bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

N.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

It 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

Cystatins 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.
The Difference in the Carboxy-Terminal Sequence Is Responsible for the Difference in the Activity of Chicken and Rat Liver Fructose-2,6-Bisphosphatase
Authors: Zhu Zheng , Ling Song , Yang Qi-Heng , Li Lin
Published: October 2004

The fructose-2,6-bisphosphatase domain of the bifunctional chicken liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase shares approximately 95% amino acid sequence homology with that of the rat enzyme. However, these two enzymes are significantly different in their phosphatase activities. In this report, we show that the COOH-terminal 25 amino acids of the two enzymes are responsible for the different enzymatic activities. Although these 25 amino acids are not required for the phosphatase activity, their removal diminishes the differences in the activities between the two enzymes. In addition, two chimeric molecules (one consisting of the catalytic core of the chicken bisphosphatase domain and the rat COOH-terminal 25 amino acids, and the other consisting of most of the intact chicken enzyme and the rat COOH-terminal 25 amino acids) showed the same kinetic properties as the rat enzyme. Furthermore, substitution of the residues Pro456pro457Ala458of the chicken enzyme with GluAlaGlu, the corresponding sequence in the rat liver enzyme, yields a chicken enzyme that behaves like the rat enzyme. These results demonstrate that the different bisphosphatase activities of the chicken and rat liver bifunctional enzymes can be attributed to the differences in their COOH-terminal amino acid sequences, particularly the three residues.
Superoxide Reactivates Nitric Oxide-Inhibited Catalase
Authors: Kim Yu Shin , Han Sanghwa
Published: October 2004

Catalase binds nitric oxide (NO) to generate ferricatalase-NO, an inhibited form of the enzyme. Superoxide
(O2
−) is also an inactivator of the enzyme. We
found, however, that O2
− efficiently converted the inhibited
ferricatalase-NO to the active ferricatalase
without producing detectable intermediates. The reaction
slowed down when O2
− was disproportionated
to H2O2 and O2 by superoxide dismutase, but H2O2
could displace the heme-bound NO slowly to regenerate
ferricatalase. Reactivation was observed even
under simultaneous generation of NO and O2
− suggesting
that ferricatalase-NO reacts with O2
− fast
enough to compete with the rapid reaction of O2
− and
NO. Formation of peroxynitrite by the simultaneous
generation of NO and O2
− was only partially inhibited by
ferricatalase, presumably due to slow binding of NO to
catalase in comparison with the reaction of NO and O2
−.
Classification of Kinin Receptors
Authors: Regoli Domenico , Rizzi Anna , Perron Stephan I. , Gobeil Fernand
Published: October 2004

This minireview is divided into three parts: the first part refers to the characterization and classification of kinin receptors using agonists and antagonists in isolated tissues (classical pharmacology). Two kinin receptors have been considered on the basis of their distinct pharmacology, namely the B1receptor of the rabbit aorta (rank order of potency of agonists: LysdesArg9BK > desArg9BK >= LysBK>BK; apparent affinities of antagonists Lys[Leu8]desArg9BK (pIC508.4 ) > [Leu8]desArg9BK (pIC507.4)>>>HOE 140, a B2receptor antagonist, pIC50<5.0), and the B2receptor of the rabbit jugular vein (potency of agonists: LysBK = BK>>> LysdesArg9BK = desArg9BK and HOE 140 (pIC509.0)>>>Lys[Leu8]desArg9BK, pIC50<5.0). The second part describes speciesrelated B1receptor subtypes, demonstrated by different pharmacological profiles of agonists and antagonists: human, rabbit and pig subtypes (LysdesArg9BK >> desArg9B K and Lys[Leu8]desArg9BK > [Leu8]desArg9BK) and dog, rat, mouse and hamster B1receptors (desArg9BK = LysdesArg9BK and [Leu8]desArg9BK = Lys[Leu8]desArg9BK). Affinities of agonists and antagonists in some species (man, rabbit, pig) are significantly increased (at least 10-fold) by the presence of a Lys at their Nterminus. The last part describes species-related B2receptor subtypes supported by results obtained with non-peptide receptor agonists (FR 190997) and antagonists (FR 173657). While BK acts as a full agonist in man, rabbit and pig, FR 190997 behaves as a full agonist on human, as partial agonist on rabbit, and as pure antagonist on pig B2receptors. Various hypotheses are considered to interpret these findings.
Molecular Cloning and Pharmacological Characterization of the Canine B1 and B2 Bradykinin Receptors
Authors: Hess J. Fred , Hey Patricia J. , Chen Tsing-Bau , OBrien Julie , Omalley Stacey S. , Pettibone Douglas J. , Chang Raymond S. L.
Published: October 2004

