bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of biliary secretion of chylomicron remnant cholesterol (esters) in the rat
Authors: van DIJK Marc C. M. , PIETERS Moniek , van BERKEL Theo J. C.
Published: March 2005

Chylomicrons labelled with [3H]cholesterol/[3H]cholesterol esters in a ratio of 25.5: 74.5, were rapidly removed from rat serum in vivo, and taken up predominantly by the parenchymal liver cells (88.2% of the hepatic uptake at 15 min after injection). Lactoferrin reduced the liver uptake of chylomicron remnants by 72%, at 20 min after injection. It appeared that the free cholesterol which is present in the chylomicrons is not readily exchanged within the used time period with other cholesterol pools in the animal. Between 10–60 min after injection of 3H‐labelled chylomicrons, cholesterol esters are hydrolysed in the liver. Appearance of radioactivity in bile was rapid and at 3, 24 and 72 h after injection, 13.4 %, 44.0 % and 70.0 %, respectively, of the injected dose appeared in bile, mainly as bile acids (> 90 %). Lactoferrin reduced the biliary secretion of radioactivity, especially during the first hour after injection. The total amount of radioactivity recovered was 58.0 % of the injected dose at 72 h after injection. After injection of β‐migrating very low‐density lipoprotein labelled with [3H]cholesterol/[3H]cholesterol esters in a ratio of 23.5:76.5, the maximum amount of radioactivity secreted in bile was much lower than with chylomicrons (2.6 % cf. 5.2% at 1 h after injection), although the kinetics of the initial liver asociation and cholesterol ester hydrolysis were even more rapid. Biliary accumulation of radioactivity was also lower with 50.5 % of the injected dose recovered at 72 h after injection. It can be concluded from these studies that the processing of chylomicron remnant cholesterol components in the liver and the subsequent secretion in the bile mainly as bile acids is very efficient. The efficient liver uptake of chylomicron remnants by the liver remnant receptor is thereby essential to achieve this high percentage of removel, thus protecting against extrahepatic cholesterol (ester) deposition.
Developmental and hormonal regulation of the Xenopus liver‐type arginase gene
Authors: XU Qiling , BAKER Betty S. , TATA Jamshed R.
Published: March 2005

Liver‐type l‐arginase is a major urea‐cycle enzyme which is strongly induced during amphibian metamorphosis, but little is known about the molecular mechanisms underlying this induction. As a first step towards elucidating the possible mechanisms, we have isolated a cDNA clone for l‐arginase from an adult Xenopus laevis liver cDNA library. Sequence comparison of Xenopus liver‐type l‐arginase cDNA shows a strong conservation at the amino acid level with those of human, rat and yeast. Using a Xenopus arginase cDNA fragment as a hybridization probe, we have shown by Northern blotting that the gene is highly expressed in the liver, and very slightly in kidney and spleen, of adult Xenopus. The expression is developmentally regulated. Only traces of arginase mRNA can be detected in pre‐metamorphic tadpoles, but its accumulation increases very markedly at the onset of natural metamorphosis, being maintained at a high concentration constitutively upon completion of this developmental process. Amphibian metamorphosis is under the strict control of thyroid hormones. It is therefore significant that exposure of pre‐metamorphic tadpoles (at stages before endogenous thyroid hormone secretion) to exogenous hormone (1 nM triiodothyronyne) precociously activated the l‐arginase gene. The time course of this precocious hormonal induction paralleled that of serum albumin gene in the liver. Polyclonal antibodies were raised against recombinant Xenopusl‐arginase expressed in Escherichia coli as a fusion protein with glutathione S‐transferase in the plasmid expression vector pGEX. Western blotting using this antibody showed that, although arginase mRNA is present in high concentration in Xenopus tadpole liver at the onset of natural metamorphosis, the protein is detected only upon its completion. Our results show a complex transcriptional and post‐transcriptional regulation of the Xenopus liver‐type l‐arginase gene during post‐embryonic development. They also demonstrate that this gene can be exploited as a target for thyroid hormones in further studies to analyze the mechanisms underlying the establishment of the adult phenotype during amphibian metamorphosis.
Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilusα‐amylase
Authors: TAKASE Kenji
Published: March 2005

