bluuebunny/crossref_metadata_2025_split huggingface dataset:
Urethanyl-3-Amidinophenylalanine Derivatives as Inhibitors of Factor Xa. X-Ray Crystal Structure of a Trypsin/Inhibitor Complex and Modeling Studies
Authors: Sperl Stefan , Bergner Andreas , Stürzebecher Jörg , Magdolen Viktor , Bode Wolfram , Moroder Luis 
Published: October 2004

AbstractHydrophobic urethanyl derivatives of 3-amidinophenylalanine methyl ester were found to be relatively potent and selective factor Xa inhibitors. These compounds consist of the argininemimetic 3-benzamidino group as P1 residue and of hydrophobic residues as potential interaction partners for the S3/S4 aryl binding site of the enzyme. Attempts to possibly identify their binding mode to factor Xa via the Xray crystal structure of a trypsin/inhibitor complex and analogy modeling on the crystal structure of factor Xa failed. However, synthesis of enantiomerically pure (R) and (S)derivatives, combined with modeling experiments, led to an hypothetical nonsubstrate like binding mode, which was fully confirmed by the remarkably inhanced inhibitory potency of derivatives in which the methyl ester was replaced by arylamides for interactions with the S3/S4 enzyme binding subsites. With adamantyloxycarbonyl(R)-3-amidinophenylalaninephenethylamide a nanomolar inhibiton was obtained, thus indicating this new class of factor Xa inhibitors as a highly promising lead structure.
The pH-Dependent Interaction of Cinnamomin with Lipid Membranes Investigated by Fluorescence Methods
Authors: Hu Rong-gui , Tang Shuang , Liu Wang-yi 
Published: October 2004

AbstractCinnamomin, a new type II ribosomeinactivating protein (RIP), was found to be able to induce the release of calcein loaded in lecithin small unilamellar vesicles and the fusion or aggregation of the lecithin liposomes. Such induction could be promoted several fold by a pH 5.0 environment, a condition similar to that in endocytic vesicles. Lowering the pH from 7.5 to 5.0 evoked conformational changes of cinnamomin and unmasked its hydrophobic areas, including the exposure of 1-anilino-8-naphthalenesulfonate (1,8-ANS) binding sites of the molecule. Some tryptophan residues with affinity to acrylamide were demonstrated to participate in the lipidprotein interaction. The pH dependent fusogenicity of type II RIP might suggest its in vivo function as a fusogen to exert its cytotoxicity.
Nitric Oxide Detection and Visualization in Biological Systems. Applications of the FNOCT Method
Authors: Meineke Petra , Rauen Ursula , Groot Herbert de , Korth Hans-Gert , Sustmann Reiner 
Published: October 2004

Abstract
Fluorescent Nitric Oxide Cheletropic Traps (FNOCTs)
were applied to specifically trap nitric oxide (NO) with
high sensitivity. The fluorescent oquinoid ?electron
system of the FNOCTs (? = 460 nm, ? = 600 nm) reacts
rapidly with NO to a fluorescent phenanthrene
system (? = 380 nm, ? = 460 nm). The cyclic nitroxides
thus formed react further to nonradical products
which exhibit identical fluorescence properties. Using
the acid form of the trap (FNOCT-4), NO release by
spermine NONOate and by lipopolysaccharide (LPS)
activated alveolar macrophages were studied. A maximum
extracellular release of NO of 37.5 nmol h[-1] (10[6]
cells)[-1] from the macrophages was determined at 11 h
after activation. Furthermore, intracellular NO release
by LPSactivated macrophages and by microvascular
omentum endothelial cells stimulated by the Ca[2+]
ionophore A-23187, respectively, was monitored on
the single cell level by means of fluorescence microscopy.
After loading the cells with the membranepermeating
acetoxymethylester derivative FNOCT-5,which is hydrolyzed to a nonpermeating dicarboxylate
by intracellular hydrolases, NO formation by the
endothelial cells started immediately upon stimulation,
whereas start of NO production by the macrophages
was delayed with a variation between 4 and 8 h
for individual cells. These results demonstrate that the
FNOCTs can be used to monitor NO release from
single cells, as well as from NOdonating compounds,
with high sensitivity and with temporal and spatial
resolution.
Multiple Promoters Direct the Tissue-Specific Expression of Rat Mitochondrial Glycerol-3-Phosphate Dehydrogenase
Authors: Weitzel Joachim M. , Grott Stefan , Radtke Christiane , Kutz Sabine , Seitz Hans J. 
Published: October 2004

