bluuebunny/crossref_metadata_2025 huggingface dataset:
Rough and smooth forms of fluorescein‐labelled bacterial endotoxin exhibit CD14/LBP dependent and independent binding that is influencedby endotoxin concentration
Authors: Triantafilou Martha , Triantafilou Kathy , Fernandez Nelson
Published: March 2003

Lipopolysaccharide (LPS, or endotoxin), is a major constituent of the outer membrane of Gram‐negative bacteria. Bacteria express either smooth LPS, which is composed of O‐antigen (O‐Ag), complete core oligosaccharides, and the lipid A, or rough LPS which lack O‐Ag but possess lipid A and progressively shorter core oligosaccharides. CD14 has been described as the receptor for complexes of LPS with LPS‐binding protein (LBP). Using flow cytometry we have compared the binding of Salmonella minnesota rough LPS (ReLPS) and Escherichia coli smooth LPS labelled with fluorescein isothiocyanate (FITC‐LPS) to Chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14‐CHO), to MonoMac 6 cells and to endothelial cells. Our results showed that both forms of LPS display the same binding characteristics, and that the binding of FITC‐LPS to cells was both CD14‐ and LBP‐dependent for LPS concentrations up to 100 ng·mL−1. At LPS concentrations higher than 100 ng·mL−1 we observed CD14/LBP‐independent binding. CD14/LBP‐dependent binding was dose dependent, saturable, and enhanced in the presence of human pooled serum (HPS), and the monoclonal anti‐CD14 antibody (MY4) or unlabelled LPS could outcompete it.
The C1–C2 interface residue lysine 50 of pig kidney fructose‐1,6‐bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition
Authors: Cárcamo Juan G. , Yañez Alejandro J. , Ludwig Heide C. , León Oscar , Pinto Rodrigo O. , Reyes Alejandro M. , Slebe Juan C.
Published: March 2003

To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig‐kidney fructose‐1,6‐bisphosphatase, Arg49 and Lys50 residues located at the C1–C2 interface of this enzyme were replaced using site‐directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild‐type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose‐2,6‐bisphosphate that were similar to those of the wild‐type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild‐type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose‐1,6‐bisphosphate changed from noncompetitive (wild‐type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000‐fold lower AMP affinity relative to the wild‐type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1–C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.
Diphospho‐myo‐inositol phosphates during the life cycle of Dictyostelium and Polysphondylium
Authors: Laussmann Tim , Pikzack Claudia , Thiel Ulrich , Mayr Georg W. , Vogel Günter
Published: March 2003

The intracellular amounts of diphospho‐myo‐inositol phosphates and InsP6 were determined in Dictyostelium discoideum AX2 throughout the life cycle, including exponential growth, starvation, differentiation, sporulation and spore germination. Similar experiments were performed with the closely related species Polysphondylium pallidum under conditions resulting in microcyst formation. A distinct accumulation of these compounds is observed during the early starvation phase of the cell population before the onset of the actual differentiation program. When exponentially growing D. discoideum cells were shifted to starvation conditions, a 25‐fold accumulation of 5,6‐bis‐PP‐InsP4 within 3 h was observed. In P. pallidum, the 5,6‐bis‐PP‐InsP4 pool rises around 20‐fold within 8 h during the formation of microcysts from vegetative cells. Finally, the diphosphoinositol phosphates are deposited in spores or microcysts and are degraded when spores or microcysts germinate at low cell density.
Induction of intracellular signalling by cyclic glycerophosphates and their deoxy analogues
Authors: Shinitzky Meir , Haimovitz Rachel , Nemas Mara , Cahana Nava , Mamillapalli Ramanaiah , Seger Rony
Published: March 2003

Cyclic glycerophosphates can be formed by enzymic degradation of phospholipids. They have only recently attracted attention, and their physiological function is still obscure. In this study, we have searched for signalling functions of the natural 1,3‐cyclic and 1,2‐cyclic glycerophosphates, their deoxy analogues, and the phenyl esters of the 1,3‐cyclic phosphates. Linear sn‐glycerol 3‐phosphate and glycerol 2‐phosphate served as the control compounds. Each of the six‐membered ring cyclic phosphates tested induced rapid intracellular tyrosine phosphorylation in CHO and NIH‐3T3 cells when applied extracellularly at a concentration of 0.5–4 µm. The phosphorylated intracellular proteins had molecular masses of ≈ 35 kDa, ≈ 45 kDa, 60–70 kDa and ≈ 120 kDa. The five‐membered ring cyclic phosphates had a similar effect, but at an external concentration of 2–10 µm, while sn‐glycerol 3‐phosphate and glycerol 2‐phosphate had no effect. The six‐membered cyclic phosphates also induced rapid threonine phosphorylation in CHO cells of ≈ 18‐kDa, ≈ 35‐kDa, and ≈ 38‐kDa proteins. Further experiments indicated that the cyclic phosphates partition rapidly into the cell cytosol where they activate kinases, including mitogen‐activated protein kinase. When their intracellular level increases, dephosphorylation presumably takes place. This pattern may account for the signalling profile of cyclic phosphates and suggests that they may take part in processes associated with cell differentiation.
Solution structure of ileal lipid binding protein in complex with glycocholate
Authors: Lücke Christian , Zhang Fengli , Hamilton James A. , Sacchettini James C. , Rüterjans Heinz
Published: March 2003