The dog is a valuable animal model in the study of the physiological role of both the B1 and B2 bradykinin receptors. To more thoroughly characterize the pharmacological properties of the canine kinin receptors we isolated the cDNA sequence encoding the B1 and B2 bradykinin receptor subtypes and overexpressed them in Chinese hamster ovary (CHO) cells. The cDNA sequence of the canine B1 bradykinin receptor encodes a protein comprised of 350 amino acids that is 76% identical to the human B1 bradykinin receptor. The cDNA sequence of the canine B2 bradykinin receptor encodes a protein of 392 amino acids that is 81% identical to the human B2 bradykinin receptor. The amino acid sequence of the canine B1 and B2 receptors are 35% identical. Pharmacological studies of the cloned receptors revealed that the agonist affinity of the dog B1 receptor is similar to the rodent B1 receptors, and differs from the human form in that there is no preference for the presence of the Nterminal Lys residue of [desArg10]Lysbradykinin. Significantly, the B1 receptor antagonist [desArg9,Leu8]BK behaves as partial agonist on the cloned dog B1 receptor. The dog B2 receptor exhibits the classical pharmacological properties of this receptor subtype.
Structural Characterization of Fucose-Containing Oligosaccharides by High-Performance Liquid Chromatography and Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry
Authors: Suzuki Minoru , Suzuki Akemi
Published: October 2004

Eight pyridylamino (PA) derivatives of fucosecontaining oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by highperformance liquid chromatography (HPLC) on normalphase and reversedphase columns, and by matrixassisted laser desorption/ionization timeofflight MALDITOF) mass spectrometry. Six out of eight PAoligosaccharides were clearly separated by both normal and reversedphase HPLC at a column temperature of 40 C, but two PAoligosaccharides, lactoNfucopentaose II [Gal?1-3(Fuc?1-4)GlcNAc?1- 3Gal?1-4GlcPA] and lactoNfucopentaose III Gal?1-4(Fuc?1-3)GlcNAc?1-3Gal?1-4GlcPA], were not separated. The two unresolved PAoligosaccharides were finally separated by reversedphase HPLC at a column temperature of 11 C. MALDITOF mass spectra of PAoligosaccharides demonstrated pseudomolecular ions as the predominant signals, therefore information about the molecular mass of each PAoligosaccharide was easily obtained. Postsource decay (PSD) MALDITOF mass spectra of PAoligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PAoligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PAoligosaccharides by HPLC, MALDITOF mass spectrometry, and PSD MALDITOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDITOF mass spectrometry or HPLC and PSD MALDITOF mass spectrometry is quite useful for the structural characterization of subpmol or pmol levels of fucosecontaining oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.
Biosynthesis of N-Acetylneuraminic Acid in Cells Lacking UDP-N-Acetylglucosamine 2-Epimerase/ N-Acetylmannosamine Kinase
Authors: Hinderlich Stephan , Berger Markus , Keppler Oliver T. , Pawlita Michael , Reutter Werner
Published: October 2004

The first two steps in mammalian biosynthesis of Nacetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase. A subclone of the human B lymphoma cell line BJAB K20, lacking UDPNacetylglucosamine 2- epimerase/Nacetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains Nacetylmannosamine kinase activity. A gel filtration analysis of BJAB K88 control cells, which express UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase, revealed two Nacetylmannosamine kinase activity peaks, one coeluting with UDPNacetylglucosamine 2-epimerase activity and one coeluting with Nacetylglucosamine kinase. For this enzyme previous studies already showed ManNAc kinase activity in vitro. In contrast, the hyposialylated BJAB K20 subclone displayed only the Nacetylmannosamine kinase peak, comigrating with Nacetylglucosamine kinase. The CMPNacetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supple menting the medium with Nacetylmannosamine. This Nacetylmannosamineinduced increase was drastically reduced by cosupplementation with Nacetylglucosamine only in K20 cells. We therefore propose the phosphorylation of Nacetylmannosamine as a hitherto unrecognized role of Nacetylglucosamine kinase in living cells.
Heuristic Statistical Analysis of Fluorescence Fluctuation Data with Bright Spikes: Application to Ligand Binding to the Human Serotonin Receptor Expressed in Escherichia coli Cells
Authors: Craenenbroeck Elke Van , Vercammen Jo , Matthys Gunther , Beirlant Jan , Marot Christophe , Hoebeke Johan , Strobbe Rik , Engelborghs Yves
Published: October 2004

