bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional analysis of alternatively spliced transcripts of the human histidine decarboxylase gene and its expression in human tissues and basophilic leukemia cells
Authors: MAMUNE‐SATO Ruriko , YAMAUCHI Kohei , TANNO Yasuo , OHKAWARA Yuichi , OHTSU Hiroshi , KATAYOSE Dai , MAEYAMA Kazutaka , WATANABE Takehiko , SHIBAHARA Shigeki , TAKISHIMA Tamotsu
Published: March 2005

L‐Histidine decarboxylase (HisDC) is the enzyme catalyzing the formation of histamine from L‐histidine. HisDC activity is expressed specifically in mast cells/basophils, endocrine cells in stomach, and histaminergic neurons in brain. As a first step in the analysis of the regulation of HisDC gene expression, we have cloned the cDNA coding for HisDC from a cDNA library of a human basophilic leukemia cell line, KU‐812‐F. We identified two types of HisDC cDNA, representing the 2.4‐kb and 3.4‐kb HisDC mRNA constitutively expressed in these cells. Sequence analysis of these cDNA revealed that the 3.4‐kb mRNA contains the insert sequence of 824 bases and suggests that both 2.4‐kb and 3.4‐kb mRNA may represent the alternatively spliced transcripts of the HisDC gene. Using expression plasmids containing a cDNA for each HisDC mRNA, we analyzed the function of possible HisDC isoforms. We show that only the 2.4‐kb mRNA encodes functional HisDC and is expressed in human brain and lung. However, we were unable to detect the 3.4‐kb mRNA in these tissues. Thus, the 3.4‐kb mRNA may be generated by KU‐812‐F cell‐specific splicing of the HisDC gene transcripts. Furthermore, we demonstrated the increase in the level of 2.4‐kb HisDC mRNA and HisDC activity in KU‐812‐F cells following treatment with phorbol 12‐myristate 13‐acetate.
Concerted activities of the RNA recognition and the glycine‐rich C‐terminal domains of nucleolin are required for efficient complex formation with pre‐ribosomal RNA
Authors: GHISOLFI Laurence , KHARRAT Abelhakim , JOSEPH Gérard , AMALRIC Francois , ERARD Monique
Published: March 2005

Nucleolin is an abundant nucleolar protein which is involved in the early stages of ribosome assembly. The central 40‐kDa domain of nucleolin comprises four RNA recognition motifs (RRM) which are presumed to be involved in specific interactions with pre‐rRNA. In order to examine in detail the role of this central domain and the contribution of the N‐terminal and C‐terminal domains of nucleolin to RNA binding, we have used an Escherichia coli expression system to synthezise polypeptides corresponding to various combinations of the three domains and their subdomains. By means of an in‐vitro binding assay and a synthetic RNA corresponding to a specific recognition site in pre‐rRNA we have been able to demonstrate conclusively that the central 40‐kDa domain is indeed responsible for the specificity of RNA recognition and that the N‐terminal domain can be removed without affecting RNA binding. Most interestingly, it appears that the C‐terminal 10‐kDa domain, which is rich in glycine and arginine residues, is essential for efficient binding of nucleolin to RNA, but does not itself contribute to the specificity of the interaction. Circular dichroic spectroscopic probing of the RNA component shows that the C‐terminal domain significantly modifies the RNA‐binding properties of the central RRM core. Finally, infrared spectroscopic studies reveal that the central 40‐kDa domain is structured in α helices and β sheets and that the interaction with the specific pre‐rRNA site induces subtle changes in the β sheet conformation.
Heme‐pocket‐hydration change during the inactivation of cytochrome P‐450camphor by hydrostatic pressure
Authors: DI PRIMO Carmelo , HUI BON HOA Gaston , DOUZOU Pierre , SLIGAR Stephen G.
Published: March 2005

Hydrostatic pressure has been used to convert cytochrome P‐450camphor to cytochrome P‐420. The latter is an inactivated but soluble and undenaturated form of cytochrome P‐450camphor. Using camphor analogues as probes of the active site we show that the inactivation volume change is directly correlated to the initial degree of hydration of the heme pocket. The values range between −73 ml/mol and −197 ml/mol [Di Primo, C., Hui Bon Hoa, G., Douzou, P. & Sligar, S. G. (1990) Eur. J. Biochem. 193, 383–386] for a totally hydrated (substrate‐free, low‐spin, six coordinated heme iron) and a non‐hydrated (camphor‐bound, high‐spin, five coordinated heme iron) heme pocket. These results suggest that the larger value, −197 ml/mol, for the inactivation volume change is due to a hydration change of the heme pocket resulting from the displacement of the substrate during the compression and the subsequent entrance of water molecules. Similarly, the stability of the protein against compression is correlated with water accessibility to the active site. Increase in substrate mobility by loss of specific interactions with both regions of well defined secondary structure of cytochrome P‐450camphor results in an increase of water accessibility and decrease of stability. Thus for camphor and adamantanone which strongly interact with the protein and exclude water from the active site [Poulos, T. L., Finzel, B. C. & Howard, A. J. (1987) J. Mol. Biol. 195, 687–700; Raag, R. & Poulos, T. L. (1989) Biochemistry 28, 917–922] the increase in stability compared to the free protein is roughly 30 kJ/mol at 20°C. With smaller substrates such as norcamphor, which loosely fits into the active site and does not completely exclude water [Raag, R. & Poulos, T. L. (1989) Biochemistry 28, 917–922], the increase in stability is only 7 kJ/mol. Finally these results suggest that cytochrome P‐420 induced by hydrostatic pressure is a unique form where the active site is hydrated and camphor is displaced from its binding site.
Substrate specificity and properties of the aryl‐alcohol oxidase from the ligninolytic fungus Pleurotus eryngii
Authors: GUILLÉN Francisco , MARTÍNEZ Angel T. , MARTÍNEZ Maria Jesús
Published: March 2005

