγRIIb in the activation of SHP‐2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p‐ITIM and the N‐terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP‐2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcγRII‐SHIP complexes formed in the intact cells may also activate SHP‐2. Grb2‐associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine‐phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP‐2, SHIP and the p85 subunit of phosphatidyl inositol 3‐kinase (PtdIns3‐K) and may regulate their activity. Gab1 is a potential substrate of SHP‐2, thus its binding to FcγRIIb may modify the Gab1‐bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcγRIIb upon its co‐clustering with BCR. Gab1 may recruit SH2 domain‐containing molecules to the phosphorylated FcγRIIb. SHP‐2, activated upon the binding to FcγRIIb‐SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3‐K and ultimately in the inhibition of downstream activation pathways in BCR/FcγRIIb co‐aggregated cells.
Implications for the Maintenance of Androdioecy in the Freshwater Shrimp, Eulimnadia texana Packard: Encounters between Males and Hermaphrodites are not Random
Authors: Medland Vicki L. , Zucker Naida , Weeks Stephen C. 
Published: March 2003

AbstractAndrodioecy is a mixed‐mating system in which there are males and hermaphrodites but no pure females. Few species exhibit such a mating system. Eulimnadia texana is a branchiopod crustacean that has recently been identified as an androdioecious species. This system is ideal for testing questions related to the evolution of sexual reproduction. We are testing a model that predicts androdioecy to be a stable mixed‐mating system under certain conditions. Specifically, we investigated whether encounters between males and hermaphrodites are random or if either sex seeks out the other for mating. Focal male or hermaphrodite clam shrimp were presented with stimulus shrimp of the other sex or kept alone. Swimming speed and time spent within different areas of a test chamber were recorded. Males did not alter mean swimming speed or spend more time than expected by chance near partitioned hermaphrodites. Hermaphrodites, however, decreased mean swimming speed in the presence of males and also spent more time than expected by chance near partitioned males, suggesting that hermaphrodites respond to male chemical and/or visual stimuli. Modified swimming behaviour probably facilitates inter‐sexual contact, thereby increasing opportunities for out‐crossing above that expected by random encounters.
Parental Feeding Rates in the House Sparrow, Passer domesticus: Are Larger‐Badged Males Better Fathers?
Authors: Voltura Karen M. , Schwagmeyer P. L. , Mock Douglas W. 
Published: March 2003

AbstractElaborated secondary sexual characteristics may reflect genetic quality or good health, either of which may be associated with an individual's competence as a parent. We examined whether female house sparrows (Passer domesticus) paired to large vs. small‐badged mates gain benefits in the form of increased parental care or improved nestling welfare. House sparrow nests where the male had been trapped and banded were observed for 1 h on at least 5 d during the peak growth period of nestlings. Male feeding shares, measured as the proportion of total feeds per chick made by the male, were marginally positively correlated with male badge size. Moreover, higher male shares of nestling feeding were associated with improved prospects for offspring survival, and a greater proportion of chicks fledged from the nests of larger‐badged males. These results suggest that females paired to large‐badged males gain direct benefits in the form of enhanced nestling survival, which presumably stem from factors associated with increases in the proportion of nestling feeding contributed by their mates.
Subordinate Males in Harem Groups of Jamaican Fruit‐Eating Bats (Artibeus jamaicensis): Satellites or Sneaks?
Authors: Ortega Jorge , Arita Héctor T. 
Published: March 2003

AbstractIn the caves of Yucatan, Mexico, the Jamaican fruit‐eating bat, Artibeus jamaicensis, forms harems consisting of four to 18 females and a dominant male that defends the group against foreign males. Large groups (>14 females) contain an additional subordinate male. In theory, subordinate males can associate with harem groups either as satellites, if they provide at least some benefits to the dominant male, or as sneaks, if they only impose costs on the dominant male. We assessed the costs and benefits of subordinate males in three removal experiments. In the first experiment, when a dominant male was removed from its group, its role was occupied by the subordinate male (in large groups) or by a foreign male (in small groups). Former subordinate males took less time to gain control of the harems and stayed longer with the groups than foreign males. In the second experiment, when a subordinate male was removed, the rate of visitation by foreign males and the number of agonistic displays by the dominant male both increased. In the third experiment, when the number of females in large groups was reduced, subordinate males spent less time with their groups and the rate of visitation by foreign males increased. However, the frequency of agonistic displays by dominant males towards subordinate males did not change. Dominant males invest large amounts of energy in defending the harems, but obtain direct and immediate benefits from the presence of subordinate males in the form of access to a larger number of females, and suffer no obvious costs. Subordinate males apparently invest little energy in defending the harems, obtain no obvious immediate benefit, but gain long‐term benefits by having priority access to vacant positions left by dominant males. Subordinate males in harem groups of the Jamaican fruit‐eating bat can be considered satellites because their presence brings immediate benefits to the dominant males.
When to Come Out from a Refuge: Balancing Predation Risk and Foraging Opportunities in an Alpine Lizard
Authors: Martín José , López Pilar , Cooper William E. 
Published: March 2003

AbstractPrey often respond to predator presence by increasing their use of refuges, but because this strategy may be costly, the decision regarding when to come out from a refuge should be optimized. The loss of foraging opportunities may be one of the main costs when safer microhabitats (i.e. refuges) are also the poorest in terms of their foraging profitability. We present the results of an experimental field study to test whether emergence times from a refuge of the Iberian rock lizard, Lacerta monticola, vary as a function of expected foraging opportunities and level of satiation of the lizard. As predicted, short‐term fluctuations in availability of food influenced emergence times; when a lizard had just detected some food in the recent past, emergence times decreased greatly, because the loss of opportunities for foraging increased costs of refuge use. Furthermore, the characteristics and success of the encounter with food, nutritional state of lizards, and the added possibility of capturing new food items influenced the duration of hiding times. Therefore, foraging requirements and avoidance of predators may be conflicting demands that L. monticola lizards balance by modifying the duration of time spent in refuges.