A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore coated beads that mimic the behaviour of multiliganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.
Determination of the Net Exchange Rate of Tubulin Dimer in Steady-State Microtubules by Fluorescence Correlation Spectroscopy
Authors: Neumann Tobias , Kirschstein Steffen O. , Gomez Juan A. Camacho , Kittler Leonhard , Unger Eberhard
Published: October 2004

The microtubule cytoskeleton plays an important role
in eukaryotic cells, e. g., in cell movement or morphogenesis.
Microtubules, formed by assembly of tubulin
dimers, are dynamic polymers changing randomly
between periods of growing and shortening, a property
known as dynamic instability. Another process
characterizing the dynamic behaviour is the socalled
treadmilling due to different binding constants of
tubulin at both microtubule ends.
In this study, we used tetramethylrhodamine
(TMR)labeled tubulin added to microtubule suspensions
to determine the net exchange rate (NER) of
tubulin dimers by fluorescence correlation spectroscopy
(FCS) as a measure for microtubule dynamics.
This approach, which seems to be suitable as
screening system to detect compounds influencing
the NER of tubulin dimers into microtubules at
steadystate, showed that taxol, nocodazole,
colchicine, and vinblastine affect microtubule dynamics
at concentrations as low as 10[-9] 10[-10] M.
Nucleotide-Binding Sites in the Functional Unit of Sarcoplasmic Reticulum Ca2+-ATPase as Studied by Photoaffinity Spin-Labeled 2-N3-SL-ATP
Authors: Palm Thomas , Coan Carol , Trommer Wolfgang E.
Published: October 2004

2-N[3]SLATP [2-azido-2,3O(1-oxyl-2,2,5,5-tetramethyl 3-carbonylpyrroline) adenosine triphosphate], a photoaffinity spinlabeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotidebinding site stoichiometry of sarcoplasmic reticulum (SR) Ca[2+]ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found. The ESR spectra showed signals from spinspin interactions between two radicals corresponding to a distance of about 15 å between labels bound to adjacent sites on the enzyme. This indicates that the minimal functional unit of the Ca[2+]ATPase is a dimer with the nucleotidebinding sites in close proximity.
Synergistic Inhibition of the Glucocorticoid Receptor by Radicicol and Benzoquinone Ansamycins
Authors: Rosenhagen Marcus C. , Young Jason C. , Wochnik Gabriela M. , Herr Alexandra S. , Schmidt Ulrike , Hartl F. Ulrich , Holsboer Florian , Rein Theo
Published: October 2004

Radicicol (RAD) and the benzoquinone ansamycin
geldanamycin (GA) are potential anticancer drugs
known to inhibit heat shock protein 90 (hsp90) and,
therefore, the activation of proteins dependent on its
function such as protooncogenic kinases and nuclear
receptors. Using the glucocorticoid receptor
(GR) as a model system we analysed the effects of
RAD and various benzoquinone ansamycins. All
compounds efficiently abolished GRdependent
transactivation. Surprisingly, whenever one of the
ansamycins was applied in combination with RAD,
synergistic inhibition of GRdependent transcription
and of hormone binding of GR was observed. In contrast,
combination of two ansamycins showed no
synergy. These findings suggest synergism within
the hsp90 dimer and may open new ways to explore
hsp90 as therapeutic target.
C-Terminal Peptides of Interleukin-6 Modulate the Expression of junB Protooncogene and the Production of Fibrinogen by HepG2 Cells
Authors: Igaz Péter , Bösze Szilvia , Tóth Sára , Falus András , Hudecz Ferenc
Published: October 2004