Site‐directed mutagenesis of a thermostable α‐amylase from Bacillus stearothermophilus was performed to assess the role of amino acid residues near the catalytic site in catalysis. Asn 329 is presumed to be adjacent to the proposed catalytic residue Asp331. Its mutation to Lys, which is found at the corresponding position in pullulanase, resulted in the loss of 99.7% of the activity, while the mutation to Asp or Val did not drastically reduce the activity. The mutation to Val altered the temperature/activity profile so that the activity was reduced to 25% of wild‐type α‐amylase at 60°C but was over twofold greater at 5°C. This effect could be ascribed to a decrease in the activation enthalpy by 32%. The mutation to Asp or Lys altered the pH/activity profile concomitant with possible changes in the ionization state of the groups introduced. These results show the feasibility of altering and possibly improving the enzyme activity by mutagenesis of residues near the catalytic groups.
Role of sugar chains in the in‐vitro activity of recombinant human interleukin 5
Authors: KODAMA Shiho , TSUJIMOTO Masafumi , TSURUOKA Nobuo , SUGO Tsukasa , ENDO Tamao , KOBATA Akira
Published: March 2005

The structures of O‐linked sugar chains of recombinant human interleukin 5 (rhIL‐5) produced by Chinese hamster ovary cells were determined employing high‐performance anion‐exchange column chromatography under high pH conditions. The core sequence was elucidated to be Galβ1→3‐GalNAc by its susceptibility to endo‐α‐N‐acetylgalactosaminidase and sialic acid linkages were determined using sialidase of different specificities.

To investigate the role of sugar chains in the in‐vitro activity of rhIL‐5, it was digested with various glycosidases. While removal of N‐linked sugar chains resulted in 2.8‐fold increase of the activity, de‐O‐glycosylated rhIL‐5 showed 10‐fold higher activity than intact rhIL‐5, suggesting that the presence of O‐linked sugar chains suppresses the activity more effectively than that of N‐linked chains. While incubation of de‐N‐glycosylated rhIL‐5 at 70°C for 30 min decreased the activity, intact and de‐O‐glycosylated rhIL‐5 lost little activity, suggesting that N‐linked sugar chains contributed to the thermostability of the molecule.
The formation of methylglyoxal from triose phosphates
Authors: PHILLIPS Susan A. , THORNALLEY Paul J.
Published: March 2005

In Krebs‐Ringer phosphate buffer, the rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3‐phosphate was first order with respect to the triose phosphate with rates constant values of 1.94 ± 0.02 × 10−5 s−1 (n= 18) and 1.54 ± 0.02 × 10−4 s−1 (n= 18) at 37°C, respectively. The rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3‐phosphate in the presence of red blood cell lysate was not significantly different from the nonenzymatic value (P > 0.05). Methylglyoxal formation from glycerone phosphate was increased in the presence of triose phosphate isomerase but this may be due to the faster non‐enzymatic formation from the glyceraldehyde 3‐phosphate isomerisation product. For red blood cells in vitro, the predicted non‐enzymatic rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3‐phosphate may account for the metabolic flux through the glyoxalase system. The reactivity of glycerone phosphate and glyceraldehyde 3‐phosphate towards the non‐enzymatic formation of methylglyoxal under physiological conditions suggests that methylglyoxal formation is unavoidable from the Embden‐Meyerhof pathway.
Identification of a nuclear protein that constitutively recognizes the sequence containing a heat‐shock element
Authors: OKINAGA Shoji , SHBAHARA Shigeki
Published: March 2005

Heme oxygenase is an essential enzyme in heme catabolism, and also known as a 32‐kDa heat‐shock protein in rat. The rat heme‐oxygenase gene promoter contains a functional heat‐shock element (HSE) designated as HSE1 (−290 to −276 from the transcriptional initiation site), which consists of three copies of a 5‐bp unit (5′‐NGAAN‐3′; →) in alternating orientation. Here we identified a putative HSE (−221 to −212), designated as HSE2, consisting of an inverted repeat of this 5‐bp unit (←→). Using transient expression assays, we show that HSE1 is sufficient to confer the heat‐inducibility (a three fold to fourfold increase) on the reporter gene located downstream from the rat heme‐oxygenase gene promoter, but HSE2 alone is not, suggesting that HSE2, a HSE of a tail‐to‐tail configuration, is not functional in vivo. However, the presence of both HSE1 and HSE2 in the promoter region increased the heat‐mediated induction of the reporter‐gene expression by more than 15‐fold, Gel mobility‐shift assays indicate that both HSE1 and HSE2 are recognized by activated heat‐shock factor present only in heat‐shocked rat glioma cells. Interestingly, the sequence containing HSE2 is also bound by a protein that is present in nuclear extracts prepared from either heat‐shocked or non‐shocked glioma cells, but this nuclear protein is nuable to bind to HSE1. We suggest that a protein binding to the sequence containing HSE2 may be involved in transcriptional regulation of the rat heme oxygenase gene under thermal stress.
Cloning of the fourth functional gene for Protein Phosphatase 1 in Drosophila melanogaster from its chromosomal location
Authors: DOMBRÁDI Viktor , MANN David J. , SAUNDERS Robert D. C. , COHEN Patricia T. W.
Published: March 2005

