bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the Characterization of the Putative S20-Thx Operon of Thermus thermophilus
Authors: Leontiadou Fotini , Triantafillidou Dimitra , Choli-Papadopoulou Theodora 
Published: October 2004

Abstract
A putative operon of the ribosomal proteins S20 and
Thx has been determined in a 1.4 kb sequenced region of T. thermophilus genomic
DNA. Both genes have a promoter sequence 29 nt upstream of ORF1, possess their
own ShineDalgarno motifs (GGAG) and are separated by only 9 nucleotides, a
feature characteristic of the compact Thermus thermophilus genome. This is a
novel arrangement, since Thx is unique to the Thermus bacteria and in all other
prokaryotes the S20 gene is monocistronic. Our results, in conjunction with the
recent finding that Thx is located on the top of the head of the 30S subunit in
a cavity between multiple RNA elements stabilizing them with its positive
charge, corroborate the observation that thermophilic ribosomes require
constituents with special features for their stabilization at high
temperatures.
Analysis of Structural Signals Conferring Localisation of Pig OST48 to the Endoplasmic Reticulum
Authors: Hardt Birgit , Aparicio Raquel , Breuer Wilhelm , Bause Ernst 
Published: October 2004

Abstract
Pig liver oligosaccharyltransferase (OST) is a
heterooligomeric protein complex responsible for the cotranslational transfer
of GlcNAc[2]Man[9]Glc[3] from Dol PP onto specific asparagine residues in the
nascent polypeptide. OST48, one of the catalytic subunits in this complex,
exerts a typical type I membrane topology, containing a large luminal domain, a
hydrophobic transmembrane domain and a short cytosolic peptide tail. Because
OST48 is found within the endoplasmic reticulum (ER) when overexpressed in
COS-1 cells, we carried out experiments to identify structural signals
potentially capable of directing ERtargeting, using OST48 mutants and hybrid
proteins consisting of individual OST48 domains and Man[9] mannosidase.
Immunofluorescence microscopy showed that OST48 mutants in which the Cterminal
lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48?K)
could be detected on the cell surface. This indicates that these two lysine
residues are sufficient for conferring ERresidency on OST48. The doublelysine
motif operates only when exposed cytosolically, where it acts as a relocation
signal rather than causing retention. OST48?K-3, when coexpressed in COS-1
cells together with myctagged ribophorin I, was quantitatively retained in the
ER. By contrast, coexpression in the presence of ribophorin I resulted in no
reduction of cell surface fluorescence for the OMO?K-5 chimera containing the
cytosolic and transmembrane domain of OST48 attached to the Cterminus of the
Man[9]mannosidase luminal domain. Thus ERlocalisation of OST48 is probably
brought about by complex formation with ribophorin I and this most likely
involves the luminal domains of both proteins. Consequently, the doublelysine
motif in the cytosolic domain of OST48 is unlikely to have a primary function
except being involved in recapture of molecules which have escaped from the
ER.
Singlet Molecular Oxygen Triggers the soxRS Regulon of Escherichia coli
Authors: Agnez-Lima Lucymara F. , Mascio Paolo Di , Demple Bruce , Menck Carlos F.M. 
Published: October 2004

AbstractThe electronically excited molecular oxygen (singlet oxygen, [1]O[2]) can be detrimental to cells in several ways, although recent reports indicate that it may play a role as an intercellular signal in eukaryotes. Here we present evidence that [1]O[2], generated by thermodissociation of disodium 3,3(1,4-naphthylidene) diproprionate endoperoxide, activates transcription of genes of the soxRS regulon, and that this induction is paralleled by induction of a soxS::lacZ operon fusion. The inductions were dependent on a functional soxR gene. These data imply that protective responses, such as induction of the soxRS regulon, may be triggered by diverse environmental oxidative stresses, and that [1]O[2] may also function as a signal molecule in prokaryotes.
Divergent Evolution of (??)8-Barrel Enzymes
Authors: Henn-Sax M. , Höcker B. , Wilmanns M. , Sterner R. 
Published: October 2004