Interleukin-6 (IL-6) is a 185 amino acid residue helical cytokine with various biological activities (e. g. B cell development, acute phase reaction). We have investigated the role of the 168 185 Cterminal region of IL-6 in the induction of fibrinogen synthesis and expression of junB mRNA using synthetic peptides corresponding to this region. Circular dichroism spectroscopy data suggest that even truncated peptides have a strong tendency to adopt an ordered conformation. Peptides were tested alone or in combination with recombinant hIL-6 on an IL-6 responsive human hepatoma HepG2 cell line. The expression of the protooncogene junB monitored by competitive RTPCR represents an early, while the fibrinogen production detected by sandwich ELISA a late, marker of IL-6 initiated events. We found that peptides depending on their structure modulate spontaneous as well as IL-6 induced fibrinogen production and/or mRNA expression of junB by exhibiting inhibition (in the presence of IL-6) or stimulation (in the absence of IL-6).
Local Variability of the Phosphoglycerate Kinase-Triosephosphate Isomerase Fusion Protein from Thermotoga maritima MSB 8
Authors: Wassenberg Doris , Wuhrer Manfred , Beaucamp Nicola , Schurig Hartmut , Wozny Manfred , Reusch Dietmar , Fabry Stefan , Jaenicke Rainer
Published: October 2004

The pgktpi gene locus of Thermotoga maritima encodes both phosphoglycerate kinase (PGK) and a bienzyme complex consisting of a fusion protein of PGK with triosephosphate isomerase (TIM). No separate tpi gene for TIM is present in T. maritima. A frameshift at the end of the pgk gene has been previously proposed as a mechanism to regulate the expression of the two protein variants [Schurig et al., EMBO J. 14 (1995), 442 451]. Surprisingly, the complete T. maritima genome was found to contain a pgktpi sequence not requiring the proposed frameshift mechanism. To clarify the apparent discrepancy, a variety of DNA sequencing techniques were applied, disclosing an anomalous local variability in the pgktpi fusion region. The comparison of different DNA samples and the mass spectrometric analysis of the amino acid sequence of the natural fusion protein from T. maritima MSB8 confirmed the local variability of the DNA variants. Since not all peptide masses could be assigned, further variations are conceivable, suggesting an even higher heterogeneity of the T. maritima MSB8 strain.
Towards Specific Functions of Lysosomal Cysteine Peptidases: Phenotypes of Mice Deficient for Cathepsin B or Cathepsin L
Authors: Reinheckel T. , Deussing J. , Roth W. , Peters C.
Published: October 2004

The lysosomal cysteine peptidases cathepsin B and cathepsin L are abundant and ubiquitously expressed members of the papain family, and both enzymes contribute to the terminal degradation of proteins in the lysosome. However, there is accumulating evidence for specific functions of lysosomal proteases in health and disease. The generation of knock out mouse strains that are deficient in lysosomal proteases provides a valuable tool for evaluation of existing hypotheses and gaining new insights into the in vivo functions of these proteases. In this minireview, we summarise and discuss the findings obtained by analysis of mice that are devoid of cathepsin B or cathepsin L. In brief, cathepsin L appears to be critically involved in epidermal homeostasis, regulation of the hair cycle, and MHC class IImediated antigen presentation in cortical epithelial cells of the thymus. Cathepsin B plays a major role in pathological trypsinogen activation in the early course of experimental pancreatitis and contributes significantly to TNFα induced hepatocyte apoptosis.
Coenzymes Q9 and Q10, Vitamin E and Peroxidation in Rat Synaptic and Non-Synaptic Occipital Cerebral Cortex Mitochondria during Ageing
Authors: Battino Maurizio , Bompadre Stefano , Leone Luciana , Villa Roberto F. , Gorini Antonella
Published: October 2004

Great attention has been devoted both to ageing phenomena at the mitochondrial level and to the antioxidant status of membrane structures. These kinds of investigations are difficult to perform in the brain because of its heterogeneity. It is known that synaptic heavy mitochondria (HM) may represent an aged mitochondrial population characterized by a partial impairment of their typical mitochondrial function. We arranged a novel system requiring no extraction procedure, very limited handling of the samples and their direct injection into the HPLC apparatus, to carry out, for the first time, a systematic and concomitant determination of vitamin E, Coenzyme Q[9] (CoQ[9]) and Coenzyme Q[10] (CoQ[10]) contents in rat brain mitochondria. The trends found for CoQ[9] and CoQ[10] levels in synaptic and nonsynaptic occipital cerebral cortex mitochondria during rat ageing are consistent with previous data. Hydroperoxides (HP) differed with age and it was confirmed that in the HM fraction the summation of contributions results in an oxidatively jeopardized subpopulation. We found that vitamin E seems to increase with age, at least in nonsynaptic free (FM) and synaptic light (LM) mitochondria, while it was inclined to remain substantially constant in HM.
Membrane Activity of (Cys48Ser) Lung Surfactant Protein B Increases with Dimerisation
Authors: Zaltash Shahparak , Griffiths William J. , Beck David , Duan Chen-Xia , Weaver Timothy E. , Johansson Jan
Published: October 2004