Ileal lipid binding protein (ILBP) is a cytosolic lipid‐binding protein that binds both bile acids and fatty acids. We have determined the solution structure of porcine ILBP in complex with glycocholate by homonuclear and heteronuclear two‐dimensional NMR spectroscopy. The conformation of the protein–ligand complex was determined by restrained energy minimization and simulated annealing calculations after docking the glycocholate ligand into the protein structure. The overall tertiary structure of ILBP is highly analogous to the three‐dimensional structures of several other intracellular lipid binding proteins (LBPs). Like the apo‐structure, the bile‐acid complex of ILBP is composed of 10 anti‐parallel β‐strands that form a water‐filled clam‐shell structure, and two short α‐helices. Chemical shift data indicated that the bile acid ligand is bound inside the protein cavity. Furthermore, 13C‐edited heteronuclear single‐quantum correlation‐NOESY experiments showed NOE contacts between several aromatic residues located in the proposed bile acid portal region and the 13C‐labeled ligand. A single bile acid molecule is bound inside the protein, with the steroid moiety penetrating deep into the water‐accessible internal cavity, such that ring A is located right above the plane of the Trp49 indole ring. The carboxylate tail of the ligand is protruding from the proposed bile acid portal into the surrounding aqueous solution. The body of the steroid moiety is oriented with the nonpolar face in contact with the mostly hydrophobic residues of β‐strands C, D and E, while the polar face shows contacts with the side‐chains of Tyr97, His99, Glu110 and Arg121 in β‐strands H, I and J. Thus, the conformational arrangement of the ligand complex suggests that the binding affinity of ILBP for bile acid molecules is based mainly on strong hydrophobic interactions inside the protein cavity. Furthermore, this binding mode explains how ILBP can transport unconjugated and conjugated bile acids.
Prolyl isomerases in a minimal cell
Authors: Bang Holger , Pecht Anja , Raddatz Guenter , Scior Tom , Solbach Werner , Brune Kay , Pahl Andreas
Published: March 2003

Peptidyl‐prolyl cis/trans isomerases (PPIases) catalyze the isomerization of prolyl peptide bonds. Distinct families of this class of enzymes are involved in protein folding in vitro, whereas their significance in free living organisms is not known. Previously, we inspected the smallest known genome of a self‐replicating organism and found that Mycoplasma genitalium is devoid of all known PPIases except the trigger factor. Despite the extensive sequence information becoming available, most genes remain hypothetical and enzyme activities in many species have not been assigned to an open reading frame. Therefore, we studied the PPIase activity in crude extracts of M. genitalium. We showed that this is solely attributed to a single enzyme activity, the trigger factor. Characterization of this enzyme revealed that its PPIase activity resides in a central 12‐kDa domain. Only the complete trigger factor is able to cis/trans isomerize extended peptide substrates, while the PPIase domain alone can not. The N‐ and the C‐terminal domains of the trigger factor seem to function in binding of proteins as substrates, as demonstrated by protein refolding experiments, in which the complete trigger factor catalyzed protein refolding towards a model protein 500‐fold more efficiently than the isolated central PPIase domain. Protein modeling studies suggest that the PPIase domain can fold in a similar way as the PPIase domain of FK506 binding proteins (FKBPs), one class of PPIases, despite only very limited sequence homology. Differences at the active site explain why this enzyme is not inhibited by FK506 in contrast with FKBPs. Trigger factor expressed in Escherichia coli confirms its additional chaperone functions, as shown by its association with chaperones GroEL and GroES after induction of misfolding. In contrast, the isolated PPIase‐domain lacks any association with chaperones from E. coli. In summary, trigger factor of M. genitalium is the single folding isomerase of this organism, which harbors an enzymatically active PPIase domain with structural homology to FKBPs. Its additional domains confer its ability to be an efficient catalyst of protein folding. The protein folding machinery is conserved and shows a dual function as a chaperone and a prolyl isomerase.
Lck protein tyrosine kinase is a key regulator of T‐cell activation and a target for signal intervention by Herpesvirus saimiri and other viral gene products
Authors: Isakov Noah , Biesinger Brigitte
Published: March 2003

Protein tyrosine kinases (PTKs) are critically involved in signaling pathways that regulate cell growth, differentiation, activation, and transformation. It is not surprising, therefore, that viruses acquire effector molecules targeting these kinases to ensure their own replication and/or persistence. This review summarizes our current knowledge on Lck, a member of the Src family of PTK, and its viral interaction partners. Lck plays a key role in T lymphocyte activation and differentiation. It is associated with a variety of cell surface receptors and is critical for signal transduction from the T‐cell antigen receptor (TCR). Consequently, Lck is targeted by regulatory proteins of T‐lymphotropic viruses, especially by the Herpesvirus saimiri (HVS) tyrosine kinase interacting protein (Tip). This oncoprotein physically interacts with Lck in HVS transformed T cells and has an impact on its catalytic activity. However, while Tip inhibits Lck activity in stably expressing cell lines, opposite effects were observed in several in vitro systems. At least in part, this complex situation may be related to the bipartite nature of the interaction surface of the two proteins. Studies on the interrelationships between Lck and its viral partners contribute to the understanding of the mechanisms of T‐cell growth regulation, in general, and of viral pathogenicity in particular. In addition, understanding the regulation of Lck activity by viral proteins may serve as a basis for the development of new drugs capable of modifying Lck activity in different pathological situations.
Ceramide mimics tumour necrosis factor‐α in the induction of cell cycle arrest in endothelial cells
Authors: López‐Marure Rebeca , Ventura José L. , Sánchez Luis , Montaño Luis F. , Zentella Alejandro
Published: March 2003