The production in a 5‐1 fermenter of the extracellular enzymes laccase and aryl‐alcohol oxidase by the fungus Pleurotus eryngii was studied. The latter enzyme has been purified 50‐fold by Sephacryl S‐200 and Mono Q chromatography. Purified aryl‐alcohol oxidase is a unique flavoprotein with 15% carbohydrate content, a molecular mass of 72.6 kDa (SDS/PAGE) and a pI of 3.9. The enzyme presents wide specificity, showing activity on benzyl, cinnamyl, naphthyl and aliphatic unsaturated alcohols. Neither activity nor inhibition of veratryl alcohol oxidation was found with saturated alcohols, but competitive inhibition was produced by aromatic compounds which were not arylalcohol oxidase substrates, such as phenol or 3‐phenyl‐1‐propanol. From these results, it was apparent that a double bond conjugated with a primary alcohol is necessary for substrate recognition by arylalcohol oxidase, and that activity is increased by the presence of additional conjugated double bonds and electron donor groups. Both affinity and maximal velocity during enzymic oxidation of methoxybenzyl alcohols were affected in a similar way by ring substituents, increasing from benzyl alcohol (Km= 0.84 mM, Vmax= 52 U/mg) to 4‐methoxybenzyl alcohol (Km= 0.04 mM, Vmax= 208 U/mg). Aryl‐alcohol oxidase presents also a low oxidase activity with aromatic aldehydes, but the highest activity was found in the presence of electron‐withdrawing groups.
Purificication and properties of Aspergillus nigerβ‐glucosidase
Authors: WATANABE Takashi , SATO Toshie , YOSHIOKA Shin , KOSHIJIMA Tetsuo , KUWAHARA Masaaki
Published: March 2005

β‐Glucosidase was purified from a crude cellulase preparation from Aspergillus niger by affinity chromatography on a methacrylamide‐N‐methylene‐bis‐methacrylamide copolymer bearing cellobiamine. The purified enzyme was a dimer with an isoelectric point of 4.0. The molecular mass of the enzyme was estimated to be 240 kDa by gel‐permeation chromatography. The enzyme hydrolyzed specificically β‐glucosidic bonds and catalyzed transglucosylation of the β‐glucosyl group of cellobiose to yield 4‐O‐β‐gentiobiosylglucose in the presence of organic solvents or under neutral conditions.
Bacterial nitrite‐reducing enzymes
Authors: BRITTAIN Thomas , BLACKMORE Richard , GREENWOOD Colin , THOMSON Andrew J.
Published: March 2005

The enzymic reduction of nitrite takes place in a wide range of bacteria and is found to occur in denitrifying, assimilatory and dissimilatory pathways. In this review we describe the major molecular characteristics of the various enzymes employed in each of these processes.
Characterization of [3H]acifluorfen binding to purified pea etioplasts, and evidence that protoporphyrinogen oxidase specifically binds acifluorfen
Authors: MATRINGE Michel , MORNET René , SCALLA René
Published: March 2005

It is now generally accepted that protoporphyrinogen oxidase is the target‐enzyme for diphenylether‐type herbicides. Recent studies [Camadro, J‐M., Matringe, M., Scalla, R. & Labbe, P. (1991) Biochem. J. 277, 17–21] have revealed that in maize, diphenyl ethers competitively inhibit protoporphyrinogen oxidase with respect to its substrate, protoporphyrinogen IX.

In this study, we show that, in purified pea etioplast, [3H]acifluorfen specifically binds to a single class of high‐affinity binding sites with an apparent dissociation constant of 6.2 ± 1.3 nM and a maximum density of 29 ± 5 nmol/g protein. [3H]Acifluorfen binding reaches equilibrium in about 1 min at 30°C. Half dissociation occurs in less than 30 s, indicating that the binding is fully reversible.

The specificity of [3H]acifluorfen binding to protoporphyrinogen oxidase is examined. [3H] Acifluorfen binding is inhibited by all the peroxidizing molecules tested. The phthalimide derivative, N(4‐chloro‐2‐fluoro‐5‐isopropoxy)phenyl‐3,4,5,6‐tetra hydrophthalimide, exerts a mixed‐competitive inhibition on this binding. The effects of all these molecules on the binding of [3H]acifluorfen are tightly linked to their capacity to inhibit pea etioplast protoporphrinogen oxidase activity. Furthermore, protoporphyrinogen IX, the substrate of the reaction catalyzed by protoporphyrinogen oxidase, was able to competitively inhibit the binding of [3H]acifluorfen. In contrast, protoporphyrin IX, the product of the reaction, did not inhibit this binding.