Abstract
The (?[alpha)8-barrel is the most versatile and most frequently encountered fold among enzymes. It is an interesting question how the contemporary (?[alpha)8-barrels are evolutionarily related and by which mechanisms they evolved from more simple precursors. Comprehensive comparisons of amino acid sequences and threedimensional structures suggest that a large fraction of the known (?[alpha)8-barrels have divergently evolved from a common ancestor. The mutational interconversion of enzymatic activities of
several (?[alpha)8-barrels further supports their common evolutionary origin. Moreover, the high structural similarity between the N and Cterminal (?[alpha)4 units of two (?[alpha)8-barrel enzymes from histidine biosynthesis indicates that the contemporary proteins evolved by tandem duplication and fusion of the gene of an ancestral halfbarrel precursor. In support of this hypothesis, recombinantly produced halfbarrels were shown to be folded, dimeric proteins.
RNA-Catalyzed Carbon-Carbon Bond Formation
Authors: Jäschke A. 
Published: October 2004

Abstract
RNA molecules with catalytic properties have been isolated by in vitro selection from combinatorial libraries. A broad range of chemical reactions can be catalyzed, and nucleic acids can accelerate bond formation between small organic substrates. The catalytic performance of nucleic acids can be enhanced by incorporation of additional functional groups. This minireview focuses on carboncarbon bond formation accelerated by in vitro selected ribozymes.
Fragment-Based Flexible Ligand Docking by Evolutionary Optimization
Authors: Budin N. , Majeux N. , Caflisch A. 
Published: October 2004

AbstractA new computational approach for the efficient docking of flexible ligands in a rigid protein is presented. It exploits the binding modes of functional groups determined by an exhaustive search with solvation. The search in ligand conformational space is performed by a genetic algorithm whose scoring function approximates steric effects and intermolecular hydrogen bonds. Ligand conformations generated by the genetic algorithm are docked in the protein binding site by optimizing the fit of their fragments to optimal positions of chemically related functional groups. We show that the use of optimal binding modes of molecular fragments allows to dock known inhibitors with about ten rotatable bonds in the active site of the uncomplexed and complexed conformations of thrombin and HIV-1 protease.
Arginine-Specific Cysteine Proteinase from Porphyromonas gingivalis as a Convenient Tool in Protein Chemistry
Authors: Banbula A. , Mak P. , Smoluch M. , Travis J. , Potempa J. 
Published: October 2004

AbstractRgpB, a cysteine proteinase produced by Porphyromonas gingivalis, exhibits proteolytic activity selectively directed against peptide bonds containing an arginine residue in the P1 position. Here we show that this enzyme can be used for very efficient and specific protein cleavage. RgpB is highly active even at high concentrations of denaturing agents, including urea (up to 6 M) and SDS (0.1%), both of them being commonly used for solubilization of insoluble proteins and peptides. Moreover, RgpB is able to digest polypeptide chains in buffers supplemented with 1% Triton X-100, 1% octyl or decylpyranoside, detergents employed for the enzymatic digestion of proteins transferred onto nitrocellulose membranes. These features render RgpB a suitable tool for use in protein chemistry.
Protein-DNA Interaction and CpG Methylation at rep*/vIL-10p of Latent Epstein-Barr Virus Genomes in Lymphoid Cell Lines
Authors: Niller H.H. , Salamon D. , Takacs M. , Uhlig J. , Wolf H. , Minarovits J. 
Published: October 2004

AbstractThe viral interleukin-10 promoter (vIL-10p), overlapping the rep* element in the EpsteinBarr virus (EBV) genome, is a promoter element active mostly in the late phase of the lytic cycle and immediately upon infection of B cells. rep* was, through transfection experiments with small plasmids, characterised as a cis element supporting oriP replicative function. In this study, in vivo protein binding and CpG methylation at rep*/vIL-10p were analysed in five cell lines that harbour strictly latent EBV genomes. Contrary to the invariably unmethylated dyad symmetry element (DS) of oriP, rep*/vIL-10p was highly methylated and showed only traces of protein binding in all examined cell lines. This result is in agreement with vIL-10p being an inactive promoter of EBV genomes, and makes it less likely that rep* functions as a replicative element of latent EBV genomes.
In Vitro and in Vivo Processing of Cyanelle tmRNA by RNase P
Authors: Gimple O. , Schön A. 
Published: October 2004