Tumour necrosis factor (TNF)‐α induces a transient increase in N‐octanoylsphingosine (C8‐ceramide) which has been postulated as an intracellular mediator in TNF‐α signalling. We tested the ability of C8‐ceramide to reproduce the TNF‐α‐mediated interference with endothelial cell proliferation and DNA synthesis. TNF‐α (10 ng·mL−1) and C8‐ceramide (20 µm) inhibited the incorporation of [3H]thymidine into DNA and led to an accumulation of cells in the G1 phase of the cell cycle. When the responses of the tumour suppressors p53 and RB were analysed, it was found that TNF‐α and C8‐ceramide induced increased expression of p53. Treatment with TNF‐α or C8‐ceramide lead to a significant decrease in total retinoblastoma protein (RB) content that correlated with high levels of p53. These results suggest that p53 and RB may complement each other in their contribution to cell cycle arrest. TNF‐α prevented RB phosphorylation whereas C8‐ceramide did not interfere with this process, suggesting that it follows a ceramide‐independent pathway.
Evidence that phosphate specific transporter is amplified in a fluoroquinolone resistant Mycobacterium smegmatis
Authors: Bhatt Kamlesh , Banerjee Sanjiban K. , Chakraborti Pradip K.
Published: March 2003

We reported in an earlier study that active efflux of drug has a predominant role in conferring resistance in a laboratory‐generated ciprofloxacin‐resistant mutant of Mycobacterium smegmatis. This mutant exhibited mRNA level overexpression, as well as chromosomal amplification, of the gene pstB, encoding the putative ATPase subunit of phosphate specific transport (Pst) system. We demonstrate here that this mutant shows enhanced phosphate uptake and that inactivation of pstB in the parental strain results in loss of high affinity phosphate uptake and hypersensitivity to fluoroquinolones. These findings suggest a novel role of the Pst system in active efflux, in addition to its involvement in phosphate transport.
Cloning, characterization and expression of complete coding sequences of three IgE binding Malassezia furfur allergens, Mal f 7, Mal f 8 and Mal f 9
Authors: Rasool Omid , Zargari Arezou , Almqvist Jenny , Eshaghi Hojjat , Whitley Paul , Scheynius Annika
Published: March 2003

Malassezia furfur, formerly known as Pityrosporum orbiculare or P. ovale, is a yeast that colonizes human skin. Normally, this yeast is nonpathogenic but under the influence of predisposing factors it may induce IgE reactivity in patients with atopic dermatitis. Approximately 40–65% of atopic dermatitis patients have IgE antibodies and/or skin reactivity against M. furfur and a higher T‐cell response against this yeast is found in atopic dermatitis patients than in healthy individuals. By making a cDNA library displayed on a phage surface, we previously cloned five different IgE‐binding proteins, Mal f 5, Mal f 6, MF 7, MF 8 and MF 9, from this yeast. The cDNAs encoding these allergens were sequenced and expressed in Escherichia coli. The sequences of MF 7, MF 8 and MF 9 were not full length (missing their 5′‐ends) giving only partial gene products. To obtain complete cDNA sequences, we performed RACE‐PCR to amplify the 5′‐ends of each cDNA. These PCR products were sequenced and analyzed. The coding sequences of Mal f 7, Mal f 8 and Mal f 9 encode proteins with ORFs of 141 (16.2 kDa), 179 (19.2 kDa) and 126 (14.0 kDa) amino‐acid residues, respectively. None of the putative proteins showed significant sequence homology with other known proteins in the searched database. The proteins encoded by the complete cDNA sequences were expressed in E. coli as recombinant proteins. Immunoblotting and radioallergosorbant test data showed that all of the expressed recombinant proteins have the ability to bind serum IgE from atopic dermatitis patients and furthermore, the M. furfur extract could specifically inhibit this IgE binding.
Physical characterization of plakophilin 1 reconstituted with and without zinc
Authors: Hofmann Ilse , Mücke Norbert , Reed Jennifer , Herrmann Harald , Langowski Jörg
Published: March 2003

Plakophilin 1 (PKP1) belongs to the arm‐repeat protein family which is characterized by the presence of a conserved 42‐amino‐acid motif. Despite individual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions. PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes. In surface plasmon resonance detection experiments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium. Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The α helix content of PKP1 was considerably higher when reconstituted with zinc than without. By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild‐type PKP1. The zinc‐reconstituted protein formed globular particles of 21.9 ± 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s20,w) for zinc‐containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 × 10−7 cm2·s−1. The molecular mass of 2.44 × 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation.
HNF‐4 plays a pivotal role in the liver‐specific transcription of thechipmunk HP‐25 gene
Authors: Kojima Miho , Takamatsu Nobuhiko , Ishii Tomoko , Kondo Noriaki , Shiba Tadayoshi
Published: March 2003