AbstractThe mitochondrial FADdependent glycerol-3-phosphate dehydrogenase (mGPDH) is an essential component of the glycerol phosphate shuttle which transfers reduction equivalents from the cytoplasm into the mitochondria. We analyzed the distribution of different exon 1-containing transcripts by RTPCR in various tissues in vivo. Exon 1a was predominantly expressed in brain, brown adipose tissue and pancreas, exon 1b was ubiquitously expressed, and exon 1c was exclusively expressed in testis. In transient transfection assays the ubiquitous promoter B showed a detectable activity, whereas promoters A and C were completely silent. A deletion mutational analysis located the basal promoter B activity to a 316 bp core sequence upstream of the transcription start site.
Characterization of SNP, a Novel Tissue- and Phase-Specific Nuclear Protein Expressed during the Proliferative Phase in the Oviduct of the Lizard Podarcis sicula Raf.
Authors: Quesada Piera , Atorino Luigia , Parente Augusto , Blanco Francesca Del Vecchio , Maro Antimo Di , Ciarcia Gaetano , Farina Benedetta 
Published: October 2004

AbstractThe amino acid sequence of a novel tissueand phasespecific nuclear protein (SNP) has been determined, after purification from the nuclei of the oviduct of the lizard Podarcis sicula Raf. during the reproductive period of the seasonal growth. SNP has a pI of 9.0 and contains 81 amino acid residues with a molecular weight of 9211.88 ± 0.09. It shows a bipartite organization as the first 40 amino acids contain all 8 cysteinyl residues, while the last 41 amino acids contain 16 prolyl residues. Two more components have also been identified and characterized, with the first 79 amino acids matching SNP and missing one or two residues at the Cterminus. They have thus been named [des(Ala[81]) SNP1] and [des(Lys[80]Ala[81]) SNP2], respectively. The molecular weights are 9140.21 ± 0.83 for [des(Ala[81]) SNP1] and 9011 ± 0.09 for [des(Lys[80]Ala[81]) SNP2].
Re-Evaluation of Amino Acid Sequence and Structural Consensus Rules for Cysteine-Nitric Oxide Reactivity
Authors: Ascenzi Paolo , Colasanti Marco , Persichini Tiziana , Muolo Massimo , Polticelli Fabio , Venturini Giorgio , Bordo Domenico , Bolognesi Martino 
Published: October 2004

Abstract
Nitric oxide (NO), produced in different cell types through
the conversion of Larginine into Lcitrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events.
The great propensity for nitrosothiol formation and
breakdown represents a mechanism which modulates
the action of macromolecules containing NOreactive
Cys residues at their active centre and/or allosteric
sites. Based on the human haemoglobin (Hb)
structure and accounting for the known acidbase
catalysed Cys?93-nitrosylation and Cys?93NOdenitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites 1,
0, and + 1, respectively) has been proposed as the minimum consensus motif for CysNO reactivity. Although
not found in human Hb, the presence of a polar amino
acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the 2
position has been observed in some NOreactive protein
sequences (e.g., NMDA receptors). However, the most important component of the tri or tetrapeptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18,
691 696]. Here, we analyse the threedimensional
structure of several proteins containing NOreactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the CysS? atomic structural microenvironment rather than on the tri or tetrapeptide sequence consensus motif.
Expression of Human Tissue Kallikrein in Differentiated HL-60 Cells
Authors: Podlich Dirk , Kemme Michael 
Published: October 2004

