bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide binding of an ADP analog to cooperating sites of chloroplast F1‐ATPase (CF1)
Authors: Günther Stephan , Huchzermeyer Bernhard
Published: March 2003

Pre‐steady state nucleotide binding to the chloroplast F1‐ATPase (CF1) was measured in a stopped‐flow apparatus by monitoring the change of fluorescence intensity of TNP‐ADP upon binding. The analysis of the time courses led to the proposal of a mechanism of nucleotide binding with the following characteristics. (a) It involves three sites binding nucleotides in a concerted manner. (b) Each binding site is able to undergo a conformational change from a loose binding state into a state refraining from any direct release of the bound nucleotide into the medium. Only the reverse reaction via the loose binding state enables release out of the tight binding state. (c) Due to very strong negative cooperativity, a maximum of two of the three sites can be found in the state of tight binding. (d) Cooperativity between the three sites leads to a slower nucleotide binding of the second nucleotide compared to the first nucleotide. Furthermore, the conformational change from the loose binding state to the tight binding state is slowed down if one of the other sites already is in the tight binding state.

Three‐sites mechanisms in which rotation leads to an exchange of the properties of the binding sites failed to simulate the observed time courses of nucleotide binding. However, as the experimental set up was designed to prevent catalysis taking place, our results entirely agree with the current finding that rotation requires catalytic turnover of the enzyme.
Purification and some properties of serine hydroxymethyltransferase from Trypanosoma cruzi
Authors: Capelluto Daniel G. S. , Hellman Ulf , Cazzulo Juan J. , Cannata Joaquín J. B.
Published: March 2003

A single form of serine hydroxymethyltransferase (SHMT) was detected in epimastigotes of Trypanosoma cruzi, in contrast to the three isoforms of the enzyme characterized from another trypanosomatid, Crithidia fasciculata[Capelluto D.G.S., Hellman U., Cazzulo J.J. & Cannata J.J.B. (1999) Mol. Biochem. Parasitol.98, 187–201]. The T. cruzi SHMT was found to be highly unstable in crude extracts. In the presence of the cysteine proteinase inhibitors N‐α‐p‐tosyl‐l‐lysine chloromethyl ketone and ltrans‐3‐carboxyoxiran‐2‐carbonyl‐l‐leucylagmatine, however, the enzyme could be purified to homogeneity. Digitonin treatment of intact cells suggested that the enzyme is cytosolic. T. cruzi SHMT presents a monomeric structure shown by the apparent molecular masses of 69 kDa (native) and 55 kDa (subunit) determined by Sephadex G‐200 gel filtration and SDS/PAGE, respectively. This is in contrast to the tetrameric SHMTs described in C. fasciculata and other eukaryotes. The enzyme was pyridoxal phosphate‐dependent after l‐cysteine and hydroxylamine treatments and it was strongly inhibited by the substrate analog folate, which was competitive towards tetrahydrofolate and noncompetitive towards l‐serine. Partial sequencing of tryptic internal peptides of the enzyme indicate considerable similarity with other SHMTs, particularly from those of plant origin.
Membrane fusion by proline‐rich Rz1 lipoprotein, the bacteriophage λRz1 gene product
Authors: Bryl Krzysztof , Keôdzierska Sabina , Laskowska Magdalena , Taylor Alina
Published: March 2003

The fusogenic properties of Rz1, the proline‐rich lipoprotein that is the bacteriophage λRz1 gene product, were studied. Light scattering was used to monitor Rz1‐induced aggregation of artificial neutral (dipalmitoylphosphatidylcholine/cholesterol) and negatively charged (dipalmitoylphosphatidylcholine/cholesterol/dioleoylphosphatidylserine) liposomes. Fluorescence assays [the resonance energy transfer between N‐(7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl)phosphatidylethanolamine and N‐(lissamine rhodamine B sulfonyl)dihexadecanol‐sn‐glycero‐3‐phosphoethanolamine lipid fluorescent probes, as well as fluorescent complex formation between terbium ions and dipicolinic acid encapsulated in two liposome populations and calcein fluorescence] were used to monitor Rz1‐induced lipid mixing, contents mixing and leakage of neutral and negatively charged liposomes. The results demonstrated that Rz1 caused adhesion of neutral and negatively charged liposomes with concomitant lipid mixing; membrane distortion, leading to the fusion of liposomes and hence their internal content mixing; and local destruction of the membrane accompanied by leakage of the liposome contents. The use of artificial membranes showed that Rz1 induced the fusion of membranes devoid of any proteins. This might mean that the proline stretch of Rz1 allowed interaction with membrane lipids. It is suggested that Rz1‐induced liposome fusion was mediated primarily by the generation of local perturbation in the bilayer lipid membrane and to a lesser extent by electrostatic forces.
The membrane‐bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species
Authors: Diefenbach Jörg , Kindl Helmut
Published: March 2003

DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane‐anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane‐bound co‐chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His‐tagged DnaJ proteins and glutathione S‐transferase–Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His‐tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome‐bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP‐related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase‐modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.
A model of a gp120 V3 peptide in complex with an HIV‐neutralizing antibody based on NMR and mutant cycle‐derived constraints
Authors: Zvi Anat , Tugarinov Vitali , Faiman Gabriel A. , Horovitz Amnon , Anglister Jacob
Published: March 2003

