bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification by Phage Display of the Linear Continuous MRPr1 Epitope in the Multidrug Resistance-Associated Protein (MRP1)
Authors: Flego M. , Mennella V. , Moretti F. , Poloni F. , Dupuis M.L. , Ascione A. , Barca S. , Felici F. , Cianfriglia M. 
Published: September 2004

Abstract
In order to study the structure of the multidrug resistance-associated protein (MRP1), which is one of the
most important members of the ATP-binding cassette
(ABC) protein family acting as drug-efflux systems, we
have developed an epitope mapping-based strategy.
By means of the mAb MRPr1, we have immunoselected clones from two distinct random peptide libraries
displayed on phages and have identified several peptide sequences mimicking the internal conformation
of this 190 kDa multidrug transporter protein. Phage
clones able to block the immunolabeling of the MRPr1
antibody to MRP1-overexpressing multidrug resistance (MDR) H69/AR cells were isolated and, after sequencing the corresponding inserts, their amino acid
sequence was compared to that of MRP1. This analysis led to the identification of the consensus sequence
L.SLNWED, corresponding to the MRP1 segment
LWSLNKED (residues 241–248). This MRP1sequence
is partially overlapping with the MRPr1 epitope GSDL
WSLNKE (residues 238–247) previously mapped using peptide scanning techniques. These results
demonstrate the high reliability of phage display technology to study not only the topography of complex
integral membrane proteins such as MRP1, but also to
help identify critical residues participating in the formation of the epitope structure.
The Cellular Control Enzyme PolyADP Ribosyl Transferase Is Eliminated in Cultured Fanconi Anemia Fibroblasts at Confluency
Authors: Ramirez M.H. , Adelfalk C. , Kontou M. , Hirsch-Kauffmann M. , Schweiger M. 
Published: September 2004

AbstractFanconi anemia (FA) is a hereditary disease of unknown pathogenic mechanisms, although mutations in seven different genes can be causative. Six of these genes have been cloned and sequenced. Only slight homology to the DNA of any other known gene has been found with the exception of FANCG which is identical to XRCC9. The function of these genes, including XRCC9, is presently unknown. Since pADP ribosyl transferase (pADPRT) plays a role in apoptosis, and apoptosis is affected in FA cells, we studied the correlation between pADPRT and FA cells. We reinvestigated the previously reported lack of pADPRT activity in fibroblasts from patients with Fanconi anemia. Here we describe the role of the lower redox potential of FA cells and demonstrate that this is an efficient strategy in the prevention of cell death due to the lack of energy under oxidative stress. This strategy is advantageous for the cells under the nonreplicative condition of confluency in which the risk of mutation is low and the prevention of apoptosis permits cell survival. pADPRT is not diminished to the same extent in all complementation groups of FA. It is prominent in FANCA, FANCG and FANCF cells, indicating that these genes control pADPRT diminution. Our experiments suggest that the pADPRT level is linked with the oxidoreduction reactions seen in FA.
‘Piggy-Back’ Transport of Xenopus Hyaluronan Synthase (XHAS1) via the Secretory Pathway to the Plasma Membrane
Authors: Müllegger J. , Rustom A. , Kreil G. , Gerdes H.-H. , Lepperdinger G. 
Published: September 2004

AbstractHyaluronan is the sole glycosaminoglycan whose biosynthesis takes place directly at the plasma membrane. The mechanism by which hyaluronan synthase (HAS) becomes inserted there, as well as the question of how the enzyme discriminates between particular membrane species in polarized cells, are largely unknown. In vitro translation of HAS suggested that the nascent protein becomes stabilized in the presence of microsomal membranes, but would not insert spontaneously into membranes after being translated in the absence of those. We therefore monitored the membrane attachment of enzymatically active fusion proteins consisting of Xenopus HAS1 and green fluorescent protein shortly after de novo synthesis in Vero cells. Our data strongly suggest that HAS proteins are directly translated on the ER membrane without exhibiting an N-terminal signal sequence. From there the inactive protein is transferred to the plasma membrane via the secretory pathway. For unknown reasons, HAS inserted into membranes other than the plasma membrane remains inactive.
A Novel Influenza A Virus Activating Enzyme from Porcine Lung: Purification and Characterization
Authors: Sato M. , Yoshida S. , Iida K. , Tomozawa T. , Kido H. , Yamashita M. 
Published: September 2004

AbstractProteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gln-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsintype serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-α and -β. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.
A Homodimeric Sporamin-Type Trypsin Inhibitor with Antiproliferative, HIV Reverse Transcriptase-Inhibitory and Antifungal Activities from Wampee (Clausena lansium) Seeds
Authors: Ng T.B. , Lam S.K. , Fong W.P. 
Published: September 2004