Complementary DNA encoding a catalytic subunit of protein phosphatase 1, PP1 87B, hybridises at four positions (87B, 9C, 13C and 96A) to Drosophila melanogaster polytene chromosomes, three of which are known to be expressed [Dombrádi, V., Axton, J. M., Brewis, N. D., Da Cruz e Silva, E. F., Alphey, L. & Cohen, P. T. W. (1990) Eur. J. Biochem. 194, 739–745]. The fourth gene has been isolated by screening a genomic library of cosmid clones, representing division 13 of the X‐chromosome of D. melanogaster, with a PP1 87B probe. This library was constructed as part of the Drosophila genome mapping project [Sidén‐Kiamos, I., Saunders, R. D. C., Spanos, L., Majerus, T., Trenear, J., Savakis, C., Louis, C., Glover, D. M., Ashburner, M. & Kafatos, F. C. (1990) Nucleic Acids Res. 18, 6261–6270]. The 5′ non‐coding region of the isolated gene hybridised to cytological position 13C1–2. By combining reverse transcription and the polymerase chain reaction, the gene was shown to be expressed at a very low level.

The PP1 13C gene encodes a protein of 302 amino acids with a predicted molecular mass of 34.5 kDa. It shows 85–94% amino acid identity to the other three protein phosphatase 1 catalytic subunits (PP1 87B, PP1 96A and PP1 9C) described previously, being most closely related to the isoform PP1 87B, which is involved in the control of chromosome separation at cell division and the regulation of chromosome condensation at interphase.
Reduction potentials and their pH dependence in site‐directed‐mutant forms of azurin from Pseudomonas aeruginosa
Authors: PASCHER Torbjörn , KARLSSON B. Göran , NORDLING Margareta , MALMSTRÖM Bo G. , VÄNNGÅRD Tore
Published: March 2005

A spectroelectrochemical method has been used to determine the reduction potential of the copper site in wild‐type and 22 mutant forms of azurin from Pseudomonas aeruginosa at 25 °C and in the range pH 4–8; the effect of buffers and ionic strength on the potentials has also been studied. Amino‐acid residues changed include Met121, which provides an S atom at a distance of about 0.3 nm from the metal, some amino acids in the hydrophobic patch, other residues believed to be important in electron transfer with physiological partners and some internal amino acids. The observed potentials span a range of about 300 mV. In all cases the potentials increase with decreasing pH, but the pKa values describing the pH dependence are essentially unchanged except in three mutants, where they change by pH 0.6–1.1 (up in one and down in two). The largest potential changes were found in some Met121 mutants, at which position large hydrophobic residues raise the potential, whereas negatively charged residues lower it; a decreased potential is also found in the Met121 → End mutant, which probably has H2O coordinated to the metal. Gly45 has its carbonyl group coordinated to copper, but the potential of Gly45 → Ala is close to that of the wild type. Some substitutions in the hydrophobic patch cause an increase in the potential, whereas substitutions involving His35 and Glu91 do not result in significant changes. No single mechanism for tuning the potential of the copper site can be discerned, but in many cases there are probably indirect effects of the protein conformation causing changes in metal‐ligand interactions.
Disruption of TPS2, the gene encoding the 100‐kDa subunit of the trehalose‐6‐phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose‐6‐phosphate and loss of trehalose‐6‐phosphate phosphatase activity
Authors: DE VIRGILIO Claudio , BÜRCKERT Niels , BELL Walter , JENÖ Paul , BOLLER Thomas , WIEMKEN Andres
Published: March 2005