The 0.5β monoclonal antibody is a very potent strain‐specific HIV‐neutralizing antibody raised against gp120, the envelope glycoprotein of HIV‐1. This antibody recognizes the V3 loop of gp120, which is a major neutralizing determinant of the virus. The antibody–peptide interactions, involving aromatic and negatively charged residues of the antibody 0.5β, were studied by NMR and double‐mutant cycles. A deuterated V3 peptide and a Fab containing deuterated aromatic amino acids were used to assign these interactions to specific V3 residues and to the amino acid type and specific chain of the antibody by NOE difference spectroscopy. Electrostatic interactions between negatively charged residues of the antibody Fv and peptide residues were studied by mutagenesis of both antibody and peptide residues and double‐mutant cycles. Several interactions could be assigned unambiguously: F96(L) of the antibody interacts with Pro13 of the peptide, H52(H) interacts with Ile7, Ile9 and Gln10 and D56(H) interacts with Arg11. The interactions of the light‐chain tyrosines with Pro13 and Gly14 could be assigned to either Y30a(L) and Y32(L), respectively, or Y32(L) and Y49(L), respectively. Three heavy‐chain tyrosines interact with Ile7, Ile20 and Phe17. Several combinations of assignments involving Y32(H), Y53(H), Y96(H) and Y100a(H) may satisfy the NMR and mutagenesis constraints, and therefore at this stage the interactions of the heavy‐chain tyrosines were not taken into account. The unambiguous assignments [F96(L), H52(H) and D56(H)] and the two possible assignments of the light‐chain tyrosines were used to dock the peptide into the antibody‐combining site. The peptide converges to a unique position within the binding site, with the RGPG loop pointing into the center of the groove formed by the antibody complementary determining regions while retaining the β‐hairpin conformation and the type‐VI RGPG turn [Tugarinov, V., Zvi, A., Levy, R. & Anglister, J. (1999) Nat. Struct. Biol.6, 331–335].
Carotenoid binding sites in LHCIIb
Authors: Hobe Stephan , Niemeier Hildrun , Bender Alexander , Paulsen Harald
Published: March 2003

The major light‐harvesting complex of photosystem II can be reconstituted in vitro from its bacterially expressed apoprotein with chlorophylls a and b and neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyll. Reconstitution of these one‐carotenoid complexes requires low‐stringency conditions during complex formation and isolation. Neoxanthin complexes (containing 30–50% of the all‐trans isomer) disintegrate during electrophoresis, exhibit a largely reduced resistance against proteolytic attack; in addition, energy transfer from Chl b to Chl a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin or lutein contain nearly two xanthophylls per 12 chlorophylls and are more resistant against trypsin. Lutein–LHCIIb also exhibits an intermediate maintenance of energy transfer at higher temperature. Violaxanthin complexes approach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in recombinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin–LHCIIb exhibits a low thermal stability like neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein–LHCIIb and zeaxanthin–LHCIIb. Binary competition experiments were performed with two xanthophylls at varying ratios in the reconstitution. Analysis of the xanthophyll contents in the reconstitution products yielded information about relative carotenoid affinities of three assumed binding sites. In lutein/neoxanthin competition experiments, two binding sites showed a strong preference (> 200‐fold) for lutein, whereas the third binding site had a higher affinity (25‐fold) to neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36‐fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: two binding sites had a fivefold higher affinity for lutein and one has a threefold higher affinity to zeaxanthin.
Biosynthesis of β1,4‐ and β1,β1‐galactopyranosyl xylopyranosides in the mammary gland of lactating cow
Authors: Hara Yoshihiro , Suyama Kyozo
Published: March 2003

Lactose is a principal carbohydrate in nearly all species of mammalian milk. In order to examine the acceptor substrate specificity of lactose synthase in vivo, d‐xylose as an acceptor substrate was injected into the jugular vein of a Holstein cow during lactation, then a milk sample obtained by milking. β1,β1‐galactopyranosyl xylopyranoside, a nonreducing disaccharide, was separated from the bovine milk sample after elimination of reducing sugars, identified by fast‐atom bombardment (FAB)‐MS and 1H‐NMR analysis. A mixture of β1,β1‐ and β1,4‐galactopyranosyl xylopyranoside fractions was also obtained by thin layer chromatography from the milk sample and elucidated by electrospray ionization (ESI)‐MS and 1H NMR analysis. Comparison of the integrated intensity of the products shows that the β1,β1 and β1,4 isomers are present in a ratio of 1.0 : 1.4, suggesting that d‐xylose, transported from capillary blood across the plasma membrane of the mammary gland, was recognized by lactose synthase in its normal and reverse orientation owing to high symmetry of its structure. While the β1,4‐isomer is known as a fragment of the linkage region between the protein and the polysaccharide chain of proteoglycans, the β1,β1‐isomer has not been identified in vivo. Here, we demonstrate that galactosylation of d‐xylose transported from the capillary blood can occur by lactose synthase catalysis in the mammary gland while the usual galactosylation of d‐glucose proceeds. In addition, these results suggest that the possibility of endogenous occurrence of the β,β‐trehalose type disaccharide in the mammary gland of lactating mammals may not be ruled out.
Transforming growth factor β induction of insulin gene expression is mediated by pancreatic and duodenal homeobox gene‐1 in rat insulinoma cells
Authors: Sayo Yoshitaka , Hosokawa Hitoshi , Imachi Hitomi , Murao Koji , Sato Makoto , Wong Norman C. W. , Ishida Toshihiko , Takahara Jiro
Published: March 2003