Abstract
A homodimeric trypsin inhibitor with a molecular
mass of 54 kDa was isolated from the seeds of
Clausena lansium (Lour) Skeels with a very simple
procedure comprising extraction with an aqueous
buffer and ion exchange chromatography on CM-cellulose.
It inhibited trypsin with an IC50 of 2.2 nM but was
without any inhibitory effect on chymotrypsin and proteinase
K. The uptake of MTT by human leukemia
HL60 and hepatoma Hep G2 cells was inhibited with
an IC50 of 100 uM. Translation in the cellfree rabbit
reticulocyte lysate system was inhibited with an IC50
of 3.6 uM. The activity of HIV-1 reverse transcriptase
was reduced in the presence of the trypsin inhibitor.
The trypsin inhibitor exerted antifungal activity toward
Physalospora piricola but not Mycosphaerella
arachidicola, Botrytis cinerea, Fusarium oxysporum
or Coprinus comatus.
A Bioinformatic Approach to the Identification of Candidate Genes for the Development of New Cancer Diagnostics
Authors: Musumarra G. , Barresi V. , Condorelli D.F. , Scirè S. 
Published: September 2004

Abstract
A multivariate analysis of the National Cancer Institute
gene expression database is reported here. The
soft independent modelling of a class analogy approach
achieved cell line classification according to
histological origin. With the PCA method, based on
the expression of 9605 genes and ESTs, classification
of colon, leukaemia, renal, melanoma and CNS cells
could be performed, but not of lung, breast and ovarian
cells. Another multivariate procedure, called partial
least squares discriminant analysis (PLS-DA),
provides bioinformatic clues for the selection of a limited
number of gene transcripts most effective in discriminating
different tumoral histotypes. Among
them it is possible to identify candidates in the development
of new diagnostic tests for cancer detection
and unknown genes deserving high priority in further
studies. In particular, melan-A, acid phosphatase 5,
dopachrome tautomerase, S100-β and acid ceramidase
were found to be among the most important
genes for melanoma. The potential of the present
bioinformatic approach is exemplified by its ability to
identify differentiation and diagnostic markers already
in use in clinical settings, such as protein S-100,
a prognostic parameter in patients with metastatic
melanoma and a screening marker for melanoma
metastasis.
Recombinant RNase Z Does Not Recognize CCA as Part of the tRNA and Its Cleavage Efficieny Is Influenced by Acceptor Stem Length
Authors: Schiffer S. , Rösch S. , Marchfelder A. 
Published: September 2004

AbstractOne of the essential maturation steps to yield functional tRNA molecules is the removal of 3'-trailer sequences by RNase Z. After RNase Z cleavage the tRNA nucleotidyl transferase adds the CCA sequence to the tRNA 3terminus, thereby generating the mature tRNA. Here we investigated whether a terminal CCA triplet as 3'-trailer or embedded in a longer 3'- trailer influences cleavage site selection by RNase Z using three activities: a recombinant plant RNase Z, a recombinant archaeal RNase Z and an RNase Z active wheat extract. A trailer of only the CCA trinucleotide is left intact by the wheat extract RNase Z but is removed by the recombinant plant and archaeal enzymes. Thus the CCA triplet is not recognized by the RNase Z enzyme itself, but rather requires cofactors still present in the extract. In addition, we investigated the influence of acceptor stem length on cleavage by RNase Z using variants of wildtype tRNATyr. While the wild type and the variant with 8 base pairs in the acceptor stem were processed efficiently by all three activities, variants with shorter and longer acceptor stems were poor substrates or were not cleaved at all.
Retinoids Inhibit Human Epidermal Keratinocyte RNase P Activity
Authors: Papadimou E. , Pavlidou D. , Séraphin B. , Tsambaos D. , Drainas D. 
Published: September 2004

AbstractRibonuclease P (RNase P) is a ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5'-end. We have investigated the effect of synthetic rertinoids (all-trans retinoic acid, acitretin) and arotinoids (Ro 13-7410, Ro 15-0778, Ro, 13-6298 and Ro 15-1570) on RNase P activity isolated for the first time from normal human epidermal keratinocytes (NHEK). All tested compounds but one (Ro 15-1570) revealed a dose-dependent inhibition of RNase P activity, indicating that they may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classic competitive inhibitors. On the basis of the Ki values Ro 13-7410 was found to be the strongest inhibitor among all compounds tested.
Synthesis and Characterization of EMPO-Derived 5,5-Disubstituted 1-Pyrroline N-Oxides as Spin Traps Forming Exceptionally Stable Superoxide Spin Adducts
Authors: Stolze K. , Udilova N. , Rosenau T. , Hofinger A. , Nohl H. 
Published: September 2004