Abstract
Ribonuclease P, the ubiquitous endonuclease required for generating mature tRNA 5 ends, is a ribonucleoprotein in most organisms and organelles, with the exception of mitochondria and chloroplasts of multicellular organisms. The cyanelle of the primitive alga Cyanophora paradoxa is the only photosynthetic organelle where the ribonucleoprotein nature of this enzyme has been functionally proven. tmRNA is another highly structured RNA: it can be aminoacylated with alanine, which is then incorporated into a tag peptide encoded on the same RNA molecule. This dualfunction RNA has been found in bacteria, and its gene is also present in mitochondria and plastids from primitive organisms. Since nothing is known about the expression of this RNA in organelles, we have performed processing studies and determined the promoter of cyanelle pretmRNA. This RNA is transcribed as a precursor molecule in vivo. Synthetic transcripts of cyanelle pretmRNA, including or lacking the mature 3 CCAend, are efficiently and correctly processed in vitro by bacterial RNase P ribo and holoenzymes and by the homologous cyanelle RNase P. In addition to these experimental data, we propose a novel secondary structure model for this organellar tmRNA, which renders it more similar to its bacterial counterpart.
Identification of a Gephyrin-Binding Motif in the GDP/GTP Exchange Factor Collybistin
Authors: Grosskreutz Y. , Hermann A. , Kins S. , Fuhrmann J.C. , Betz H. , Kneussel M. 
Published: October 2004

AbstractThe brainspecific GDP/GTP exchange factor collybistin interacts with the receptoranchoring protein gephyrin and activates the Rholike GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with heterooligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pulldown assays and in the yeast twohybrid system. Sitedirected mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyRβsubunit binding motif. Our data are consistent with collybistingephyrin interactions occuring during inhibitory postsynaptic membrane formation.
Tissue Kallikrein KLK1 Is Expressed de Novo in Endothelial Cells and Mediates Relaxation of Human Umbilical Veins
Authors: Dedio J. , Wiemer G. , Rütten H. , Dendorfer A. , Schölkens B.A. , Müller-Esterl W. , Wohlfart P. 
Published: October 2004

AbstractBradykinin released by the endothelium is thought to play an important local role in cardiovascular regulation. However, the molecular identity of endothelial proteases liberating bradykinin from its precursors remained unclear. Using RTPCR and Southern blotting techniques we detected mRNA for tissue kallikrein (KLK1) in human umbilical vein endothelial cells and in bovine aortic endothelial cells. Protein expression was confirmed by precipitation of KLK1 from lysates of endothelial cells prelabeled with [35S]cysteine/methionine. Partial purification of tissue kallikrein from total endothelial cell extracts resulted in a protein triplet of about 50 kDa in Western blots using specific antiKLK1 antibodies. The immunodetection of tissue kallikrein antigen in the fractions from ion exchange chromatography correlated with the presence of amidolytic tissue kallikrein activity. Stimulation of endothelial cells with angiotensin II (ANGII), which recently has been shown to activate the vascular kinin system and to cause vasodilation, resulted in the release of bradykinin and kallidin. ANGIIdependent relaxation of preconstricted rings from human umbilical veins was abolished in the presence of a specific tissue kallikrein inhibitor. We conclude that endothelial cells de novo express significant amounts of tissue kallikrein, which likely serves in the local generation of vasoactive kinins.
Secretion of Gelatinases and Activation of Gelatinase A (MMP-2) by Human Rheumatoid Synovial Fibroblasts
Authors: Smolian H. , Aurer A. , Sittinger M. , Zacher J. , Bernimoulin J.-P. , Burmester G.-R. , Kolkenbrock H. 
Published: October 2004