Although transforming growth factor‐beta (TGF‐β) stimulates pancreatic islet cells to synthesize and secret insulin, the mechanism underlying this effect is not known. To investigate this question, we examined the insulin promoter activity focusing on a transcription factor, pancreatic and duodenal homeobox gene‐1 (PDX‐1) that binds to the A3 element of the rat insulin promoter. Studies performed using the rat insulinoma cell line, INS‐1 showed that TGF‐β stimulation of endogenous insulin mRNA expression correlated with increased activity of a reporter construct containing the insulin promoter. A potential mechanism for this increase arose from, electrophoretic mobility shift assay showing that the nuclear extract from TGF‐β treated cells contained higher levels of A3 binding activity. Western blot analysis confirmed that PDX‐1 was increased in the nuclear extract from INS‐1 cells treated with TGF‐β. As expected, a mutant insulin promoter that lacked the PDX‐1 binding site was not stimulated by TGF‐β. In summary, the results of these studies show that TGF‐β stimulates the transcription of insulin gene and this action is mediated by the transcription factor, PDX‐1.
Activity and stability of a neutral protease from Vibrio sp. (vimelysin) in a pressure‐temperature gradient
Authors: Ikeuchi Hidekazu , Kunugi Shigeru , Oda Kohei
Published: March 2003

The apparent second‐order rate constant of hydrolysis of Fua‐Gly‐LeuNH2 by vimelysin, a neutral protease from Vibrio sp. T1800, was measured in a variable pressure–temperature gradient (0.1–400 MPa and 5–40 °C). The apparent maximum rate was observed at approximately 15 °C and 150–200 MPa; the pressure‐activation ratio (kcat/Km(max)/kcat/Km(0.1 MPa)) was reached about sevenfold. The pressure dependence of the kcat and Km parameters at constant temperature (25 °C) revealed that the pressure‐activation below 200 MPa was mainly caused by a change in the kcat parameter.

The change in the intrinsic fluorescence intensity of vimelysin was also measured in a pressure‐temperature plane (0.1–400 MPa and −20 to +60 °C). The fluorescence intensity was found to decrease by increasing pressure and temperature, and the isointensity contours were more or less circular. The tangential lines to the contours at high temperatures and low to medium pressures seem to have slightly positive slopes, which was reflected by the higher residual activities left after incubations at higher temperatures and medium pressure (200 MPa and 50 °C) and by the almost intact secondary structure left after 1 h of incubation at 200 MPa and 40 °C, as studied by circular dichroism.

These results were compared with the corresponding results for thermolysin, a moderately thermostable protease from Bacillus thermoproteolyticus. Apparent differences that might be related to the temperature adaptations of the respective source microbes are also discussed.
A diminished activation capacity of the interferon‐inducible protein kinase PKR in human T lymphocytes
Authors: Li Suiyang , Nagai Kazushige , Koromilas Antonis E.
Published: March 2003

The double‐stranded (ds) RNA activated protein kinase PKR is an interferon (IFN)‐inducible serine/threonine protein that regulates protein synthesis through the phosphorylation of the α subunit of translation initiation factor 2 (eIF‐2α). PKR activation in cells is induced by virus infection or treatment with dsRNA and is modulated by a number of viral and cellular factors. To better understand the mechanisms of PKR action we have analyzed and compared the mode of PKR activation in a number of cell lines of different histological origin. Here we show that PKR activation and phosphorylation of eIF‐2α are both diminished in various virus‐transformed and nontransformed human T cells. Priming of T cells with IFN does not restore PKR activation. In vitro kinase assays show that the diminished PKR activation in T cells correlates with the presence of a 60‐kDa (p60) phosphoprotein coimmunoprecipitated with PKR. P60 is absent from PKR immunoprecipitates from non T cells. Incubation of active PKR with T cell extracts results in inhibition of PKR autophosphorylation, which is proportional to the amount of phosphorylated p60 in the kinase reactions. Treatment of T cells with proteasome inhibitors or incubation of PKR immunoprecipitates with phosphatase inhibitors does not restore PKR activation. However, phosphorylation of p60 is enhanced upon treatment with the phosphatase inhibitor microcystin. These data show that the impaired activation capacity of PKR in human T cells is exerted at the post‐translational levels in a manner that is independent of cell transformation or virus infection.
Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I‐937 and expression in Pichia pastoris
Authors: Otterbein Ludovic , Record Eric , Longhi Sonia , Asther Marcel , Moukha Serge
Published: March 2003