Abstract
EMPO [5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide]
is a highly hydrophilic cyclic nitrone spin trap,
whose superoxide adduct is considerably more stable
(t1/2=8.6 min) than DMPO (5,5-dimethyl-1-pyrroline
Noxide, t1/2=45 s). EPR spectra of spin adducts of
EMPO and its derivatives are very similar to those of
the respective DMPO spin adducts, in contrast to the
rather complex spectra obtained using DEPMPO [5-
(diethoxyphosphoryl)-5-methyl-1-pyrroline Noxide].
Several EMPO derivatives, with both the ethoxycarbonyl
group and the methyl group at position 5 of the
pyrroline ring being replaced by other substituents,
were synthesized and characterized by 1H and 13C
NMR spectroscopy. Thus, a series of derivatives was
obtained that exhibit large differences in the stability
of their superoxide adducts, ranging from less than
one to more than 25 min. The stability of the superoxide
adducts was mainly determined by the steric environment
of the nitroxyl group: in compounds with
less bulky 5-alkoxycarbonyl substituents the nitroxyl
group is sterically less shielded, which resulted in a
lower stability of the superoxide adducts. The spin
density distribution, as obtained from DFT computations,
was found to be nearly identical for all compounds,
so that in contrast to the steric influences the
spin density did not seem to be a crucial factor for the
stability of the superoxide adducts.
Modulation of the Chymotrypsin-Like Activity of the 20S Proteasome by Intracellular Redox Status: Effects of Glutathione Peroxidase-1 Overexpression and Antioxidant Drugs
Authors: Kretz-Remy C. , Arrigo A.-P. 
Published: September 2004

AbstractATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsinlike activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IκBα half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or proreducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.
Testis-Specific Expression of the Nuclear Form of Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx)
Authors: Moreno S. G. , Laux G. , Brielmeier M. , Bornkamm G. W. , Conrad M. 
Published: September 2004

Abstract
The selenoprotein phospholipid hydroperoxide glutathione
peroxidase (PHGPx) is present in at least
three different isoforms in testis: as a cytosolic, as a
mitochondrial, and as a nuclear protein. We have recently
shown that a sperm nucleus-specific glutathione
peroxidase (snGPx) is identical to the mitochondrial
and cytosolic forms of PHGPx apart from
its N-terminus. This arginine-rich N-terminus of snGPx, reminiscent of protamines, is encoded by an alternative
exon located in the first intron of the PHGPx gene and is responsible for nuclear localisation and chromatin binding of snGPx [Pfeifer et al., FASEB J.
15 (2001), pp. 1236-1238]. By using a combination of
techniques including selective cloning of mRNA 5'
ends, RT-PCR, and S1 analyses, we provide evidence
that the transcript encoding the nuclear form is generated
by transcription initiation at an alternative promoter
and not by alternative splicing. We show that
the major transcription start region is located at 12
to 14 upstream of the AUG translation initiation site
of the sperm nucleus-specific exon and lacks a TATA
box. Two minor TATA-less transcription initiation sites
are located at around -30 and -45. We have shown by
in situ hybridisation that snGPx expression in testis,
like protamine expression, is restricted to late stages
of spermatogenesis whereas PHGPx expression is
only found in spermatocytes and early spermatids.
These findings have to be taken into account when
studying either the differential regulation of PHGPx
and snGPx expression in testis or the impact of putative
mutations in snGPx on male fertility in man.
Regulation of Gene Transcription by a Constitutively Active Mutant of Activating Transcription Factor 2 (ATF2)
Authors: Steinmüller L. , Thiel G. 
Published: September 2004

AbstractActivating transcription factor 2 (ATF2) belongs to the family ofbasic region leucinezipper (bZIP) proteins that are characterized by the presence of a basic domain that functions as the DNAbinding domain and a leucine zipper domain that is required for dimerization. Together with bZIP proteins of the Fos and Jun families, ATF2 constitutes the AP-1 transcription factor complex. The biological activity of ATF2 is controlled by phosphorylation of two threonine residues within the N-terminal activation domain. Unphosphorylated ATF2 is trancriptionally silent, excluding simple overexpression studies to identify transcriptional targets of ATF2. We therefore decided to construct a constitutively active ATF2 mutant that would allow us to uncouple the investigation of transcriptional targets and biological functions of ATF2 from the variety of signaling pathways that lead to an activation of ATF2. We exchanged the phosphorylationdependent activation domain of ATF2 with the constitutively active transcriptional activation domain of the transcription factor CREB2. In transient transfection experiments, this constitutively active ATF2 mutant stimulated c-jun, tumor necrosis factor α, and Fas ligand promoter activities. The transcriptional activity of the constitutively active ATF2 mutant could be impaired by dominantnegative forms of ATF2 or c-jun, indicating that ATF2 and c-jun utilize a similar dimerization code. In contrast, a dominantnegative CREB2 mutant did not impair ATF2-mediated transcriptional activation, suggesting that CREB2 exhibits a different dimerization specificity than ATF2 or c-jun.
Solvent Isotope Effect on the Reaction Catalysed by the Pyruvate Dehydrogenase Complex from Escherichia coli
Authors: Liu X. , Bisswanger H. 
Published: September 2004