All these results provide clear evidence that in pea etioplasts, [3H]acifluorfen exclusively binds to protoporphyrinogen oxidase, that the protoporphyrinogen oxidase inhibitors tested so far bind to the same region of the enzyme and that this region overlaps the catalytic site of the enzyme.
Mutational replacements in subtilisin 309
Authors: BECH Lene M. , SØRENSEN Steen Bech , BREDDAM Klaus
Published: March 2005

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site‐directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position‐104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate‐induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4
Structure of the acidic N‐linked carbohydrate chains of the 55‐kDa glycoprotein family (PZP3) from porcine zona pellucida
Authors: NOGUCHI Satoru , NAKANO Minoru
Published: March 2005

N‐linked carbohydrate chains of the major 55‐kDa family, PZP3, of porcine zona pellucida glycoproteins are composed of neutral (28%) and acidic (72%) complex‐type chains. The structures of the main components of the neutral chain have been established [Noguchi, S., Hatanaka, Y., Tobita, T. & Nakano, M. (1992) Eur. J. Biochem. 204, 1089–1100]. Here we report the structures of the acidic chains. Only two kinds of acidic fragments were released from PZP3 by endo‐β‐galactosidase digestion following β‐elimination of O‐linked chains. 500‐MHz one‐dimensional and two‐dimensional 1H‐NMR spectroscopy revealed their structures to be Siaα(2–3)Galβ(1–4)[HSO3‐6]GlcNAcβ(1–3)Gal and HSO3‐6GlcNAcβ(1–3)Gal, showing that the sulfate‐containing acidic chains are constructed with non‐branched N‐acetyllactosamine repeats which have sialic acid(s) at the non‐reducing end(s) and sulfate at the C‐6 position of GlcNAc residues. The acidic N‐linked chains obtained from PZP3 by hydrazinolysis were separated into diantennary chains (34%) and tri‐ and tetra‐antennary chains (66%) by concanavalin‐A–agarose gel chromatography. The diantennary chains and their sialidase digests were fractionated by DEAE‐HPLC. From the analyses of the endo‐β‐galactosidase digests of each fraction, structures of the diantennary acidic chains were determined. They are classified into four groups. The first group is the sialylated chains without the sulfated N‐acetyllactosamine repeating unit. The other three groups have the chains of various lengths differing in the number of monosulfated N‐acetyllactosamine unit. These chains are extended from the Manα(1–3) branch of the trimannosyl core in the second group, from the Manα(1–6) branch in the third group, and from both branches in the fourth group. The structural features of the tri‐ and tetra‐antennary acidic chains are also presented.
The Asn‐linked carbohydrate chains of human Tamm‐Horsfall glycoprotein of one male
Authors: HÅRD Karl , VAN ZADELHOFF Guus , MOONEN Peter , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G.
Published: March 2005

Human Tamm‐Horsfall glycoprotein has been purified from the urine of one male. The Asn‐linked carbohydrate chains were enzymically released by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F, and separated from the remaining protein by gel‐permeation chromatography on Bio‐Gel P‐100. Fractionation of the intact (sulfated) sialylated carbohydrate chains was achieved by a combination of three liquid‐chromatographic techniques, namely, anion‐exchange FPLC on Q‐Sepharose, amine‐adsorption HPLC on Lichrospher‐NH2, and high‐pH anion‐exchange chromatography on CarboPac PA1. In total, more than 150 carbohydrate‐containing fractions were obtained, some of which still contained mixtures of oligosaccharides. The primary structure of 30 N‐glycans, including 10 novel oligosaccharides, were determined by one‐ and two‐dimensional 1H‐NMR spectroscopy at 500 MHz or 600 MHz. The types of compounds identified range from non‐fucosylated, monosialylated, diantennary to fucosylated, tetrasialylated, tetraantennary carbohydrate chains, possessing the following terminal structural elements:
Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells
Authors: FERNANDO M. Rohan , NANRI Hiroki , YOSHITAKE Shinichirou , NAGATA‐KUNO Kazue , MINAKAMI Shigeki
Published: March 2005

The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde‐3‐phosphate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2–14 μM) of reduced thioredoxin, with 0.125 μM thioredoxin reductase and 0.25 mM NADPH, regenerated H2O2‐inactivated glyceraldehyde‐3‐phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37°C. Although the treatment of endothelial cells with 0.2–12 mM H2O2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde‐3‐phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial cells at control state was in the reduced form and 70–85% remained in the reduced form even after the H2O2 treatment. The inactivated glyceraldehyde‐3‐phosphate dehydrogenase in a cell lysate prepared from the H2O2‐treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37°C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13‐cis‐retinoic acid resulted in a decrease in the regeneration of glyceraldehyde‐3‐phosphate dehydrogenase in the H2O2‐treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells.
On the stoichiometry of the iron‐sulphur clusters in mitochondrial NADH: ubiquinone oxidoreductase
Authors: van BELZEN Ronald , Mariette A. , ALBRACHT Simon P. J.
Published: March 2005