The gene for chipmunk hibernation‐specific protein HP‐25 is expressed specifically in the liver. To understand the transcriptional regulation of HP‐25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5′ flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5′ flanking sequence was sufficient for the liver‐specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5′ flanking sequence from −74 to −46 showed a shifted band. All cDNA clones isolated by a yeast one‐hybrid system for a protein capable of binding to this 5′ flanking sequence encoded HNF‐4. HNF‐4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti‐(HNF‐4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF‐4. When transfected into HeLa cells, HNF‐4 transactivated transcription from the HP‐25 gene promoter, and mutation of the HNF‐4 binding site abolished transactivation by HNF‐4, indicating that HNF‐4 plays an important role in HP‐25 gene expression.
Fibrinogen interactions with ICAM‐1 (CD54) regulate endothelial cell survival
Authors: Pluskota Elzbieta , D'Souza Stanley E.
Published: March 2003

We tested hypothesis that the interaction of fibrinogen (Fg) with intercellular adhesion molecule 1 (ICAM‐1) mediates cellular adhesion and cell proliferation. Our results demonstrate that Fg : ICAM‐1 ligation mediates endothelial cell survival and has an anti‐apoptotic effect via activation of the MAP kinase pathway. Fg : ICAM‐1 ligation in endothelial cells treated with tumor necrosis factor (TNF)α resulted in the hyperphosphorylation of extracellular signal‐regulated kinase (ERK)‐1/2 (eightfold to 10‐fold) at 5–30 min. The specificity of ERK‐1/2 phosphorylation was verified using the recognition peptides Fg‐γ‐(117–133) and ICAM‐1(8–22). ERK‐1/2 hyperphosphorylation was dependent on intact cytoskeleton, as treatment with cytochalasin B and nocodazole blocked this activity. The attachment of TNFα‐treated endothelial cells to fibrinogen or Fg‐γ‐(117–133) resulted in cell survival, as assessed by an annexin V binding assay. ICAM‐1(8–22) blocked the survival process. The MEK‐1 inhibitor PD 98059 blocked ERK‐1/2 phosphorylation, and treatment of endothelial cells with PD 98059 resulted in apoptosis even upon Fg : ICAM‐1 ligation. Cells transfected with dominant‐negative ERK‐1/2 underwent apoptosis upon Fg : ICAM‐1 ligation. Cell survival factor A1 was specifically upregulated upon adhesion of TNFα‐stimulated endothelial cells to Fg. A1 expression was blocked by ICAM‐1(8–22) and PD 98059. The Fg : ICAM‐1 endothelial cell survival pathway appears to be mediated via the activation and upregulation of ERK‐1/2 and A1.
Comparative biology of the endocannabinoid system
Authors: Salzet Michel , Breton Christophe , Bisogno Tiziana , Di Marzo Vincenzo
Published: March 2003

In this review we discuss data showing that the endogenous cannabinoid system, represented by cannabinoid receptors, endogenous cannabinoid receptor ligands and enzymes for the biosynthesis and degradation of these ligands, is conserved throughout evolution from coelenterates to man. This signaling system has been suggested to play several roles in animals, including the regulation of cell development and growth, nervous functions, reproduction and feeding behavior. In this article, however, we shall describe with more detail the possible function of the endogenous cannabinoid system in the modulation of immune response in organisms from the lower to the higher levels of animal evolution.
The renaissance of mitochondrial calcium transport
Authors: Pozzan Tullio , Rizzuto Rosario
Published: March 2003

Although the capacity of mitochondria for accumulating Ca2+ down the electrical gradient generated by the respiratory chain has been known for over three decades, the physiological significance of this phenomenon has been re‐evaluated only recently. Indeed, it was long believed that the low affinity of the mitochondrial Ca2+ transporters would allow significant uptake only in conditions of cellular Ca2+ overload. Conversely, the direct measurement of [Ca2+] in the mitochondrial matrix revealed major [Ca2+] increases upon agonist stimulation. In this review, we will summarize: (a) the mechanisms that allow this large response, reconciling the biochemical properties of the transporters and the large amplitude of the mitochondrial [Ca2+] rises, and (b) the biological role of mitochondrial Ca2+ signalling, that encompasses the regulation of mitochondrial function and the modulation of the spatio‐temporal pattern of cytosolic [Ca2+] increases.
O‐glycosylation of insulin‐like growth factor (IGF) binding protein‐6 maintains high IGF‐II binding affinity by decreasing binding to glycosaminoglycans and susceptibility to proteolysis
Authors: Marinaro Joe A. , Neumann Gregory M. , Russo Vincenzo C. , Leeding Kerri S. , Bach Leon A.
Published: March 2003