Laccases are multicopper‐containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full‐length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I‐937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino‐acid signal sequence corresponding to a putative signal peptide. The deduced amino‐acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiaeα‐factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated.
Fluorescent neoglycolipids
Authors: Stoll Mark S. , Feizi Ten , Loveless R. Wendy , Chai Wengang , Lawson Alexander M. , Yuen Chun‐Ting
Published: March 2003

A second generation of lipid‐linked oligosaccharide probes, fluorescent neoglycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2‐dihexadecyl‐sn‐glycero‐3‐phosphoethanolamine (DHPE), which has been used extensively to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N‐aminoacetyl‐N‐(9‐anthracenylmethyl)‐1,2‐dihexadecyl‐sn‐glycero‐3‐phosphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHP‐neoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequence‐specific antibodies. Among O‐glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl‐Lea‐ and sialyl‐Lex‐active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary‐ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno‐detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state‐of‐the‐art technologies for carbohydrate analysis.
New functions of a long‐known molecule
Authors: Ziegler Mathias
Published: March 2003

Over the past decades, the pyridine nucleotides have been established as important molecules in signaling pathways, besides their well known function in energy transduction. Similarly to another molecule carrying such dual functions, ATP, NAD(P)+ may serve as substrate for covalent protein modification or as precursor of biologically active compounds.

Protein modification is catalyzed by ADP‐ribosyl transferases that attach the ADP‐ribose moiety of NAD+ to specific amino‐acid residues of the acceptor proteins. For a number of ADP ribosylation reactions the specific transferases and their target proteins have been identified. As a result of the modification, the biological activity of the acceptor proteins may be severely changed. The cell nucleus contains enzymes catalyzing the transfer of ADP‐ribose polymers (polyADP‐ribose) onto the acceptor proteins. The best known enzyme of this type is poly(ADP‐ribose) polymerase 1 (PARP1), which has been implicated in the regulation of several important processes including DNA repair, transcription, apoptosis, neoplastic transformation and others.

The second group of reactions leads to the synthesis of an unusual cyclic nucleotide, cyclic ADP‐ribose (cADPR). Moreover, the enzymes catalyzing this reaction may also replace the nicotinamide of NADP+ by nicotinic acid resulting in the synthesis of nicotinic acid adenine dinucleotide phosphate (NAADP+). Both cADPR and NAADP+ have been reported to be potent intracellular calcium‐mobilizing agents. In concert with inositol 1,4,5‐trisphosphate, they participate in cytosolic calcium regulation by releasing calcium from intracellular stores.
Structural analysis of two glycosphingolipids from the lipopolysaccharide‐lacking bacterium Sphingomonas capsulata
Authors: Kawahara Kazuyoshi , Moll Hermann , Knirel Yuriy A. , Seydel Ulrich , Zähringer Ulrich
Published: March 2003

Two glycosphingolipids, GSL‐1 and GSL‐3, were isolated from Sphingomonas capsulata and studied by methylation analysis, laser desorption mass spectrometry, and 1H and 13C NMR spectroscopy, including two‐dimensional 1H,1H COSY and heteronuclear 13C,1H COSY experiments. GSL‐1 and GSL‐3 differ in their carbohydrate part, their structures being α‐d‐GlcpA‐(1→1)‐Cer and α‐d‐Galp‐(1→6)‐α‐d‐GlcpN‐(1→4)‐α‐d‐GlcpA(1→1)Cer, respectively. Variations occur in the ceramide of GSL‐1 and GSL‐3, both having the same long‐chain bases, erythro‐2‐amino‐1,3‐octadecanediol (sphinganine), (13Z)‐erythro‐2‐amino‐13‐eicosene‐1,3‐diol and (13Z)‐erythro‐2‐amino‐13,14‐methylene‐1,3‐eicosanediol, in the ratios 2.6 : 1 : 3.5 in GSL‐1 and 1 : 1.2 : 1.5 in GSL‐3. All bases are quantitatively substituted by amide‐linked (S)‐2‐hydroxymyristic acid.
Pig MAP/ITIH4 and haptoglobin are interleukin‐6‐dependent acute‐phase plasma proteins in porcine primary cultured hepatocytes
Authors: González‐Ramón Nieves , Hoebe Kasper , Alava María A. , van Leengoed Leo , Piñeiro Matilde , Carmona Silvana , Iturralde María , Lampreave Fermín , Piñeiro Andrés
Published: March 2003