The concentration of the iron‐sulphur (Fe‐S) cluster 1b, present in complex I or soluble high‐molecular‐mass NADH dehydrogenase, was determined using different methods. It was found that direct double integration of the EPR signal at temperatures higher than 40 K, as is commonly used in this field of research, results in a considerable overestimation of the concentration of cluster 1b. It is demonstrated that this is caused by contributions from the relaxation‐broadened signals of the Fe‐S clusters 2–4 in the enzyme. The correct way for determining the intensity of the EPR signal of cluster 1b is by comparison with a simulated line shape. It is concluded that the concentration of cluster 1b is half that of cluster 2. This corroborates our proposal based on presteady‐state kinetic and inhibitor‐titration studies [Van Belzen, R., Van Gaalen, M. C. M., Cuypers, P. A. & Albracht S. P. J. (1990) Biochim. Biophys Acta 1017, 152–159] that the minimal functional unit of mitochondrial NADH:ubiquinone oxidoreductase must be a heterodimer.
Regulation of two rat serine‐protease inhibitor gene promoters by somatotropin and glucocorticoids
Authors: PAQUEREAU Laurent , VILAREM Marie José , ROSSI Valérie , ROUAYRENC Jean François , LE CAM Alphonse
Published: March 2005

Only two out of the three serine‐protease inhibitor genes (SPI 2.1, 2.2 and 2.3) expressed in rat liver are tightly controlled by somatotropin acting mainly at the transcriptional level, thus making this gene system particularly suitable to study its molecular mechanism of action. In these studies, we analyzed SPI promoter activities in cultured hepatocytes transfected by electroporation or in cell‐free extracts. The proximal SPI 2.1 promoter region contains two somatotropin‐responsive sites which are functional in intact cells. The more distal element that maps at positions – 175 to – 114, and is analogous to the one originally described by Yoon et al. (1990) [Yoon, J. B., Berry, S. A., Seelig, S. & Towle, H. C. (1990) J. Biol. Chem. 265, 19947–19954], behaves as a weak enhancer whose activity is strongly potentiated by proximal 5′ downstream sequences that contains potential CCAAT‐enhancer binding protein (C/EBP) sites. An additional proximal hormone‐sensitive site is located in the close vicinity of the transcription‐start site between positions −41 and +8, and also requires the first C/EBP‐binding element to be active. The distal element appears to contribute more importantly (60%) than the proximal one (40%) to the overall somatotropin stimulation of chimeric gene expression. Nonetheless, both displayed similar dose‐dependence, with half‐maximal and maximal effects occurring at 0.5–1 nM and 5–10 nM, respectively. The somatotropin refractoriness of the SPI 2.3 gene appears to be due to the presence of distal (–2300 to –200) inhibitory element(s) in the promoter. Glucocorticoids exert both positive and negative effects on SPI promoter activity. Their stimulatory action appears to involve sequences located between positions –114 and –82, together with a more distal half glucocorticoid‐responsive element, whereas their inhibitory effect is more likely mediated by sequences located between positions −41 and +8.

In vitro transcription assays, performed with promoter‐deletion mutants and competitor oligonucleotides, revealed the presence of a major functional C/EBP site located immediately upstream from the transcription‐start point. Unfortunately, the regulatory features of SPI gene expression observed in intact cells were completely obliterated by breaking down the cell structure, and could not therefore be studied using cell‐free systems.
Binding of a 100‐kDa ubiquitous factor to the human prolactin promoter is required for its basal and hormone‐regulated activity
Authors: PEERS Bernard , NALDA Asunción M. , MONGET Philippe , VOZ Marianne L. , BELAYEW Alexandra , MARTIAL Joseph A.
Published: March 2005

cAMP strongly stimulates the activity of the human prolactin (hPRL) promoter. We have previously shown that two types of cis‐element are required for this cAMP regulation; binding sites for the pituitary‐specific factor Pit‐1, and the sequence spanning nucleotides −115 to −85 (named sequence A). Sequence A contains the TGACG motif found in the consensus sequence of the cAMP‐responsive element (CRE). In this study, we show that a mutation in the TGACG motif of sequence A strongly reduces not only the cAMP regulation but also the Ca2+ regulation and basal activity of the hPRL promoter. Furthermore, gel‐shift assays indicate that the mutation prevents binding of a ubiquitous factor which is not the CRE‐binding protein. Southwestern experiments suggest that this ubiquitous factor's molecular mass is approximately 100 kDa. We conclude that binding of a 100‐kDa ubiquitous factor to sequence A is required for full basal and hormonal regulation of hPRL‐promoter activity.
EPR studies of cytochrome aa3 from Sulfolobus acidocaldarius
Authors: ANEMÜLLER Stefan , BILL Eckhard , SCHÄFER Günter , TRAUTWEIN Alfred X. , TEIXEIRA Miguel
Published: March 2005