AbstractThe kallikreinkinin system is a mediator of inflammation in humans. In order to elucidate the range of expression of human tissue kallikrein and its substrates, high and low molecular weight kininogen, in inflammatory cells in vitro, we examined their biosynthesis in the HL-60 cell line by RTPCR and Southern blot analyses. Prominent expression of tissue kallikrein mRNA occurred in untreated promyelocytic cultures as well as in HL-60 cells that were induced to differentiate toward neutrophilic, monocytic, and macrophagic cells. Under the same inducing conditions, kininogen biosynthesis was undetectable at each differentiation state of HL-60 cultures. These results indicate that the myelomonocytic lineage of human leukocytes is a source of tissue kallikrein, which may be secreted as part of the inflammatory process.
Protein Aggregation and Pathogenesis of Huntingtons Disease: Mechanisms and Correlations
Authors: Wanker Erich E. 
Published: October 2004

Abstract
The formation of insoluble protein aggregates is a hallmark
of Huntington's disease (HD) and related neurodegenerative
disorders, such as dentatorubral pallidoluysian
atrophy (DRPLA), spinal bulbar muscular
atrophy (SBMA) and the spinocerebellar ataxia (SCA)
type 1, 2, 3, 6 and 7. These disorders are caused by an
expanded polyglutamine (polyQ) tract in otherwise unrelated
proteins. They are characterized by late-onset,
selective neuropathology, a pathogenic polyQ threshold
and a relationship between polyQ length and disease
progression. Thus, molecular models of HD and
related glutamine-repeat disorders must account for
these characteristic features. During the last three
years, considerable effort has been invested in the
development of in vitro and in vivo model systems to
study the mechanisms of protein aggregation in glutamine-repeat disorders and its potential effects on disease
progression and neurodegeneration. A selection
of these studies is reviewed here. Furthermore, the
correlation between aggregate formation and development
of HD is discussed.
Molecular Genetic Analysis of Glucocorticoid Signaling Using the Cre/loxP System
Authors: Reichardt Holger M. , Tronche François , Bauer Anton , Schütz Günther 
Published: October 2004

Abstract
Glucocorticoids (GC) are involved in a plethora of
physiological processes that range from the regulation
of the stress response and the control of the immune
system to modulation of behavior. Most GC effects
are mediated by the glucocorticoid receptor (GR)
via activation and repression of gene expression.
Whereas in most cases activation requires DNA binding
of the receptor, repression is usually mediated by
protein-protein interaction with other transcription
factors. To decipher the molecular mode of action of
GR, mice were generated by gene targeting carrying a
point mutation in one of the dimerization domains, thus
abrogating DNA binding by GR. Analysis of these mice
demonstrated that thymocyte apoptosis and stress
erythropoiesis require the DNA binding-dependent
function of GR, whereas lung development and the
anti-inflammatory activity of GR are mediated by protein-protein interaction. Furthermore, to study the role
of GC in the brain, mice were generated specifically
lacking GR function in the nervous system. Using
these mice we demonstrated that GR is essential for
the regulation of the HPA-axis and the stress response,
as well as for the control of emotional behavior. Taken
together, gene targeting using the Cre/loxP system
proved to be highly valuable for the analysis of both
molecular mechanism and tissue-specific functions
of the GR.
Macromolecular Intelligence in Microorganisms
Authors: Bruggeman Frank J. , Heeswijk Wally C. van , Boogerd Fred C. , Westerhoff Hans V. 
Published: October 2004

AbstractBiochemistry and molecular biology have been focusing on the structural, catalytic, and regulatory properties of individual macromolecules from the perspective of clarifying the mechanisms of metabolism and gene expression. Complete genomes of ‘primitive’ living organisms seem to be substantially larger than necessary for metabolism and gene expression alone. This is in line with the findings of silent phenotypes for supposedly important genes, apparent redundancy of functions, and variegated networks of signal transduction and transcription factors. Here we propose that evolutionary optimization has been much more intensive than to lead to the bare minima necessary for autonomous life. Much more complex organisms prevail. Much of this complexity arises in the nonlinear interactions between cellular macromolecules and in subtle differences between paralogs (isoenzymes). The complexity can only be understood when analyzed quantitatively, for which quantitative experimentation is needed in living systems that are as simple and manipulatable as possible, yet complex in the above sense. We illustrate this for the glutamine synthetase cascade inEscherichia coli. By reviewing recent molecular findings, we show that this cascade is much more complex than necessary for simple regulation of ammonia assimilation. Simulations suggest that the function of this complexity may lie in quasi-intelligent behavior, including conditioning and learning.
Helical Tubes of FtsZ from Methanococcus jannaschii
Authors: Löwe Jan , Amos Linda A. 
Published: October 2004