Insulin‐like growth factor binding protein‐6 (IGFBP‐6) is an O‐linked glycoprotein which specifically inhibits insulin‐like growth factor (IGF)‐II actions. The effects of O‐glycosylation of IGFBP‐6 on binding to glycosaminoglycans and proteolysis, both of which reduce the IGF binding affinity of other IGFBPs were studied. Binding of recombinant human nonglycosylated (n‐g) IGFBP‐6 to a range of glycosaminoglycans in vitro was approximately threefold greater than that of glycosylated (g) IGFBP‐6. When bound to glycosaminoglycans, IGFBP‐6 had ≈ 10‐fold reduced binding affinity for IGF‐II. Exogenously added n‐gIGFBP‐6 but not gIGFBP‐6 also bound to partially purified rat PC12 phaeochromocytoma membranes. Binding of n‐gIGFBP‐6 was inhibited by increasing salt concentrations, which is typical of glycosaminoglycan interactions. O‐glycosylation also protected human IGFBP‐6 from proteolysis by chymotrypsin and trypsin. Proteolysis decreased the binding affinity of IGFBP‐6 for IGF‐II, even with a relatively small reduction in apparent molecular mass as observed with chymotrypsin. Analysis by ESI‐MS of IGFBP‐6 following limited chymotryptic digestion showed that a 4.5‐kDa C‐terminal peptide was removed and peptide bonds involved in the putative high affinity IGF binding site were cleaved. The truncated, multiply cleaved IGFBP‐6 remained held together by disulphide bonds. In contrast, trypsin cleaved IGFBP‐6 in the mid‐region of the molecule, resulting in a 16‐kDa C‐terminal peptide which did not bind IGF‐II. These results indicate that O‐glycosylation inhibits binding of IGFBP‐6 to glycosaminoglycans and cell membranes and inhibits its proteolysis, thereby maintaining IGFBP‐6 in a high‐affinity, soluble form and so contributing to its inhibition of IGF‐II actions.
ε‐Crystallin from duck eye lens
Authors: Berr Karin , Wassenberg Doris , Lilie Hauke , Behlke Joachim , Jaenicke Rainer
Published: March 2003

Taxon‐specific ε‐crystallin (εC) from duck eye lens is identical to duck heart muscle lactate dehydrogenase. It forms a dimer of dimers with a dissociation constant of 2.2 × 10−7 m, far beyond the value observed for other vertebrate lactate dehydrogenases. Comparing the characteristics of wild‐type ε‐crystallin with those of three mutants, G115N, G119F and 115N/119F, representing the only significant peripheral sequence variations between duck εC and chicken or pig heart muscle lactate dehydrogenase, no significant conformational differences are detectable. Regarding the catalytic properties, the Michaelis constant of the double mutant 115N/119F for pyruvate is found to be decreased; for wild‐type enzyme, the effect is overcompensated by the high expression level of εC in the eye lens. As taken from spectral analysis of the guanidine‐induced and temperature‐induced denaturation transitions, εC in its dimeric state is relatively unstable, whereas the native tetramer exhibits the high intrinsic stability characteristic of common vertebrate heart and muscle lactate dehydrogenases. The denaturation mechanism of εC is complex and only partially reversible. In the case of thermal unfolding, the predominant side reaction competing with the reconstitution of the native state is the kinetic partitioning between proper folding and aggregation. α‐Crystallin, the major molecular chaperone in the eye lens, inhibits the aggregation of εC by trapping the misfolded protein.
The role of Glu74 and Tyr82 in the reaction catalyzed by sheep liver cytosolic serine hydroxymethyltransferase
Authors: Rao J. V. Krishna , Prakash V. , Rao N. Appaji , Savithri H. S.
Published: March 2003

The three‐dimensional structures of human and rabbit liver cytosolic recombinant serine hydroxymethyltransferases (hcSHMT and rcSHMT) revealed that E75 and Y83 (numbering according to hcSHMT) are probable candidates for proton abstraction and Cα‐Cβ bond cleavage in the reaction catalyzed by serine hydroxymethyltransferase. Both these residues are completely conserved in all serine hydroxymethyltransferases sequenced to date. In an attempt to decipher the role of these residues in sheep liver cytosolic recombinant serine hydroxymethyltransferase (scSHMT), E74 (corresponding residue is E75 in hcSHMT) was mutated to Q and K, and Y82 (corresponding residue is Y83 in hcSHMT) was mutated to F. The specific activities using serine as the substrate for the E74Q and E74K mutant enzymes were drastically reduced. These mutant enzymes catalyzed the transamination of d‐alanine and 5,6,7,8‐tetrahydrofolate independent retroaldol cleavage of lallo threonine at rates comparable with wild‐type enzyme, suggesting that E74 was not involved directly in the proton abstraction step of catalysis, as predicted earlier from crystal structures of hcSHMT and rcSHMT. There was no change in the apparent Tm value of E74Q upon the addition of l‐serine, whereas the apparent Tm value of scSHMT was enhanced by 10 °C. Differential scanning calorimetric data and proteolytic digestion patterns in the presence of l‐serine showed that E74Q was different to scSHMT. These results indicated that E74 might be required for the conformational change involved in reaction specificity. It was predicted from the crystal structures of hcSHMT and rcSHMT that Y82 was involved in hemiacetal formation following Cα‐Cβ bond cleavage of l‐serine and mutation of this residue to F could lead to a rapid release of HCHO. However, the Y82F mutant had only 5% of the activity and failed to form a quinonoid intermediate, suggesting that this residue is not involved in the formation of the hemiacetal intermediate, but might be involved indirectly in the abstraction of the proton and in stabilizing the quinonoid intermediate.
Paullones are potent inhibitors of glycogen synthase kinase‐3β and cyclin‐dependent kinase 5/p25
Authors: Leost Maryse , Schultz Christiane , Link Andreas , Wu Yong‐Zhong , Biernat Jacek , Mandelkow Eva‐Maria , Bibb James A. , Snyder Gretchen L. , Greengard Paul , Zaharevitz Daniel W. , Gussio Rick , Senderowicz Adrian M. , Sausville Edward A. , Kunick Conrad , Meijer Laurent
Published: March 2003