The purified cytochrome aa3‐type oxidase from Sulfolobus acidocaldarius (DSM 639) consists of a single subunit, containing one low‐spin and one high‐spin A‐type hemes and copper [anemüller, S. and Schäfer, G. (1990) Eur. J. Biochem. 191, 297–305]. The enzyme metal centers were investigated by electron paramagnetic resonance spectroscopy (EPR), coupled to redox potentiometry. The low‐spin heme EPR signal has the following g‐values: gz= 3.02, gy= 2.23 and gx= 1.45 and the highspin heme exhibits an almost axial spectrum (gy= 6.03 and gx= 5.97, E/D < 0.002). In the enzyme as isolated the low‐spin resonance corresponds to 95 ± 10% of the enzyme concentration, while the high‐spin signal accounts for only 40 ± 5%. However, taking into account the redox potential dependence of the high‐spin heme signal, this value also rises to 95 ± 10%. The high‐spin heme signal of the Sulfolobus enzyme shows spectral characteristics distinct from those of the Paracoccus denitrificans one: it shows a smaller rhombicity (gy= 6.1 and gx= 5.9, E/D= 0.004 for the P. denitrificans enzyme) and it is easier to saturate, having a half saturation power of 148 mW compared to 360 mW for the P. denitrificans protein, both at 10 K. The EPR spectrum of an extensively dialyzed and active enzyme sample containing only one copper atom/enzyme molecule does not display CuA‐like resonances, indicating that this enzyme contains only a CuB‐type center. The EPR‐redox titration of the high‐spin heme signal, which is assigned to cytochrome a3, gives a bell shaped curve, which was simulated by a non‐interactive two step redox process, with reduction potentials of 200 ± 10 mV and 370 ± 10 mV at pH = 7.4. The decrease of the signal amplitude at high redox potentials is proposed to be due to oxidation of a CuB(I) center, which in the CuB(II) state is tightly spin‐coupled to the heme a3 center. The reduction potential of the low‐spin resonance was determined using the same model as 305 ± 10 mV at pH = 7.4 by EPR redox titration. Addition of azide to the enzyme affects only the high‐spin heme signal, consistent with the assignment of this resonance to heme a3. The results are discussed in the context of the redox center composition of quinol and cytochrome c oxidases.
Control of the metabolic flux in a system with high enzyme concentrations and moiety‐conserved cycles
Authors: KHOLODENKO Boris N. , LYUBAREV Arkadii E. , KURGANOV Boris I.
Published: March 2005

In a number of metabolic pathways enzyme concentrations are comparable to those of substrates. Recently it has been shown that many statements of the ‘classical’ metabolic control theory are violated if such a system contains a moiety‐conserved cycle. For arbitrary pathways we have found: (a) the equation connecting coefficients CJEi (obtained by varying the Ei concentration) and CJvi (obtained by varying the Kcati), and (b) modified summation equations. The sum of the enzyme control coefficients (equal to unity under the ‘classical’ theory) appears always to be below unity in the systems considered. The relationships revealed were illustrated by a numerical example where the sum of coefficients CJEi reached negative values. A method for experimental measurements of the above coefficients is proposed.
Interleukin‐1β and transforming growth factor‐β2 enhance cytosolic high‐molecular‐mass phospholipase A2 activity and induce prostaglandin E2 formation in rat mesangial cells
Authors: SCHALKWIJK Casper G. , de VET Edwin , PFEILSCHIFTER Josef , van den BOSCH Henk
Published: March 2005

Interleukin‐1β induces gene expression and secretion of group‐II phospholipase A2 and release of prostaglandin E2 from rat mesangial cells. The interleukin‐1β‐induced synthesis of group‐II phospholipase A2 is prevented by transforming growth factor‐β2, whereas transforming growth factor‐β2 potentiated the interleukin‐1β‐evoked prostaglandin E2 production. Transforming growth factor‐β2 itself did not induce synthesis of group‐II phospholipase A2, although it stimulated prostaglandin E2 formation. Here we describe the effect of interleukin‐1β and transforming growth factor‐β2 on a cytosolic phospholipase A2 activity and prostaglanding E2 formation in rat mesangial cells. Based on the resistance to dithiothreitol and migration profiles on a Mono‐Q anion‐exchange column and a Superose 12 gel‐filtration column, the cytosolic phospholipase A2 activity was assigned to a high‐molecular‐mass phospholipase A2. Measured with 1‐stearoyl‐2‐[1‐14C]arachidonoylglycerophosphocholine as substrate, both interleukin‐1β and transforming growth factor‐β2 enhanced the high‐molecular‐mass phospholipase A2 activity. The stimulation of ratmesangial cells with interleukin‐1β and transforming growth factor‐β2 was time‐ and dose‐dependent with maximal cytosolic phospholipase A2 activities at 10 nM and at 10ng/ml respectively, after 24 h of stimulation. Under these conditions, interleukin‐1β and transforming growth factor‐β2 enhanced the cytosolic phospholipase A2 activity 2.2 ± 0.6‐fold and 2.5 ± 0.6‐fold, respectively. These results strongly suggest that an enhanced cytosolic high‐molecular‐mass phospholipase A2 activity is involved in the formation of prostaglandin E2 mediated by transforming growth factor‐β2. Whether interleukin‐1β induced group‐II phospholipase A2 and/or interleukin‐1β‐enhanced cytosolic phospholipase A2 activity is involved in prostaglandin E2 formation in rat mesangial cells is discussed.
Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain
Authors: STANGE Nicole , BEIER Dagmar , BEIER Hildburg
Published: March 2005

It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice‐site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre‐tRNATyr species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre‐tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre‐tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre‐tRNATyr species were spliced. In order to identify the elements essential for pre‐tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNATyr domains from yeast and Xenopus, respectively. The chimeric pre‐tRNA comprising the mature tRNATyr domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNATyr domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme‐substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of matated pre‐tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre‐tRNA molecule, are recognition points for plant splicing endonuclease.
Study of polynucleotide conformation by resolution‐enhanced ultraviolet spectroscopy
Authors: GARRIGA Pere , GARCIA‐QUINTANA David , MANYOSA Joan
Published: March 2005

Self‐deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single‐stranded and double‐helix structures of different cytosine‐containing polynucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2u band plus possibly some n→π* transition) of the absorption spectra in the cytosine‐containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280–310 nm region are attributed mainly to cytosine‐cytosine stacking interactions. In particular, the fourth‐derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth‐derivative peaks is discussed.