One of the possible functions of lung surfactant protein B (SPB), an hydrophobic membraneassociated saposinlike protein, is to reduce the alveolar surface tension by promoting insertion of phospholipids into the air/liquid interface of the lung. SPB is a covalent homodimer; Cys48 of two polypeptides form an intermolecular disulphide bond. In order to test whether dimerisation of SPB is important for surfactant function, transgenic mice which express (Cys48Ser) human SPB in a mouse SPB null background were generated. In previous studies (Cys48Ser)SPB showed a concentrationdependent in vitro activity, suggesting that it may form noncovalent dimers. Here (Cys48Ser)SPB isolated from bronchoalveolar lavage of transgenic mice was studied at different concentrations by circular dichroism (CD) spectroscopy, pulsating bubble surfactometry, mass spectrometry and reversedphase HPLC. The results indicate that (Cys48Ser)SPB, both in a phospholipid environment and in organic solvents, is largely monomeric and exhibits low activity at concentrations lower than 1 2 M, while at higher concentrations it forms noncovalent dimers, which are nearly functionally equivalent to native SPB in vitro. Furthermore, electrospray mass spectrometry showed that more dimers were found relative to the monomer when the polarity of the solvent was decreased, and when the concentration of SPB increased. (Cys48Ser)SPB also eluted earlier than native SPB in reversedphase HPLC. Taken together, these results indicate that a polar surface is buried upon dimerisation, thereby promoting formation of interchain ion pairs between Glu51 Arg52 and Glu51Arg52.
A Selenocysteine-Containing Peroxiredoxin from the Strictly Anaerobic Organism Eubacterium acidaminophilum
Authors: Söhling Brigitte , Parther Tina , Rücknagel Karl Peter , Wagner Matthias , Andreesen Jan R.
Published: October 2004

A strongly [75]Selabeled 22 kDa protein detected previously showed in its Nterminal sequence the highest similarity to the family of thioldependent peroxidases, now called peroxiredoxins. The respective gene prxU was cloned and analyzed. prxU encodes a protein of 203 amino acids (22470 Da) and contains an inframe UGA codon (selenocysteine) at the position of the so far strictly conserved and catalytically active Cys47. The second conserved cysteine present in 2-Cys peroxiredoxins was replaced by alanine. Heterologous expression of the Eubacterium acidaminophilum PrxU as a recombinant selenoprotein in Escherichia coli was not possible. A cysteineencoding mutant gene, prxU47C, containing UGC instead of UGA was strongly expressed. This recombinant PrxU47C mutant protein was purified to homogeneity by its affinity tag, but was not active as a thioldependent peroxidase. The identification of prxU reveals that the limited class of natural selenoproteins may in certain organisms also include isoenzymes of peroxiredoxins, previously only known as nonselenoproteins containing catalytic cysteine residues.
Characterization of C-Terminally Truncated Human Tissue Inhibitor of Metalloproteinases-4 Expressed in Pichia pastoris
Authors: Stratmann Bernd , Farr Martin , Tschesche Harald
Published: October 2004

The tight regulation of extracellular matrix remodeling
and degradation is of great importance in physiological
processes like development and morphogenesis,
as well as in pathological situations like tumor
invasion and metastasis. Tissue inhibitors of metalloproteinases (TIMPs) are the naturally occuring inhibitors of matrix metalloproteinases, which are involved
in matrix turnover.
In this report we describe the cloning of human
TIMP-4 from a human adenocarcinoma and an osteosarcoma cell line and the expression of the inhibitory domain in the methylotrophic yeast Pichia
pastoris. The inhibition of MMP-8, -9, -12, -13 and -14
by the Nterminal domain of TIMP-4 was analysed.
Using a fluorescent MCApeptide, K values for each
subclass of MMPs were determined. With dissociation
constants in the nanomolar range, TIMP-4 seems
to be a good inhibitor for all classes of MMPs without
remarkable preference for special MMPs.
On the Characterization of the Putative S20-Thx Operon of Thermus thermophilus
Authors: Leontiadou Fotini , Triantafillidou Dimitra , Choli-Papadopoulou Theodora
Published: October 2004

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

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

The 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

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

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

A 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

RgpB, 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

The 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

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

The 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

Bradykinin 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

In 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

Human 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

Protein 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

The 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

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.