Paullones constitute a new family of benzazepinones with promising antitumoral properties. They were recently described as potent, ATP‐competitive, inhibitors of the cell cycle regulating cyclin‐dependent kinases (CDKs). We here report that paullones also act as very potent inhibitors of glycogen synthase kinase‐3β (GSK‐3β) (IC50: 4–80 nm) and the neuronal CDK5/p25 (IC50: 20–200 nm). These two enzymes are responsible for most of the hyperphosphorylation of the microtubule‐binding protein tau, a feature observed in the brains of patients with Alzheimer’s disease and other neurodegenerative ‘taupathies’. Alsterpaullone, the most active paullone, was demonstrated to act by competing with ATP for binding to GSK‐3β. Alsterpaullone inhibits the phosphorylation of tau in vivo at sites which are typically phosphorylated by GSK‐3β in Alzheimer’s disease. Alsterpaullone also inhibits the CDK5/p25‐dependent phosphorylation of DARPP‐32 in mouse striatum slices in vitro. This dual specificity of paullones may turn these compounds into very useful tools for the study and possibly treatment of neurodegenerative and proliferative disorders.
Regulation of Gadd45γ expression by C/EBP
Authors: Jung Neoncheol , Yi Yong‐Weon , Kim Daegun , Shong Minho , Hong Seung‐Suh , Lee Hyun‐Soo , Bae Insoo
Published: March 2003

The Gadd45γ (growth arrest and DNA damage‐inducible) gene is activated transcriptionally by at least two kinds of agents: DNA damaging agent such as methyl methanesulfonate (MMS) and UV radiation, or cytokines such as interleukin (IL)‐6, IL‐2 and granulocyte colony‐stimulating factor (G‐CSF). To investigate the sequences and transcription factors involved in induction of Gadd45γ after treatment with IL‐6, the human gene was cloned and sequenced. We found C/EBP (CCAAT/enhancer‐binding protein) family proteins, major transcription factors in the IL‐6 signal transduction pathway, could regulate the transcriptional activity of the Gadd45γ promoter. In addition, a noncanonical C/EBP‐binding site within the Gadd45γ promoter where C/EBPβ and C/EBPδ could bind, was identified by electrophoretic mobility shift assay (EMSA) and reporter gene analysis. Furthermore, we found a coordinated expression profile between Gadd45γ mRNA and C/EBPs (β and δ) protein during the differentiation of M1 cells: the amount of Gadd45γ transcripts became maximal when both C/EBPβ and C/EBPδ levels were high, on day 1 of differentiation of M1 cells after treatment with IL‐6. These findings suggest that mitotic growth arrest coupled to M1 cell differentiation is mediated by C/EBPs stimulation of growth arrest‐associated genes such as Gadd45γ.
Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids
Authors: Solstad Therese , Flatmark Torgeir
Published: March 2003

The microheterogeneity of recombinant human phenylalanine hydroxylase (hPAH) was investigated by isoelectric focusing and 2D electrophoresis. When expressed in Escherichia coli four main components (denoted hPAH I‐IV) of ≈ 50 kDa were observed on long‐term induction at 28–37 °C with isopropyl thio‐β‐d‐galactoside (IPTG), differing in pI by about 0.1 pH unit. A similar type of microheterogeneity was observed when the enzyme was expressed (1 h at 37 °C) in an in vitro transcription‐translation system, including both its nonphosphorylated and phosphorylated forms which were separated on the basis of a difference in mobility on SDS/PAGE. Experimental evidence is presented that the microheterogeneity is the result of nonenzymatic deamidations of labile amide containing amino acids. When expressed in E. coli at 28 °C, the percentage of the acidic forms of the enzyme subunit increased as a function of the induction time with IPTG, representing about 50% on 8 h induction. When the enzyme obtained after 2 h induction (containing mainly hPAH I) was incubated in vitro, its conversion to the acidic components (hPAH II–IV) revealed a pH and temperature dependence characteristic of a nonenzymatic deamidation of asparagine residues in proteins, with the release of ammonia. Comparing the microheterogeneity of the wild‐type and a truncated form of the enzyme expressed in E. coli, it is concluded that the labile amide groups are located in the catalytic domain as defined by crystal structure analysis [Erlandsen, H., Fusetti, F., Martínez, A., Hough, E., Flatmark, T. & Stevens, R. C. (1997) Nat. Struct. Biol. 4, 995–1000]. It is further demonstrated that the progressive deamidations which occur in E. coli results in a threefold increase in the catalytic efficiency (Vmax/[S]0.5) of the enzyme and an increased susceptibility to limited tryptic proteolysis, characteristic of a partly activated enzyme. The results also suggest that deamidation may play a role in the long term regulation of the catalytic activity and the cellular turnover of this enzyme.
Characterization of five new low‐molecular‐mass trypsin inhibitors from white mustard (Sinapis alba L.) seed
Authors: Ruoppolo Margherita , Amoresano Angela , Pucci Piero , Pascarella Stefano , Polticelli Fabio , Trovato Maurizio , Menegatti Enea , Ascenzi Paolo
Published: March 2003