The acute‐phase expression of pig MAP (major acute‐phase protein)/ITIH4 (inter‐α‐trypsin inhibitor heavy chain 4) and haptoglobin were analysed in primary cultures of isolated pig hepatocytes in response to recombinant human (rh) cytokines: tumor necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1), interleukin‐6 (IL‐6), as well as to bacterial lipopolysaccharide (LPS). Analysis of pig MAP/ITIH4 and haptoglobin mRNAs was carried out by RT‐PCR amplification. Secreted proteins from the cytokine‐treated hepatocytes were quantified by immunochemical techniques. Time‐course and dose–response experiments show that pig MAP/ITIH4 and haptoglobin belong to the type II acute‐phase proteins, as they are specifically induced by rhIL‐6 and not by rhTNF‐α or rhIL‐1. Stimulation of cultured pig hepatocytes with rhIL‐6 for 48 h at doses of 1000 U·mL−1showed a fourfold to fivefold increase in pig MAP/ITIH4 concentration in the medium, while the concentration of haptoglobin only increased twofold. A similar increase in the concentration of pig MAP/ITIH4 was also observed in media of LPS‐treated hepatocytes with the simultaneous generation of IL‐6 by the Kupfer cells present in the cultures. Albumin secretion decreased after stimulation with doses of 100 or 1000 U·mL−1rhTNF‐α, rhIL‐1 or rhIL‐6. Therefore, it can be concluded that pig MAP/ITIH4 behaves as a major acute‐phase protein produced by porcine hepatocytes under the effect of inflammatory cytokines.
Evidence against a direct role of the integrin α2β1 in collagen‐induced tyrosine phosphorylation in human platelets
Authors: Hers Ingeborg , Berlanga Oscar , Tiekstra Margriet J. , Kamiguti Aura S. , Theakston R. David G. , Watson Steve P.
Published: March 2003

In the present study we have investigated whether the collagen receptor α2β1 (GPIa‐IIa; GP, glycoprotein) regulates protein tyrosine phosphorylation in platelets directly through activation of tyrosine kinases or indirectly through modification of the response to GPVI. The interaction of collagen with α2β1 was inhibited in two distinct ways, using the metalloprotease jararhagin, which cleaves the β1 subunit, or the antibody P1E6 which competes with binding of collagen to the integrin. The two inhibitors caused a shift to the right in the collagen concentration response curves for protein tyrosine phosphorylation and platelet activation consistent with a causal relationship between the two events. There was no change in the overall pattern of tyrosine phosphorylation in response to high concentrations of collagen in the presence of α2β1 blockade demonstrating that the integrin is not required for this event. In contrast, jararhagin and P1E6 had a small, almost negligible inhibitory effect against responses to the GPVI‐selective agonist collagen‐related peptide (CRP) and the G protein‐coupled receptor agonist thrombin. Crosslinking of α2β1 in solution or by adhesion to a monolayer using a variety of antibodies to either subunit of the integrin did not induce detectable protein tyrosine phosphorylation in whole cell lysates. The snake venom toxin trimucytin‐stimulated a similar pattern of tyrosine phosphorylation to that induced by crosslinking of GPVI which was maintained in the presence of jararhagin. Trimucytin may therefore induce activation via GPVI rather than α2β1 as previously thought. These observations show that the integrin α2β1 is not required for regulation of tyrosine phosphorylation by collagen.
The P2Y1 receptor mediates ADP‐induced p38 kinase‐activating factor generation in human platelets
Authors: Dangelmaier Carol , Jin Jianguo , Daniel James L. , Smith J. Bryan , Kunapuli Satya P.
Published: March 2003

U46619, a thromboxane A2 mimetic, but not ADP, caused activation of p38 mitogen activated protein (MAP) kinase in aspirin‐treated platelets. In nonaspirinated human platelets ADP activated p38 MAP kinase in both a time‐and concentration‐dependent manner, suggesting that ADP‐induced p38 MAP kinase activation requires generation of thromboxane A2. However, neither a thromboxane A2/prostaglandin H2 receptor antagonist SQ29548 and a thromboxane synthase inhibitor, furegrelate, either alone or together, nor indomethacin blocked ADP‐induced p38 kinase activation in nonaspirinated platelets. Other cycloxygenase products, PGE2, PGD2, and PGF2α, failed to activate p38 kinase in aspirin‐treated platelets. Hence, ADP must be generating an agonist, other than thromboxane A2, via an aspirin‐sensitive pathway, which is capable of activating p38 kinase. AR‐C66096, a P2TAC (platelet ADP receptor coupled to inhibition of adenylate cyclase) antagonist, did not inhibit ADP‐induced p38 MAP kinase activation. The P2X receptor selective agonist, α,β‐methylene ATP, failed to activate p38 MAP kinase. On the other hand, the P2Y1 receptor selective antagonist, adenosine‐2′‐phosphate‐5′‐phosphate inhibited ADP‐induced p38 kinase activation in a concentration‐dependent manner, indicating that the P2Y1 receptor alone mediates ADP‐induced generation of the p38 kinase‐activating factor. These results demonstrate that ADP causes the generation of a factor in human platelets, which can activate p38 kinase, and that this response is mediated by the P2Y1 receptor. Neither the P2TAC receptor nor the P2X1 receptor has any significant role in this response.
The antiproliferative effect of β‐carotene requires p21waf1/cip1 in normal human fibroblasts
Authors: Stivala Lucia A. , Savio Monica , Quarta Santina , Scotti Claudia , Cazzalini Ornella , Rossi Laura , Scovassi Ivana A. , Pizzala Roberto , Melli Raffaele , Bianchi Livia , Vannini Vanio , Prosperi Ennio
Published: March 2003

In normal human fibroblasts, β‐carotene induces a cell‐cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear‐bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1‐associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell‐cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1‐deficient human fibroblasts treated with β‐carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which β‐carotene inhibits cell‐cycle progression.
Modulation of the phosphatase activity of calcineurin by oxidants and antioxidants in vitro
Authors: Sommer Debbie , Fakata Keri L. , Swanson Stanley A. , Stemmer Paul M.
Published: March 2003