AbstractThe pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide. The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction. The pH profile and, accordingly, the δpK1 and δpK2 values were shifted by 0.6 units to higher pL values. A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0%. This is an indication for a single proton transfer. It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1 nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue. The proton of its carboxylic group exchanges very fast with deuterons of the solvent.
The Circadian Clock of the Unicellular Eukaryotic Model Organism Chlamydomonas reinhardtii
Authors: Mittag M. , Wagner V. 
Published: September 2004

AbstractThe green unicellular alga Chlamydomonas reinhardtii, also called 'green yeast', emerged in the past years as a model organism for specific scientific questions such as chloroplast biogenesis and function, the composition of the flagella including its basal apparatus, or the mechanism of the circadian clock. Sequencing of its chloroplast and mitochondrial genomes have already been completed and a first draft of its nuclear genome has also been released recently. In C. reinhardtii several circadian rhythms are physiologically well characterized, and one of them has even been shown to operate in outer space. Circadian expression patterns of nuclear and plastid genes have been studied. The mode of regulation of these genes occurs at the transcriptional level, although there is also evidence for posttranscriptional control. A clock-controlled, phylogenetically conserved RNA-binding protein was characterized in this alga, which interacts with several mRNAs that all contain a common cis-acting motif. Its function within the circadian system is currently under investigation. This review summarizes the current state of the knowledge about the circadian system in C. reinhardtii and points out its potential for future studies.
Identification of Rhythmic Subsystems in the Circadian Cycle of Crassulacean Acid Metabolism under Thermoperiodic Perturbations
Authors: Bohn A. , Hinderlich S. , Hütt M.-T. , Kaiser F. , Lüttge U. 
Published: September 2004

AbstractLeaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier de la Bâthie show overt circadian rhythms in net CO2 uptake, leaf conductance to water and intercellular CO2 concentration, which are entrained by periodic temperature cycles. To probe their sensitivity to thermoperiodic perturbations, intact leaves were exposed to continuous light intensity and temperature cycles with a period of 16 h, applying a set of different baseline temperatures and thermodriver amplitudes. All three overt rhythms were analyzed with respect to their frequency spectra and their phase relations with the thermodriver. For most stimulation protocols, stomatal conductance and net CO2 change were fully or partially entrained by the temperature pulses, while the internal CO2 concentration remained dominated by oscillations in the circadian range. Prolonged time series recorded for up to 22 d in continuous light underline the robustness of these circadian oscillations. This suggests that the overt circadian rhythm of net CO2 uptake in CAM results from the interaction of two coupled original systems: (i) an endogenous cycle of CO2 fixation in the mesophyll, showing very robust periodic activity, and (ii) stomatal movements that respond to environmental stimuli independently of rhythmic processes in the mesophyll, and thus modulate the gas exchange amplitude.
Age-Dependent Effects of Conditioning on Cholinergic and Vasopressin Systems in the Rat Suprachiasmatic Nucleus
Authors: Biemans B. A. M. , Zee E. A. Van der , Daan S. 
Published: September 2004

Abstract
Active shock avoidance was used to explore the impact
of behavioural stimulation on the neurochemistry
of the suprachiasmatic nucleus. We have found
previously that the expression of muscarinic acetylcholine
receptors in the suprachiasmatic nucleus of
young rats was significantly enhanced 24 hours after
fear conditioning. Here, we investigated whether this
observation is age-dependent. We used 26 month-old
Wistar rats with a deteriorated circadian system, and
compared them with young rats (4 months of age)
with an intact circadian system. Vasopressin, representing
a major output system of the suprachiasmatic
nucleus, was studied in addition to muscarinic receptors.
Young rats showed a significant increase in
immunostaining for muscarinic acetylcholine receptors
24 h after training, corroborating earlier observations.
Aged rats did not show such an increase. In
contrast, aged rats did show an increase in vasopressin
immunoreactivity 24 h after fear conditioning,
both at the level of content and cell number, while
young rats did not reveal a significant rise. Thus, it
seems that these two neurochemical systems in the
suprachiasmatic nucleus are regulated independently.
The results further demonstrate that the circadian
pacemaker is influenced by fear conditioning in an
age-dependent manner.