Five new low‐molecular‐mass trypsin inhibitors belonging to the RTI/MTI‐2 family were identified from white mustard (Sinapis alba L.; MTI‐2) seed. Purified MTI‐2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C‐terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI‐2 inhibitors originated from different genes. The presence of 5‐oxo‐proline (pyroglutamic acid; 5‐oxoPro1) and Kyn44 reflected post‐translational processing of the serine proteinase inhibitor. MTI‐2 showed ≈ 70% amino‐acid identity with low‐molecular‐mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI‐III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI‐2 was homologous to brazzein, the sweet‐tasting protein from Pentadiplandra brazzeana Baillon fruit (≈ 30% amino‐acid identity). Although snake‐venom toxins showed a low amino‐acid identity (< 20%) with MTI‐2, RTI‐III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI‐2 (Cys5–Cys27, Cys18–Cys31, Cys42–Cys52, and Cys54–Cys57) corresponded to that of RTI‐III and of snake‐venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three‐dimensional arrangement rather than to the amino‐acid sequence. Values of Ka for MTI‐2 binding to bovine β‐trypsin (trypsin) and bovine α‐chymotrypsin were 6.3 × 109m−1 and 2.0 × 106 m−1, respectively, at pH 8.0 and 21.0 °C. Moreover, values of kon for MTI‐2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 × 105 m−1·s−1 and 8.9 × 10−5 m−1·s−1, respectively, at pH 8.0 and 21.0 °C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI‐2 inhibitors were indistinguishable.
Mitochondrial membrane potential differentiates cells resistant to apoptosis in hybridoma cultures
Authors: Follstad Brian D. , Wang Daniel I. C. , Stephanopoulos Gregory
Published: March 2003

Previous research has implicated mitochondrial physiology and, by extension, respiratory capacity in the initiation and progress of apoptosis of cells in culture and tissue environments. This hypothesis was tested by separating a hybridoma cell population into subpopulations of varying mitochondrial membrane potential (MMP) using Rhodamine 123 stain and fluorescence‐activated cell sorter analysis and subjecting them to two apoptosis inducers, rotenone and staurosporin. Apoptotic death was characterized morphologically through the determination of apoptosis‐related chromatin condensation and biochemically through the measurement of caspase‐3 enzymatic activity. We found dramatic differences in the apoptotic death kinetics for the subpopulations, with the high MMP cells showing higher resistance to apoptotic death. After incubation with 30 µm rotenone, the low MMP cells exhibited one‐third of the viability of the high MMP cells and a three‐fold increase in the capsase‐3 enzymatic activity. No changes were observed in the DNA content or the cell cycle distributions of the two cell subpopulations, which maintained their mean MMP difference after 20 generations. These results suggest that heterogeneity exists in mammalian cell populations with respect to mitochondrial physiology, which correlates with resistance to apoptotic death.
Transcription inhibitors stimulate translation of 5′ TOP mRNAs through activation of S6 kinase and the mTOR/FRAP signalling pathway
Authors: Loreni Fabrizio , Thomas George , Amaldi Francesco
Published: March 2003

We have analysed the effect of transcription inhibitors on the polysomal localization of 5′ terminal oligopyrimidine (TOP‐) mRNAs. It is known that, in vertebrates, the translation of this group of mRNAs is regulated according to the growth status of the cell. Mitogenic stimulation of quiescent cells induces a rapid recruitment of TOP mRNAs from translationally inactive light messenger ribonucleoprotein particles to polysomes. It was found that administration of transcription inhibitors to resting cells causes a similar collective translational activation of TOP mRNAs, without affecting global translation. A number of transcription inhibitors were tested in amphibian and mammalian cultured cells. Actinomycin D (act D), cordycepin, and 5,6‐dichloro‐1‐β‐d‐ribofuranosylbenzimidazole caused a similar activation whereas α‐amanitin or low doses of act D did not induce the translational response. Concentrations of act D sufficient to induce TOP mRNA translation also induce 40S ribosomal protein S6 kinases 1 (S6K1) activation. Moreover at these concentrations of act D increased phosphorylation of 4E‐BP1 was also observed, indicating the involvement of FRAP/mTOR. Consistent with this observation, pretreatment of resting cells with rapamycin suppresses the activation of TOP mRNA translation induced by act D. These results indicate that the effect of act D on translation is mediated by the S6Ks through FRAP/mTOR.
Purification and characterization of a new, ubiquitously distributed class of microtubule‐associated protein with molecular mass 250 kDa
Authors: Katsuki Miho , Tokuraku Kiyotaka , Nakagawa Hiroyuki , Kotani Susumu
Published: March 2003