It is concluded that the resolution‐enhancement techniques used in this work, i.e. self‐deconvolution and fourth derivative, complement each other and are useful methods to study structural changes of single‐stranded and double‐stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.
1H‐15N‐NMR studies of bacteriorhodopsin Halobacterium halobium
Authors: OREKHOV Vladislav Yu. , ABDULAEVA Galina V. , MUSINA Larisa Yu. , ARSENIEV Alexander S.
Published: March 2005

Series of uniformly and selectively 15N‐labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a 1H‐15N‐NMR study was performed in methanol/chloroform (1:1) and 0.1 M NH4CHOO, medium which mimics that in the membrane in vivo. Less than half of the cross‐peaks expected from the amino acid sequence of uniformly 15N‐labeled bacteriorhodopsin were observed, using heteronuclear 1H‐15N coherence spectroscopy. In order to assign the observed cross‐peaks, a selective 15N‐labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and 1H‐15N‐NMR spectra of bacteriorhodopsin and its fragments C1 (residues (72–231), C2 (residues 1–71), B1 (residues 1–155) and BP2 (residues 163–231) were investigated. By this procedure, all observed 1H‐15N cross‐peaks of the entire bacteriorhodopsin were found to belong to the transmembrane segments A, B and G. The cross‐peaks from four (C, D, E and F) helical bundles (79–189 residues) were missed. These results clearly indicate that dynamic processes occur in the four helice bundle. The significance of this, in respect to bacteriorhodopsin functioning, is discussed.
Voltage dependence of liver gap‐junction channels reconstituted into liposomes and incorporated into planar bilayers
Authors: MAZET Jean‐Luc , JARRY Thérèse , GROS Daniel , MAZET Françoise
Published: March 2005

The voltage dependence of rat liver gap junctions was investigated using non‐denaturing solubilization and reconstitution of gap‐junction protein into proteoliposomes in controlled conditions of connexon aggregation. The presence of liver connexin 32 in reconstituted proteoliposomes was checked with specific antibodies. The proteoliposomes were inserted into planar lipid bilayers by fusion. The single‐channel conductance was voltage independent, and its magnitude was 700–1900 pS in 1 M NaCl, as expected from other reports, assuming that conductance is linear with ion activity. The channels were open at zero voltage and completely closed above 40 mV in either direction. This steep voltage dependence corresponded to an open/closed‐state voltage difference of 19 mV and to 3.5 gating charges moving through the field. When several channels were inserted into the bilayer, a large fraction of the membrane conductance became voltage insensitive. These results show that the isolated channel units are highly voltage dependent and are consistent with the assumption that aggregated connexons interact through links which prevent voltage‐sensitive conformational changes.
Inhibition of chloroplast ATPase by the K+ channel blocker α‐dendrotoxin
Authors: SCHULENBURG Peter , SCHWARZ Martin , WAGNER Richard
Published: March 2005

Possible structural and functional similarities between the channel part, CF0, of chloroplast ATPase (CF0CF1) and ion channels permeable to monovalent cations were investigated using high‐affinity toxins mainly targeted against voltage‐sensitive K+ channels. In particular, the effect of the K+‐channel blocker α‐dendrotoxin and the crude scorpion venom of Leiurus quinquestriatus hebraeus (LQ venom) on ATP synthesis in thylakoid membranes and in CF0CF1‐containing liposomes was characterised.

α‐dendrotoxin (Ki∼ 5.05 μM) and the LQ venom (Ki∼ 1.55 μg/ml) specifically inhibited ATP synthesis in thylakoid membranes and in CF0CF1‐containing liposomes. Our results show that α‐dendrotoxin and peptides of the LQ venom with an apparent molecular mass of about 4.0 kDa, probably isoforms of charybdotoxin, specifically bind to CF0CF1. This binding was reversible and induced a high leak conductance for H+ through CF0. The Ca2+‐dependent ATPase activity of the isolated soluble part of CF0CF1 (CF1) was completely inhibited by 1 μM α‐dendrotoxin, while the crude LQ venom, at concentrations up to 10 μg/ml, had no affect on ATPase activity. The concentration dependence of the inhibition by α‐dendrotoxin indicates that approximately 2 mol α‐dendrotoxin bind/mol CF0CF1 and 1 mol α‐dendrotoxin/mol CF1. Known inhibitors of H+‐flow‐through CF0 acted in the presence of α‐dendrotoxin synergistically. Dicyclohexylcarbodiimide and venturicidin, in contrast to their known effect of blocking H+‐flow‐through CF0, increased the leak conductance through CF0 in the presence of α‐dendrotoxin drastically. This uncoupling effect indicates that their normal mode of blocking is a secondary effect. Binding of the inhibitors to their respective sites apparently does not affect the proton pathway in CF0, but induces a conformation which closes the channel part for H+.