Previous research has indicated that oxidants, antioxidants and the intracellular redox state regulate the activities of a variety of protein tyrosine kinases, protein tyrosine phosphatases, phospholipases and transcription factors. In order to explore the redox regulation of the serine/threonine phosphatase calcineurin, we have investigated the effects of a variety of oxidants and antioxidants on calcineurin phosphatase activity in vitro. The oxidants hydrogen peroxide, superoxide and glutathione disulfide inhibited the phosphatase activity of calcineurin in a dose‐dependent manner. Incubation of purified calcineurin with the antioxidants ascorbate, ascorbate 2‐phosphate, α‐lipoic acid, N‐acetyl‐l‐cysteine and glutathione increased phosphatase activity relative to untreated controls. In contrast, several other commonly used antioxidants, including butylated hydroxytoluene, butylated hydroxyanisole, TEMPOL (4‐hydroxy‐2,2,6,6‐tetramethylpiperidine‐N‐oxyl), Trolox (6‐hydroxy‐2,5,7,8‐tetramethyl‐chroman‐2‐carboxylic acid) and dihydrolipoic acid decreased the activity of purified calcineurin, possibly through prooxidative mechanisms. Although the antioxidant pyrrolidine dithiocarbamate increased the activity of purified calcineurin, it significantly inhibited the activity of calcineurin present in crude fibroblast lysates. These results support and extend the hypothesis that redox factors modulate the phosphatase activity of calcineurin and suggest that further in vivo studies are warranted.
Methyl‐coenzyme M formation in methanogenic archaea
Authors: Sauer Karin , Thauer Rudolf K.
Published: March 2003

Methyl‐coenzyme M (2‐methylthioethane sulfonate) is the key intermediate of methane formation in methanogenic archaea. It is generated from coenzyme M (2‐mercaptoethane sulfonate) in methyl transfer reactions catalyzed by proteins containing zinc. Here, we report that, for methyltransferase MtaA from Methanosarcina barkeri, the zinc is involved in coenzyme M activation. For the experiments an inactive MtaA apoprotein was obtained by heterologous overproduction in Escherichia coli grown in the presence of 2 mm EDTA. The apoprotein was found to react with zinc or cobalt to the fully active holoenzyme. Appoximately 1 mol of transition metal was bound per mol of protein. Upon incubation of the holoenzyme with coenzyme M ≈ 1 mol of proton was released per mol of zinc or cobalt. Protons were not released upon incubation of the apoprotein with coenzyme M or of the holoprotein with other thiol compounds or with methyl‐coenzyme M. The findings are interpreted as indicating that the role of the transition metal in MtaA is to lower the microscopic pKa of the thiol group of coenzyme M by coordination to the zinc, and thus to increase its nucleophilicity for methyl group attack. The pKZn2+ of MtaA was re‐determined and found to be > 15 and not 9.6 as previously reported by us.
Alu‐DNA repeat‐binding protein p68 is a part of Alu‐RNA containing α‐RNP
Authors: Lukyanov Dmitry V. , Urusova Maria E. , Shcherba Ksenia M. , Podgornaya Olga I.
Published: March 2003

An Alu‐DNA repeat‐binding protein with a molecular mass of 68 kDa (p68) is identified in the somatic human cell nucleoplasm. Gel mobility shift assay (GMSA), South‐western blotting and affinity purification on DNA attached to the carrier were used in the identification. GMSA revealed multiple complexes with the exponential dependence of their relative mobility. A narrow binding site of the p68 was revealed using synthetic oligonucleotides. It is located between the A‐box and B‐box of the RNA polymerase III promoter and is identical to that reported for the Alu‐binding protein from human spermatozoids. The same narrow binding site, the similarity of the isolation procedure from germ and somatic cells, and similar binding properties and molecular masses suggest homology of the two proteins. Antibodies raised against Alu‐protein complexes led to hypershift of the complexes in GMSA and stained p68 in active fractions in human spermatozoids and in Alu‐RNA‐containing α‐RNP particles. Immunofluorescence of a HeLa cell monolayer revealed an intranuclear dot pattern with the dots corresponding to euchromatin areas and some dots located at the cell periphery in the cytoplasm. α‐RNP particles bound Alu‐DNAin vitroand contained p68 as shown using the immunogold procedure. Alu‐DNA binding activity was revealed in cytoplasm as well as in nucleoplasm. The possible nature of the main Alu‐DNA binding protein and its involvement in the particle structure are discussed.
Antitermination in bacteriophage λ
Authors: Schärpf Manuela , Sticht Heinrich , Schweimer Kristian , Boehm Markus , Hoffmann Silke , Rösch Paul
Published: March 2003