Preparations of the trehalose‐6‐phosphate synthase/phosphatase complex from Saccharomyces cerevisiae contain three polypeptides with molecular masses 56, 100 and 130 kDa, respectively. Recently, we have cloned the gene for the 56‐kDa subunit of this complex (TPS1) and found it to be identical with CIFI, a gene essential for growth on glucose and for the activity of trehalose‐6‐phosphate synthase. Peptide sequencing of the 100‐kDa subunit of the trehalose‐6‐phosphate synthase/phosphatase complex (TPS2) revealed one sequence to be 100% identical with the deduced amino acid sequence of the upstream region of PPH3 on the right arm of chromosome IV. This sequence was used to clone an upstream region of PPH3 containing an open reading frame of 2685 nucleotides, predicted to encode a polypeptide of 102.8 kDa. The N‐terminal sequence, as well as three internal amino acid sequences, obtained from peptide sequencing of the 100‐kDa subunit, were identical with specific regions of the deduced amino acid sequence. Thus, the sequence cloned represents TPS2, the gene encoding the 100‐kDa subunit of the trehalose‐6‐phosphate synthase/phosphatase complex. Interestingly, a stretch of about 500 amino acids from the first part of TPS2 was 33% identical with the entire TPS1 sequence. Disruption of TPS2 had no effect on trehalose‐6‐phosphate synthase activity but caused complete loss of trehalose‐6‐phosphate phosphatase activity, measured in vitro, and accumulation of excessive amounts of trehalose‐6‐phosphate instead of trehalose upon heat shock or entrance into stationary phase in vivo. These results suggest that TPS2 codes for the structural gene of the trehalose‐6‐phosphate phosphatase. Heat shock induced an increase in trehalose‐6‐phosphate phosphatase activity and this was preceded by an accumulation in TPS2 mRNA, suggesting that the trehalose‐6‐phosphate phosphatase is subjected to transcriptional control under heat‐shock conditions.
Four recombinant isoforms of Cor a I, the major allergen of hazel pollen, show different IgE‐binding properties
Authors: BREITENEDER Heimo , FERREIRA Fátima , HOFFMANN‐SOMMERGRUBER Karin , EBNER Christof , BREITENBACH Michael , RUMPOLD Helmut , KRAFT Dietrich , SCHEINER Otto
Published: March 2005

Previous studies showed that pollens from trees of the order Fagales (e. g. birch, alder, hazel and hornbeam) all contain one major allergen. These proteins are cross‐reactive between these tree species, and approximately 95% of tree‐pollen‐allergic patients display IgE binding to these allergens. Using the reported N‐terminal amino acid sequence of the hazel pollen allergen Cor a I, it was possible to amplify Cor‐a‐I cDNA by use of the polymerase chain reaction. Four clones with cDNA inserts were isolated. All four clones contained an open reading frame of 477 nucleotides (159 amino acids) but differed in length of their 3′‐non‐coding regions. Within the overlapping regions, the nucleotide sequence of the 3′‐non‐coding regions of the four clones were nearly identical. The open reading frames coded for different isoforms of the major hazel pollen allergen, Cor a I. The clones were designated Cor a I/5, 6, 11 and 16, respectively. Comparison of the deduced amino acid sequences of these Cor a I isoforms revealed identities of 96–99%. The sequence identities between the Cor a I isoforms and Bet v I, the major birch pollen allergen, were 71–73% (80.5–83% similarity). Comparing amino acid sequences of Cor a I isoforms with the published sequences of Aln g I, the major allergen from alder, and Cor a I and isoforms, the major allergen from hornbeam, 75.5–76.7% identity (83.6–85% similarity) and 83.6–89.9% sequence identity (89.3–95% similarity), respectively, was found. The four Cor a I cDNAs were subcloned into plasmid pKK223‐3 and expressed in Escherichia coli as non‐fusion proteins; their capacity to bind serum IgE from tree‐pollen‐allergic patients was investigated. The four cloned isoforms showed an apparent molecular mass of 17 kDa in SDS/PAGE, identical to the natural, pollen‐derived Cor a I. IgE antibodies from tree‐pollen‐allergic patients was reacted with all four recombinant isoforms. However, we noted marked differences in the IgE‐binding patterns of the distinct isoforms. Furthermore, Cor a I/11 was the only isoform recognized by the anti‐(Bet v I) mAb, BIP 1. Our results demonstrate that Cor a I isoforms display different antigenic properties, very likely due to few but significant changes in their amino acid sequences. These findings have implications for the development of reagents for diagnosis and immunotherapy of type I allergies.
Purification and phosphorylation of elongation factor‐2 kinase from rabbit reticulocytes
Authors: REDPATH Nicholas T. , PROUD Christopher G.
Published: March 2005