AbstractBacterial cell division depends on the formation of a cytokinetic ring structure, the Z-ring. The bacterial tubulin homologue FtsZ is required for Z-ring formation. FtsZ assembles into various polymeric formsin vitro, indicating a structural role in the septum of bacteria. We have used recombinant FtsZ1 protein fromM. jannaschiito produce helical tubes and sheets with high yield using the GTP analogue GMPCPP [guanylyl-(α,β)-methylene-diphosphate]. The sheets appear identical to the previously reported Ca++-induced sheets of FtsZ fromM. jannaschiithat were shown to consist of ‘thick’-filaments in which two protofilaments run in parallel. Tubes assembled either in Ca++or in GMPCPP contain filaments whose dimensions indicate that they could be equivalent to the ‘thick’-filaments in sheets. Some tubes are hollow but others are filled by additional protein density. Helical FtsZ tubes differ from eukaryotic microtubules in that the filaments curve around the filament axis with a pitch of ~ 430 Å for Ca++-induced tubes or 590–620 Å for GMPCPP. However, their assemblyin vitroas well-ordered polymers over distances comparable to the inner circumference of a bacterium may indicate a rolein vivo. Their size and stability make them suitable for use in motility assays.
Sec61p Is the Main Ribosome Receptor in the Endoplasmic Reticulum of Saccharomyces cerevisiae
Authors: Prinz Anke , Hartmann Enno , Kalies Kai-Uwe 
Published: October 2004

Abstract
A characteristic feature of the co-translational protein
translocation into the endoplasmic reticulum (ER) is
the tight association of the translating ribosomes with
the translocation sites in the membrane. Biochemical
analyses identified the Sec61 complex as the main
ribosome receptor in the ER of mammalian cells. Similar
experiments using purified homologues from the
yeast Saccharomyces cerevisiae, the Sec61p complex
and the Ssh1p complex, respectively, demonstrated
that they bind ribosomes with an affinity similar to that
of the mammalian Sec61 complex. However, these
studies did not exclude the presence of other proteins
that may form abundant ribosome binding sites in the
yeast ER. We now show here that similar to the situation
found in mammals in the yeast Saccharomyces
cerevisiae the two Sec61-homologues Sec61p and
Ssh1p are essential for the formation of high-affinity
ribosome binding sites in the ER membrane. The number
of binding sites formed by Ssh1p under standard
growth conditions is at least 4 times less than those
formed by Sec61p.
Noncovalent Binding of Poly(ADP-Ribose) to Nuclear Matrix Proteins: Developmental Changes and Tissue Specificity
Authors: Malanga M. , Farina B. 
Published: October 2004

AbstractPoly(ADP-ribose) is a nuclear polynucleotide involved in the regulation of chromatin functionsviacovalent and/or noncovalent modification of nuclear proteins. Using a binding assay on protein blots, we searched for poly(ADP-ribose) binding proteins in nuclear matrices from testes of differently aged rats as well as from various adult rat tissues (brain, liver, spleen). We found that nuclear matrix proteins represent a significant subset of the nuclear proteins that can establish noncovalent interactions with poly(ADP-ribose). The profiles of poly(ADP-ribose) binding nuclear matrix proteins appeared to be tissue-specific and changed during postnatal development in the testis.The isolation and analysis of endogenous poly (ADP-ribose) from rat testes showed that the ADP-ribose polymers that bind nuclear matrix proteinsin vitroare also present under physiologic conditionsin vivo. These results further substantiate the possibility that poly(ADP-ribose) may affect chromatin functions through noncovalent interaction with specific protein targets, including nuclear matrix components.
Cloning, Heterologous Expression and Kinetic Analysis of Glycerol Kinase (TbGLK1) from Trypanosoma brucei
Authors: Steinborn K. , Szallies A. , Mecke D. , Duszenko M. 
Published: October 2004