The solution structure of a 15‐mer nutRboxBRNA hairpin complexed with the 36‐mer N‐terminal peptide of the N protein (N36) from bacteriophage λ was determined by 2D and 3D homonuclear and heteronuclear magnetic resonance spectroscopy. These 36 amino acids include the arginine‐rich motif of the N protein involved in transcriptional antitermination of phage λ. Upon complex formation withboxBRNA, the synthetic N36 peptide binds tightly to the major groove of theboxBhairpin through hydrophobic and electrostatic interactions forming a bent α helix. Four nucleotides of the GAAAA pentaloop of theboxBRNA adopt a GNRA‐like tetraloop fold in the complex. The formation of a GAAA tetraloop involves a loop‐closing sheared base pair (G6‐A10), base stacking of three adenines (A7, A8, and A10), and extrusion of one nucleotide (A9) from the loop, as observed previously for the complex of N(1–22) peptide and the nutLboxBRNA [Legault, P., Li, J., Mogridge, J., Kay, L.E. & Greenblatt, J. (1998)Cell93, 289–299]. Stacking of the bases is extended by the indole‐ring of Trp18 which also forms hydrophobic contacts to the side‐chains of Leu24, Leu25, and Val26.

Based on the structure of the complex, three mutant peptides were synthesized and investigated by CD and NMR spectroscopy in order to determine the role of particular residues for complex formation. These studies revealed very distinct amino‐acid requirements at positions 3, 4, and 8, while replacement of Trp18 with tyrosine did not result in any gross structural changes.
Aspartate aminotransferase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125
Authors: Birolo Leila , Tutino M. Luisa , Fontanella Bianca , Gerday Charles , Mainolfi Katia , Pascarella Stefano , Sannia Giovanni , Vinci Floriana , Marino Gennaro
Published: March 2003

The gene encoding aspartate aminotransferase from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned, sequenced and overexpressed in Escherichia coli. The recombinant protein (PhAspAT) was characterized both at the structural and functional level in comparison with the E. coli enzyme (EcAspAT), which is the most closely related (52% sequence identity) bacterial counterpart. PhAspAT is rapidly inactivated at 50 °C (half‐life = 6.8 min), whereas at this temperature EcAspAT is stable for at least 3 h. The optimal temperature for PhAspAT activity is ≈ 64 °C, which is some 11 °C below that of EcAspAT. The protein thermal stability was investigated by following changes in both tryptophan fluorescence and amide ellipticity; this clearly suggested that a first structural transition occurs at ≈ 50 °C for PhAspAT. These results agree with the expected thermolability of a psychrophilic enzyme, although the observed stability is much higher than generally found for enzymes isolated from cold‐loving organisms. Furthermore, in contrast with the higher efficiency exhibited by several extracellular psychrophilic enzymes, both kcat and kcat/Km of PhAspAT are significantly lower than those of EcAspAT over the whole temperature range. This behaviour possibly suggests that the adaptation of this class of endocellular enzymes to a cold environment may have only made them less stable and not more efficient.

The affinity of PhAspAT for both amino‐acid and 2‐oxo‐acid substrates decreases with increasing temperature. However, binding of maleate and 2‐methyl‐l‐aspartate, which both inhibit the initial steps of catalysis, does not change over the temperature range tested. Therefore, the observed temperature effect may occur at any of the steps of the catalytic mechanism after the formation of the external aldimine.

A molecular model of PhAspAT was constructed on the basis of sequence homology with other AspATs. Interestingly, it shows no insertion or extension of loops, but some cavities and a decrease in side chain packing can be observed.
Genomic structure and chromosomal localization of the human hepatocyte growth factor activator inhibitor type 1 and 2 genes
Authors: Itoh Hiroshi , Yamauchi Masamichi , Kataoka Hiroaki , Hamasuna Ryouichi , Kitamura Naomi , Koono Masashi
Published: March 2003

Hepatocyte growth factor activator inhibitor type 1 (HAI‐1) and type 2 (HAI‐2) are recently discovered Kunitz‐type serine protease inhibitors which can be purified and cloned from human stomach cancer cell line MKN45 as specific inhibitors against hepatocyte growth factor activator (HGFA). HAI‐2 was identical with the protein originally reported as placental bikunin. Both proteins contain two Kunitz inhibitor domains (KDs), of which the first domain (KD1) is mainly responsible for the inhibitory activity against HGFA, and are expressed ubiquitously in various tissues. In this study, we cloned the genes coding for these two structurally similar proteins by screening of human genomic bacterial artificial chromosome (BAC) library and their genomic structures were compared. HAI‐1 and ‐2 genes consist of 11 and 8 exons spanning 12 kbp and 12.5 kbp, respectively. Three exons were inserted between KD1 and KD2 of each gene, of which the middle one was the low‐density lipoprotein (LDL) receptor‐like domain (HAI‐1) and the testis specific exon (HAI‐2). Apparently homologous regions between HAI‐1 and ‐2 were not found in 5′‐flanking region and neither TATA nor CAAT box was present. The genes were mapped to chromosome 15q15 (HAI‐1) and 19q13.11 (HAI‐2). These results suggested that although HAI‐1 and ‐2 genes might be derived from same ancestor gene, they acquired distinctive in vivo roles during their evolution.
Rapid formation and high diffusibility of actin–cofilin cofilaments at low pH
Authors: Bonet Carmel , Ternent Diane , Maciver Sutherland K. , Mozo‐Villarias Angel
Published: March 2003