Eukaryotic elongation‐factor‐2 kinase has been purified to homogeneity from rabbit reticulocytes through a seven‐step procedure and has been identified as a protein with a molecular mass of approximately 103 kDa as judged by SDS/PAGE. A degradation product of about 95 kDa was also evident in some preparations. The activity of the purified kinase was completely dependent on calcium and calmodulin. The kinase rapidly underwent extensive autophosphorylation, incorporating 1 mol phosphate/mol within 1 min; 5 mol phosphate/mol were incorporated within 1 h. The autophosphorylation was Ca2+/calmodulin‐dependent; phosphopeptide mapping revealed multiple phosphopeptides even after just 0.5 min of autophosphorylation, suggesting that a number of sites became rapidly phosphorylated. Autophosphorylation occurred on serine and threonine residues. Preincubation in the presence of Ca2+, Mg2+ and ATP produced a rapid 2–3‐fold activation of the kinase and also induced partial Ca2+‐independent activity. Preincubation in the absence of the ligands showed that all three were required for full activation and induction of Ca2+‐independent activity.
The 0.25‐nm X‐ray structure of the Bowman‐Birk‐type inhibitor from mung bean in ternary complex with porcine trypsin
Authors: LIN Guangda , BODE Wolfram , HUBER Robert , CHI Chengwu , ENGH Richard A.
Published: March 2005

The structure of the Bowman‐Birk‐type inhibitor from mung bean Phaseolus aureus has been determined in ternary complex with porcine trypsin. The complex formed crystals of the trigonal space group P3121 which diffracted to a resolution of 250 pm. Each of the two mung bean protease reactive sites is bound to trypsin according to the standard mechanism for serine proteinase inhibition. The binding loops thereby adopt the canonical conformation for the standard mechanism; however, the sub‐van der Waals contact between the active‐site serine Oγ (195) and the P1 carbonyl carbon of both loops is significantly smaller (210 pm) than hitherto observed, with continuous electron density connecting the two atoms. The inhibitor is formed by two double‐stranded antiparalel β‐sheets, which are connected into a moderately twisted β‐sheet by a network of hydrogen bonds involving main‐chain atoms and two water molecules. All contacts with neighbors in the crystal lattice occur between trypsin molecules. This apparently gives rise to an unusual form of disorder where the complexes pack in two orientatiions Ta: MaMb: Tb and Tb: MbMa: Ta (Ta, Tb = trypsin, Ma = mung bean loop I, Mb = mung bean loop II), such that the asymmetric unit consists of the ternary complex in two orientations, each with half occupancy. This is nearly equivalent to an asymmetric unit which has one trypsin molecule with full occupancy and one mung bean inhibitor with half occupancy and a crystallographic twofold symmetry axis through its center. Because of the approximate twofold symmetry of the inhibitor itself, however, the electron density was interpretable for most of the inhibitor (17 residues at the termini were not resolved) and shows evidence of its double orientation.
Identification of insulin‐stimulated protein kinase‐1 as the rabbit equivalent of rskmo‐2
Authors: SUTHERLAND Calum , CAMPBELL David G. , COHEN Philip
Published: March 2005

An improved procedure has been developed for the isolation of insulin‐stimulated protein kinase‐1 (ISPK‐1), an S6 kinase‐II homologue, by which 0.5 mg highly purified enzyme can be obtained within four days. The sequences of tryptic peptides from ISPK‐1 (100 residues) revealed 100% identity with the predicted protein product of rskmo‐2, a cDNA clone isolated from a mouse F2 cell line library [Alcorta, D. A., Crews, C. M., Sweet, L. J., Bankston, L., Jones, S. W. and Erikson, R. L. (1989) Mol. Cell. Biol. 9, 3850–3859], demonstrating that rskmo‐2 encodes an S6 kinase‐II. Two isoforms of mitogen‐activated protein (MAP) kinase (p42mapk and p44mapk) were the only ISPK‐1‐reactivating enzymes detected after Mono Q chromatography of extracts prepared from rabbit skeletal muscle or phaeochromocytoma 12 cells stimulated by nerve or epidermal growth factors. One of the residues on ISPK‐1 phosphorylated by p42mapk was a threonine located nine residues N‐terminal to the conserved Ala‐Pro‐Glu motif in the C‐terminal protein kinase domain, an analogous location to phosphorylation sites essential for the activity of cAMP‐dependent protein kinase, MAP kinase and p34cdc2. A further threonine located five residues N‐terminal to the same Ala‐Pro‐Glu motif was also phosphorylated, probably via autophosphorylation catalysed by ISPK‐1 itself.
The NMR solution structure of a Kunitz‐type proteinase inhibitor from the sea anemone Stichodactyla helianthus
Authors: ANTUCH Walfrido , BERNDT Kurt D. , CHÁVEZ María A. , DELFÍN Julieta , WÜTHRICH Kurt
Published: March 2005