A heat‐stable microtubule‐associated protein (MAP) with relative molecular mass 250 000, termed 250‐kDa MAP, was purified from bovine adrenal cortex. It is classified as a MAP subspecies distinct from MAP1, MAP2, tau, and MAP4, as judged from its electrophoretic mobility, heat stability and immunoreactivity. Purified 250‐kDa MAP was able to bind to taxol‐stabilized microtubules, although it lacked the ability to polymerize purified tubulin into microtubules. Western‐blot analysis showed that this MAP was expressed ubiquitously in mammalian tissues. Immunofluorescence microscopy revealed that polyclonal antibodies raised against 250‐kDa MAP stained many punctate structures in the cytoplasm of cultured cells. Blurry cytosolic staining was also observed. Judging from the result of nocodazole treatment, the punctate structures were associated with the microtubule network throughout the cytoplasm, while cytosolic 250‐kDa MAP colocalized with free tubulin. Under electron microscopy, 250‐kDa MAP has the appearance of a hollow sphere of about 12 nm diameter.
Functions of transforming growth factor‐β isoforms in the nervous system
Authors: Unsicker Klaus , Strelau Jens
Published: March 2003

This review briefly describes the cellular distribution and documented roles of the transforming growth factor (TGF)‐β isoforms TGF‐β2 and ‐β3 in the central and peripheral nervous system. TGF‐β2 and ‐β3 are coexpressed in developing radial glial and mature astroglial and Schwann cells, as well as in subpopulations of differentiated neurons, most prominently in cortical, hippocampal, and brainstem/spinal cord motor neurons. In vitro studies have suggested a number of potential, physiologically relevant functions for TGF‐βs including regulation of astroglial cell proliferation, expression of adhesion molecules, survival promoting roles for neurons in combination with established neurotrophic factors, and differentiative actions on neurons.
Signaling of transforming growth factor‐β family members through Smad proteins
Authors: Itoh Susumu , Itoh Fumiko , Goumans Marie‐José , ten Dijke Peter
Published: March 2003

Smads are pivotal intracellular nuclear effectors of transforming growth factor‐β (TGF‐β) family members. Ligand‐induced activation of TGF‐β family receptors with intrinsic serine/threonine kinase activity trigger phosphorylation of receptor‐regulated Smads (R‐Smads), whereas Smad2 and Smad3 are phosphorylated by TGF‐β, and activin type I receptors, Smad1, Smad5 and Smad8, act downstream of BMP type I receptors. Activated R‐Smads form heteromeric complexes with common‐partner Smads (Co‐Smads), e.g. Smad4, which translocate efficiently to the nucleus, where they regulate, in co‐operation with other transcription factors, coactivators and corepressors, the transcription of target genes. Inhibitory Smads act in most cases in an opposite manner from R‐ and Co‐Smads. Like other components in the TGF‐β family signaling cascade, Smad activity is intricately regulated. The multifunctional and context dependency of TGF‐β family responses are reflected in the function of Smads as signal integrators. Certain Smads are somatically mutated at high frequency in particular types of human cancers. Gene ablation of Smads in the mouse has revealed their critical roles during embryonic development. Here we review the latest advances in our understanding of the Smad mechanism of action and their in vivo functions.
Methyl‐CpG‐binding proteins
Authors: Ballestar Esteban , Wolffe Alan P.
Published: March 2003

CpG methylation, the most common epigenetic modification of vertebrate genomes, is primarily associated with transcriptional repression. MeCP2, MBD1, MBD2, MBD3 and MBD4 constitute a family of vertebrate proteins that share the methyl‐CpG‐binding domain (MBD). The MBD, consisting of about 70 residues, possesses a unique α/β‐sandwich structure with characteristic loops, and is able to bind single methylated CpG pairs as a monomer. All MBDs except MBD4, an endonuclease that forms a complex with the DNA mismatch‐repair protein MLH1, form complexes with histone deacetylase. It has been established that MeCP2, MBD1 and MBD2 are involved in histone deacetylase‐dependent repression and it is likely that this is also the case for MBD3. The current model proposes that MBD proteins are involved in recruiting histone deacetylases to methyl CpG‐enriched regions in the genome to repress transcription. The lack of selectivity for MBD association with particular DNA sequences indicates that other mechanisms account for their recruitment to particular regions in the genome.
Unmasking a hyaluronan‐binding site of the BX7B type in the H3 heavy chain of the inter‐α‐inhibitor family
Authors: Jean Laetitia , Mizon Charlotte , Larsen William J. , Mizon Jacques , Salier Jean‐Philippe
Published: March 2003

The inter‐α‐inhibitor (IαI) family gathers together several plasma protease inhibitors such as IαI and pre‐α‐inhibitor (PαI) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P. In addition to their protease inhibitory activity, a major physiological function of IαI family members is hyaluronan (HA) binding and HA‐dependent stabilization of the extracellular matrix surrounding various cell types. Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis. However, how HA and IαI family members first recognize each other has so far remained elusive. The so‐called BX7B domain found in some HA‐binding proteins is an HA‐binding site in which B represents a basic amino‐acid residue and X represents any nonacidic residue. This domain has now been identified in the N‐terminal end of H3P that is a precursor of PαI. A series of wild‐type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion. Furthermore, unmasking this HA‐binding activity required most of H3P to be trimmed off at its C‐terminal end. The latter observation was confirmed with a natural, mature H3 chain purified from human plasma. Indeed, a thermolysin‐generated, N‐terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. Therefore, in vivo, the HA‐binding activity of the mature H3 chain within PαI may vary with the folding and/or fragmentation of this protein.