AbstractIn monolayer cultures human rheumatoid synovial fibroblasts (HRSF) secrete gelatinase A (MMP-2) and, unlike other human fibroblasts, to a minor extent also gelatinase B (MMP-9) as inactive proenzymes. In this regard HRSF resemble the fibrosarcoma cell line HT-1080. Unlike HT-1080, however, HRSF do not increase the secretion of MMP-9 in response to phorbol-12-myristate-13-acetate. This indicates that in HRSF the protein kinase C pathway for an enhanced MMP-9 secretion is inactive. None of the substances used in our study increased MMP-9 secretion, but some of them inhibited MMP-9 secretion. The secretion of MMP-2 could not be enhanced either, not even by dbcAMP, which has been reported to be effective in Sertoli and peritubular cells. Activation of MMP-2 in HRSF could be induced by treatment with concanavalin A (ConA) or cytochalasin D, as was shown for other cell types. This activation was not accompanied by a significant change in the amount of secreted TIMP-1 and TIMP-2. In contrast to reports on human skin fibroblasts, however, the activation of MMP-2 could not be induced in HRSF by treatment of the cells with monensin or sodium orthovanadate. Moreover, monensin was shown to act as an inhibitor of ConA or cytochalasin Dmediated activation. Additionally, and in contrast to a report on a rat fibroblast cell line, MMP-2 activation is not mediated via the MAP kinase pathway in HRSF: PD 98059, a specific inhibitor of MAP kinase kinase, did not inhibit the activation of MMP-2. Similarly ineffective were PD 169316, an inhibitor for p38 MAP kinase, other inhibitors for protein kinases as lavendustin A, Gö 6983, wortmannin, rapamycin, as well as the protein tyrosine kinase inhibitors herbimycin A and genistein. Only staurosporin, a broad spectrum inhibitor of protein kinases, and the ionophores monensin and A 23187 effectively inhibited MMP-2 activation in HRSF. Our results demonstrate that MMP-2 can be activated by quite different pathways, and that different cells, even when belonging to the fibroblast family, do not necessarily use the same activating pathways.
Splice Variants of Human Cathepsin L mRNA Show Different Expression Rates
Authors: Abudula Abulizi , Rommerskirch Winfried , Weber Ekkehard , Günther Dagmar , Wiederanders Bernd 
Published: October 2004

AbstractHuman cathepsin L (hCATL) mRNA occurs in vivo in at least three splice variants. They differ in the length of exon 1, which comprises 278 nucleotides (hCATLA), 188 nucleotides (hCATLA2) and 132 nucleotides (hCATLA3), respectively. We describe here the shortest variant for the first time. This form is predominant in all tissues and cells examined so far, including malignant tumors. We studied the expression rate of the three mRNA variants in order to explain why malignant kidney tumors show low cathepsin L activity despite of high mRNA levels. The variant hCATLA3 showed the highest expression rate in vitro and in vivo. Based on these results, we suggest a cisacting element on human cathepsin L mRNA which can be bound by a negative transacting regulator, thus leading to reduced expression rates.
Osmolytes as Modulators of Conformational Changes in Serpins
Authors: Chow Michelle K.M. , Devlin Glyn L. , Bottomley Stephen P. 
Published: October 2004

AbstractProtein misfolding and aggregation play an integral role in many diseases. The misfolding of the serpin (SERine Proteinase INhibitor) α1-antitrypsin results in the accumulation of insoluble polymers within hepatocytes and α1-antitrypsin deficiency in plasma, predisposing patients to liver cirrhosis and emphysema. We have examined the effect of three naturally occurring osmolytes, sarcosine, glycine betaine and trimethylamine Noxide, on conformational changes in α1-antitrypsin. All three solutes protected native α1-antitrypsin against thermally induced polymerisation and inactivation in a concentrationdependent manner. Further spectroscopic analysis showed that sarcosine stabilises the native conformation of α1-antitrypsin, thus hindering its conversion to an intermediate state and subsequent polymerisation. On refolding in the presence of sarcosine, α1-antitrypsin formed a heterogeneous population, with increasing proportions of molecules adopting an inactive conformation in higher concentrations of the osmolyte. These data show that sarcosine can be used to prevent abnormal structural changes in native α1-antitrypsin, but is ineffective in facilitating the correct folding of the protein. The implications of these results in the context of conformational changes and states adopted by α1-antitrypsin are discussed.
Structural and Redox Properties of the Leaderless DsbE (CcmG) Protein: Both Active-Site Cysteines of the Reduced Form Are Involved in Its Function in the Escherichia coli Periplasm
Authors: Li Qi , Hu Hong-Yu , Wang Wei-Qing , Xu Gen-Jun 
Published: October 2004