Protein sequence comparison between the known binding site of charybdotoxin in the shaker K+ channel from Drosophila [MacKinnon, R. & Heginbotham, L. (1990) Neuron 5, 767–771] and the choroplast ATPase showed that subunit III reveals a significant similarity (64%) in parts of its sequence (Gln28–Leu53) to the helix 5 and helix 6 (S5‐S6) linker region (Ala413–Cys462; the charybdotoxin‐binding site) of the shaker K+ channel. According to secondary‐structure predictions, the homologous sequences in subunit III and the shaker K+ channel represent putative hydrophilic loops connecting two transmembrane α‐helices. Apparently the shaker K+ channel and subunit III share significant topological features in these hydrophilic loops which may be part of the respective channel entrance.
Characterization of a nitric‐oxide‐catalysed ADP‐ribosylation of glyceraldehyde‐3‐phosphate dehydrogenase
Authors: DIMMELER Stefanie , BRÜNE Bernhard
Published: March 2005

Auto‐ADP‐ribosylation of the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GraPDH) has recently been demonstrated to be dramatically stimulated in the presence of nitric oxide. In order to obtain insight into the sequence of events leading to ADP‐ribosylation of GraPDH, we studied the target amino acid, the nucleotide cofactor requirement, pH dependency and the stoichiometry of the reaction. Basal as well as stimulated ADP‐ribose transfer is inhibited by the SH‐group alkylating reagent, N‐ethylmaleimide. Furthermore, the radiolabel of auto‐[32P]ADP‐ribosylated GraPDH is removed by treatment with HgCl2, suggesting an ADP‐ribose–cysteine bond. Several indirect and direct mechanistic considerations point to NAD+ as the only cofactor for the ADP‐ribosylation reaction, excluding the possibility of a reaction sequence involving a NAD‐glycohydrolase(s) followed by nonenzymatic ADP‐ribose transfer to GraPDH. Optimal ADP‐ribosylations were carried out at alkaline pH values using 10 μM free NAD+ as the sole nucleotide cofactor. Bovine serum albumin with an S‐nitrosylated SH group can serve as a model of ADP‐ribose transfer from NAD+ and suggests that the nitric‐oxide‐modified SH group (S‐nitrosylated SH group) is a prerequisite for the reaction.
The primary sequence and the subunit structure of mouse α‐2‐macroglobulin, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA
Authors: VAN LEUVEN Fred , TORREKENS Sophie , OVERBERGH Lut , LORENT Kristin , DE STROOPER Bart , VAN DEN BERGHE Herman
Published: March 2005

Mouse plasma α‐2‐macroglobulin (mα2M) was isolated and the N‐terminal amino‐acid sequences determined after separation of the 165‐kDa and 35‐kDa subunits. These sequences were compared to the protein sequence predicted by the cDNA, which was cloned from a mouse liver library and sequenced. From these data it is evident that both subunits are encoded by one mRNA of approximately 5kb expressed predominantly in liver. The smaller subunit, with the N‐terminal sequence DLSSSDLT, comprises the C‐terminal 257 residues of mα2M and is derived from a single‐chain precursor probably by proteolytic processing at an arginine residue in the sequence PTRDLSS. Analysis of the predicted protein further showed all the salient features of a proteinase inhibitor of the macroglobulin family: a bait region that deviates from all known sequences in this family, a very conserved internal thiolester site and conserved cysteine residues and putative N‐glycosylation sites. The synthesis of mα2M in adult liver was demonstrated by Northern blotting and in fetal liver by in‐situ hybridization. Transient transfection of COS cells with the cDNA under control of a viral promoter demonstrated the secretion and partial processing of mα2M in the culture medium. In plasma the level of mα2M was found to be stable as expected for the murine counterpart of human plasma α‐2‐macroglobulin. The possibilities of using the mouse as a genetic model to study this proteinase inhibitor in vivo are discussed.
The human rhabdomyosarcoma cell line A204 lays down a highly insoluble matrix composed mainly of α1 type‐XI and α2 type‐V collagen chains
Authors: KLEMAN Jean‐Philippe , HARTMANN Daniel J. , RAMIREZ Francesco , van der REST Michel
Published: March 2005

The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin‐digested collagen showed that it is composed mainly of α1(XI) and α2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [α1(XI)]2α2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing α2(V) but not α1(XI) collagen chains.

Electron microscopy analyses of 24‐h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold‐labelled anti‐(type‐V collagen) IgG, but not with anti‐(type‐XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of β‐aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of β‐aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti‐(type‐V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti‐(type‐XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa.

These data indicate that type‐V and type‐XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.
Purification of assembly‐competent tubulin from Saccharomyces cerevisiae
Authors: BELLOCQ Christine , ANDREY‐TORNARE Isabelle , DORET Anne‐Marie PAUNIER , MAEDER Bernard , PATURLE Lawrence , JOB Didier , HAIECH Jacques , EDELSTEIN Stuart J.
Published: March 2005