AbstractWe have cloned and sequenced the gene for the glycerol kinase ofTrypanosoma brucei(TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase inE. colienabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival ofTrypanosoma bruceiunder anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.
Identification of the 50S Ribosomal Proteins from the Eubacterium Thermus thermophilus
Authors: Katsani K.R. , Tsiboli P. , Anagnostopoulos K. , Urlaub H. , Choli-Papadopoulou T. 
Published: October 2004

Abstract
The total protein mixture from the 50S subunit (TP-50)
of the eubacterium Thermus thermophilus was characterized
after blotting onto PVDF membranes from
two-dimensional polyacrylamide gel electrophoresis
(2D-PAGE) and sequencing. The proteins were numbered
according to their primary structure similarity
with their counterparts from other species. One of
them has been marked with an asterisk, namely L*23,
because unlike the other known ribosomal proteins it
shows a very low degree of homology.
A highly acidic 5S rRNA binding protein, TL5, was
characterized and compared with the available primary
structure information.
Proteins L1 and L4 migrate similarly on 2D-PAGE.
Protein L4, essential for protein biosynthesis, is N-terminally
blocked and shows a strikingly low homology
to other L4 proteins. In addition to L4, two other proteins,
namely L10 and L11, were found to be N-terminally
blocked.
In conclusion, 33 proteins from the large subunit
were identified, including TL5. Homologs to rpL25 and
rpL26 were not found.
Characterization of the Specific DNA Nicking Activity of Restriction Endonuclease N.BstNBI
Authors: Morgan R.D. , Calvet C. , Demeter M. , Agra R. , Kong H. 
Published: October 2004

AbstractN.BstNBI is a unique restriction endonuclease isolated fromBacillus stearothermophilus. We have characterized the recognition sequence and the cleavage site of N.BstNBI. Mapping of cleavage sites of N.BstNBI showed that it recognizes an asymmetric sequence, 5′ GAGTC 3′, and cleaves only on the top strand 4 base pairs away from its recognition sequence. To verify the nicking activity of N.BstNBI, we have constructed two plasmids containing a single recognition sequence (pNB1) or no recognition site (pNB0). When pNB1 and pNB0 were incubated with the enzyme, N.BstNBI nicked only the plasmid pNB1, suggesting that N.BstNBI is a specific nicking endonuclease.
Phosphatidylinositol 3-Kinase and Prostaglandylinositol Cyclic Phosphate (Cyclic PIP), a Mediator of Insulin Action, in the Signal Transduction of Insulin
Authors: Gypakis A. , Wasner H.K. 
Published: October 2004

AbstractIt has been suggested that downstream signaling from the insulin receptor to the level of the protein kinases and protein phosphatases is accomplished by prostaglandylinositol cyclic phosphate (cyclic PIP), a proposed second messenger of insulin. However, evidence points also to both phosphatidylinositol 3-kinase, which binds to the tyrosine phosphorylated insulin receptor substrate-1, and the Ras complex in insulin's downstream signaling. We have examined whether a correlation exists between these various observations. It was found that wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, prevented insulin-induced, as well as cyclic PIP-induced activation of glucose transport, indicating that PI 3-kinase action on glucose transport involves downstream signaling of both insulin and cyclic PIP. Wortmannin has no effect on cyclic PIP synthase activity nor on the substrate production for cyclic PIP synthesis either, indicating that the functional role of PI 3-kinase is exclusively downstream of cyclic PIP.
Cytokine-Inducing Activity of Family 2 Cystatins
Authors: Kato T. , Imatani T. , Miura T. , Minaguchi K. , Saitoh E. , Okuda K. 
Published: October 2004

AbstractCystatins are physiological cysteine proteinase inhibitors. Here we report a novel function for some family 2 cystatins that is not related to these activities. The release of interleukin-6 (IL-6) and interleukin-8 (IL-8) by the gingival fibroblasts and that of IL-6 by murine splenocytes were measured using ELISA systems specific for these cytokine molecules. Family 2 cystatins, including cystatins C, SA1, SA2, S, and egg white cystatin, upregulated the IL-6 production by two human gingival fibroblast cell lines or murine splenocytes and also the IL-8 production by gingival fibroblasts at physiological concentrations. After complete saturation with papain, those family 2 cystatins still upregulated IL-6 production, suggesting that the papain- inhibitory site was not involved in the cytokine-inducing activity.