AbstractThe thiol/disulfide oxidoreductases play important roles in ensuring the correct formation of disulfide bonds, of which the DsbE protein, also called CcmG, is the one implicated in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli. The soluble, Nterminally truncated DsbE was overexpressed and purified to homogeneity. Here we report the structural and redox properties of the leaderless form (DsbEL ). During the redox reaction, the protein undergoes a structural transformation resulting in a more stable reduced form, but this form shows very low reactivity in thiol/ disulfide exchange of cysteine residues and low activity in accelerating the reduction of insulin. The standard redox potential (E' 0 ) for the active thiol/ disulfide was determined to be 0.186 V; only one of the two cysteines (Cys80) was suggested to be the active residue in the redox reaction. From the aspect of biochemical properties, DsbE can be regarded as a weak reductant in the Escherichia coli periplasm. This implies that the function of DsbE in cytochrome c maturation can be ascribed to its active site cysteines and the structure of the reduced form.
Determination of NADH in Frozen Rat Brain Sections by Laser-Induced Fluorescence
Authors: Rex André , Pfeifer Lutz , Fink Heidrun 
Published: October 2004

Abstract
Methods to assess metabolism are important analytical
tools in neuroscience. The fluorophore nicotinamide
adenine dinucleotide (NADH) is a parameter
of cellular metabolism. NADH fluorescence was
measured using a laserbased fluorescence detector
with spectral and temporal filters. Distribution and intensity
of NADH fluorescence were investigated in
frozen brain sections. In sections containing hippocampus
the intensity of NADH fluorescence was
correlated to brain structures. In order to investigate
the consequences of neurotoxic lesions, 5,7-dihydroxytryptamine
was injected into the dorsal raphe
nucleus 4 to 240 days prior to the measurement.
NADH fluorescence decreased in the affected region
by 50%, indicating that no recovery in metabolic activity
had occurred.
Kinetic Study of Apo B100 Containing Lipoprotein Metabolism Using Amino Acids Labeled with Stable Isotopes: Methodological Aspects
Authors: Maugeais Cyrille , Ouguerram Khadija , Krempf Michel , Magot Thierry 
Published: October 2004

Abstract
Kinetic disturbances of lipoprotein metabolism are important to know for a better understanding of lipid diseases or effects of drugs. These kinetic aspects were previously studied with radioactive tracers. The ethical concerns related to these tracers can be now overcome at a reasonable cost with the new development of small bench top mass spectrometers and the increased production of stable isotope tracers. In this review, we will discuss some methodological aspects related to stable isotope tracers and the analysis of the data with non-compartmental or compartmental models.
Activated Immune System in Patients with Huntington's Disease
Authors: Leblhuber Friedrich , Walli Johannes , Jellinger Kurt , Tilz Gernot P , Widner Bernhard , Laccone Franco , Fuchs Dietmar 
Published: October 2004

Abstract
Abnormalities of immune system compartments were determined in 12 patients with Huntington's disease (eight males, four females; age 42.4 ± 11.7 years) and 11 controls (7 males, 4 females; age 47.0 ± 12.0). All patients were free from infectious diseases. Serum concentrations of a panel of serum soluble markers of immune activation were investigated, namely neopterin, 55-kDa-type soluble tumor necrosis factor receptor (sTNF-R), interleukin-2-receptor (sIL-2R), kynurenine, tryptophan, immunoglobulins (Ig) A, M and G as well as routine laboratory tests. Compared to controls, we found significantly higher serum levels of IgA (p < 0.01), sTNF-R, sIL-2R, neopterin, and complement component C3 (all p < 0.05), and serum tryptophan was decreased (p < 0.001). Higher concentrations of circulating immune complexes, cardiolipin antibodies, IgM, neopterin and lower tryptophan were associated with loss of cognitive function as assessed by the minimental-test. Five patients died within 1 year after measurements were performed. In these patients IgM, circulating immune complexes and neopterin concentrations were higher compared to survivors and serum tryptophan was lower. The data indicate an activation of various immune system compartments in Huntington's disease and that systemic immunological alterations might be important in the course of the disease.