We have developed a straightforward, two‐step procedure to isolate highly purified yeast tubulin that reproducibly assembles into microtubules. The starting extracts are obtained from cells genetically engineered to overproduce both the α and β subunits of tubulin, under control of the galactose promoter, to approximately 10‐times wild‐type levels. The first step of purification is carried out with the high‐speed supernatant of lysed cells loaded onto a DEAE‐Sephadex column; after this step the tubulin preparation is approximately 30% pure. In the second step, the tubulin fractions are loaded onto an immunoaffinity column prepared by coupling the anti‐(α‐tubulin) monoclonal antibody YL 1/2 to Sepharose‐4B. Following elution with 0.8 M KCl, the tubulin present in the peak is 90% pure. Upon addition of porcine brain microtubule‐associated proteins or DEAE‐dextran, this tubulin preparation is functionally active for assembly into microtubules, as visualized by electron microscopy on negatively stained samples. Virtually identical microtubule structures are produced in parallel experiments on the assembly of yeast or porcine brain tubulin, with differences observed only at acidic pH values. Overall, this relatively simple procedure provides a useful tool for the production of functional tubulin suitable both for structural studies and for investigations of the assembly process.
Comparison of biochemical properties of DNA‐topoisomerase I from normal and regenerating liver
Authors: TOURNIER Marie‐Françoise , SOBCZAK Joëlle , de NECHAUD Béatrice , DUGUET Michel
Published: March 2005

Biochemical properties of topoisomerase I from normal and regenerating rat liver were analysed using crude or fractionated nuclear extracts. We could not detect significative change in topoisomerase I content or activity (magnesium stimulation and inhibition by ATP) during the course of liver regeneration. Topoisomerase I can be resolved into two species of 97 kDa and 100 kDa, with the same pI of 8.2–8.6 as shown by two dimensional gel electrophoresis. The two polypeptides contained a non‐phosphorylated precursor and others forms with variable degrees of phosphorylation. In‐vitro dephosphorylation with alkaline phosphatase leads to the disappearance of the phosphorylated forms and inactivation of the enzyme. The affinity of topoisomerase I for chromatin (measured by salt elution) differs markedly between normal and regenerating liver: nearly 50% of topoisomerase I remained bound to the chromatin from normal liver at 250 mM NaCl whereas it was completely eluted from 24‐h‐regenerating‐liver nuclei. The biological significance of these results is discussed.
Developmental changes in hepatocyte growth factor mRNA and its receptor in rat liver, kidney and lung
Authors: KAGOSHIMA Masako , KINOSHITA Taisei , MATSUMOTO Kunio , NAKAMURA Toshikazu
Published: March 2005

Hepatocyte growth factor (HGF) is a mesenchymal‐derived factor which induces mitosis, cell movement and morphogenesis of tissue‐like structure. We analyzed changes in HGF mRNA and its receptor, the c‐met proto‐oncogene product, in the liver, kidney and lung during late fetal and postnatal development in rats. In the liver, the HGF‐mRNA level was very low during late gestation and in neonates, it increased remarkably and reached a maximum two weeks postnatally, to be followed by a decrease to 33% of the maximum. HGF mRNA in the kidney and lung was either undetectable or very low during late gestation and the neonatal period and increased markedly to reach a maximum, respectively, 3–4 weeks postnatally. HGF‐mRNA level in the adult rat lung was fivefold higher than that in the liver and kidney. The number of HGF receptors on plasma membranes of these tissues was low in neonates but there was a rapid increase after birth and a maximum was reached within three weeks. The number of HGF receptors/ng plasma membrane protein at the maximal level was highest in the liver and lowest in the lung. c‐met/HGF‐receptor mRNA in the liver was also low during late‐gestation or in early neonatal periods and increased postnatally. Since HGF‐mRNA and HCF‐receptor levels changed differently in liver, kidney and lung, the expression of HGF and its receptor may be independently regulated in each organ. However, in these organs, HGF mRNA and the HGF receptor increased within a few weeks of birth, HGF may play roles in organ growth, organ maturation and the maintenance of tissue homeostasis during the postnatal period, presumably through its potential to act as mitogen, motogen and morphogen.
Reversible shutdown of replicon initiation by transient hypoxia in Ehrlich ascites cells
Authors: RIEDINGER Hans‐Jörg , GEKELER Volker , PROBST Hans
Published: March 2005

The O2‐dependent regulation of replication in Ehrlich ascites cells, characterized by a reversible shutdown of replicon initiation during hypoxia, was scrutinized with respect to the involvement of gene expression. Synchronous and asynchronous cells were subjected to transient hypoxia and examined for expression of selected ‘late’ growth‐regulated mRNA and for the influence of inhibitors of transcription and translation on DNA replication. Irrespective of whether replicon initiation was suppressed by hypoxia or retriggered by reoxygenation, the levels of thymidine kinase mRNA and of proliferating cell‐nuclear antigen/cyclin mRNA were as high as in untreated replicating cells. The level of histone H3.1 mRNA followed, with a distinct delay, the replicative activity of the cells governed by the imposed changes of pO2. The response of replication to inhibition of transcription and translation was virtually the same as to hypoxia, i.e. a selective suppression of replicon initiation. It was demonstrated that replicon initiation‐depends on one or several short‐lived protein(s) (lifetime about 5 min) which is (are) formed under hypoxic conditions as well. The lifetime of the corresponding RNA message(s) is in the range of several hours. It is suggested that the expression of genes conditioning resting cells for DNA replication remains unaffected by hypoxia or by restoring the normal pO2. Hypoxic cells appear to rest in a state fully prepared for entering DNA replication, but a yet unknown event essential for replicon initiation is blocked. This event depends on a critical oxygen tension as well as on short‐lived protein(s).