Cofilin is a small actin‐binding protein that is known to bind both F‐actin and G‐actin, severing the former. The interaction of cofilin with actin is pH‐sensitive, F‐actin being preferentially bound at low pH and G‐actin at higher pH, within the physiological range. Diffusion coefficients of F‐actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (DLM) as well as the fraction of polymerized actin, fLM, present in solution. In the range of cofilin–actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co‐operative manner to a plateau. We interpret this as indicating co‐operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP–actin–cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F‐actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin–actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin–cofilin complexes.
On the iron–sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase
Authors: Hagen W. R. , Vanoni M. A. , Rosenbaum K. , Schnackerz K. D.
Published: March 2003

Porcine liver dihydropyrimidine dehydrogenase is a homodimeric iron–sulfur flavoenzyme that catalyses the first and rate‐limiting step of pyrimidine catabolism. The enzyme subunit contains 16 atoms each of nonheme iron and acid‐labile sulfur, which are most likely arranged into four [4Fe−4S] clusters. However, the presence and role of such Fe–S clusters in dihydropyrimidine dehydrogenase is enigmatic, because they all appeared to be redox‐inactive during absorbance‐monitored titrations of the enzyme with its physiological substrates. In order to obtain evidence for the presence and properties of the postulated four [4Fe−4S] clusters of dihydropyrimidine dehydrogenase, a series of EPR‐monitored redox titrations of the enzyme under a variety of conditions was carried out. No EPR‐active species was present in the enzyme ‘as isolated’. In full agreement with absorbance‐monitored experiments, only a small amount of neutral flavin radical was detected when the enzyme was incubated with excess NADPH or dihydrouracil under anaerobic conditions. Reductive titrations of dihydropyrimidine dehydrogenase with dithionite at pH 9.5 and photochemical reduction at pH 7.5 and 9.5 in the presence of deazaflavin and EDTA led to the conclusion that the enzyme contains two [4Fe−4S]2+,1+ clusters, which both exhibit a midpoint potential of ≈ −0.44 V (pH 9.5). The two clusters are most likely close in space, as demonstrated by the EPR signals which are consistent with dipolar interaction of two S = 1/2 species including a half‐field signal around g≈ 3.9. Under no circumstances could the other two postulated Fe–S centres be detected by EPR spectroscopy. It is concluded that dihydropyrimidine dehydrogenase contains two [4Fe−4S] clusters, presumably determined by the C‐terminal eight‐iron ferredoxin‐like module of the protein, whose participation in the enzyme‐catalysed redox reaction is unlikely in light of the low midpoint potential measured. The presence of two additional [4Fe−4S] clusters in dihydropyrimidine dehydrogenase is proposed based on thorough chemical analyses on various batches of the enzyme and sequence analyses. The N‐terminal region of dihydropyrimidine dehydrogenase is similar to the glutamate synthase β subunit, which has been proposed to contain most, if not all, the cysteinyl ligands that participate in the formation of the [4Fe−4S] clusters of the glutamate synthase holoenzyme. It is proposed that the motif formed by the Cys residues at the N‐terminus of the glutamate synthase β subunit, which are conserved in dihydropyrimidine dehydrogenase and in several β‐subunit‐like proteins or protein domains, corresponds to a novel fingerprint that allows the formation of [4Fe−4S] clusters of low to very low midpoint potential.
Regulation of expression of the rat SOCS‐3 gene in hepatocytes by growth hormone, interleukin‐6 and glucocorticoids
Authors: Paul Conception , Seiliez Iban , Thissen Jean P. , Le Cam Alphonse
Published: March 2003

Suppressors of cytokine signalling (SOCS) represent a newly discovered family of molecules that seem to play an important role in the shutting off of cytokine and possibly peptide hormone action. Thus, understanding the mechanisms controlling their expression is of cardinal importance. In the present study, we have cloned the rat SOCS‐3 gene and analyzed its expression and the functioning of its promoter in hepatocytes. Expression of SOCS‐3 mRNA, which is very weak in freshly isolated cells, tended to increase when hepatocytes were incubated without hormones. Growth hormone (GH) and, to a much larger extent, interleukin‐6 (IL‐6) rapidly activated mRNA synthesis whereas glucocorticoids (GC) strongly inhibited both basal and hormone‐dependent expressions. A short promoter fragment (−137/+35) responded maximally to GH and IL‐6 (a threefold stimulation for each effector) and to GC (a 70–80% inhibition), whereas longer promoter sequences supported higher basal activity and lower positive hormonal responses. Deletion and mutation analyses indicated that all hormonal responses were dependent on two cis‐acting sequences termed the G‐rich and the A/T‐rich elements. Only the A/T‐rich element was active in a heterologous context, thus behaving as a typical enhancer. Unexpectedly, the two signal transducer and activator of transcription (STAT) binding sites found immediately upstream of the G‐rich motif didn’t seem to participate in either GH or IL‐6 effect, despite the fact that one of them strongly responded to IL‐6 when placed in front of a heterologous promoter. Finally, the negative regulation of SOCS‐3 promoter by GC that may contribute to gene silencing in vivo, appeared to involve interactions of the GC receptor with other transcription factors and not direct binding to DNA, as no GC‐response element was found in the sequence.