The solution structure of a 55‐amino‐acid Kunitz‐type proteinase inhibitor, ShPI, purified from the Caribbean sea anemone Stichodactyla helianthus, was determined by NMR spectroscopy. Nearly complete sequence‐specific 1H‐NMR assignments were obtained at pH 4.6 and 36°C, and stereospecific assignments were determined for 23 pairs of diastereotopic substituents. A data set of 666 upper distance limit constraints and 122 dihedral angle constraints collected on this basis was used as input for a structure calculation with the program DIANA. Following energy minimization with the program OPAL, the average root‐mean‐square diviation (RMSD) of the 20 DIANA conformers used to represent the solution structure relative to the mean structure is 61 pm for all backbone atoms N, Cα and C′, and 106 pm for all heavy atoms of residues 2–53. This high‐quality solution structure of ShPI has a nearly identical molecular architecture as the bovine pancreatic trypsin inhibitor (BPTI), despite a mere 35% of sequence similarity between the two proteins. Exchange rates measured for 48 out of the 51 backbone amide protons showed that the positions of 20 slowly exchanging amide protons correlate well with hydrogen bonds involving these protons in the energyminimized solution structure. The solution structure of ShPI is compared to the four homologous proteins for which the three‐dimensional structure is also available.
Possible intermediates in the biosynthesis of mycoside B by Mycobacterium microti
Authors: THURMAN Paul F. , CHAI Wengang , ROSANKIEWICZ Jerzy R. , ROGERS Henry J. , LAWSON Alexander M. , DRAPER Philip
Published: March 2005

Two lipids were isolated from Mycobacterium microti which became labelled when the cells were grown in the presence of [2‐14C]propionate. They were purified by thin‐layer chromatography and studied by chemical degradation and mass spectrometry. The lipids were identified as phenolphthiocerol dimycocerosate and phenolphthiodiolone dimycocerosate, the aglycosyl derivatives of mycoside B, the phenolic glycolipid produced by M. microti. Cell‐free extracts of the organism were able to glycosylate the lipids to form mycoside B in vitro. It is probable that the lipids are intermediates in the biosynthesis of phenolic glycolipids by mycobacteria.
Sequencing and characterization of the sdaB gene from Escherichia coli K‐12
Authors: SHAO ZhongQi , NEWMAN E. B.
Published: March 2005

The sdaB gene which codes for the second l‐serine deaminase (L‐SD) of Escherichia coli K‐12 has been sequenced and shown to be very similar to the sdaA gene which codes for the first l‐serine deaminase. sdaB is transcribed in rich medium, particularly in the absence of glucose, and is under the control of catabolite activator protein. A mutation which established expression of the sdaB gene and synthesis of l‐serine deaminase 2 in minimal medium has been demonstrated to result in a change in the ribosome‐binding site of the sdaB gene.
Isozyme developments in mammalian class‐I alcohol dehydrogenase
Authors: HÖÖG Jan‐Olov , VAGELOPOULOS Nikolaos , YIP Ping‐Kwai , KEUNG Wing Ming , JÖRNVALL Hans
Published: March 2005

Isozyme patterns differ widely among the classical type (class I) of mammalian alcohol dehydrogenases. For the rabbit enzyme, the possibility of isozymes has been reported but structural evidence is lacking. This system was now studied at both the mRNA/cDNA and protein levels. Ten cDNA clones, coding for class‐I alcohol dehydrogenase, were isolated from a rabbit liver cDNA library using a human DNA fragment as probe. The cDNA spanned 1296 bp, including the entire coding region. All clones coded for the same polypeptide and Northern blots identified a single mRNA corresponding to about 1.5 kb. Comparison of two protein forms (CC and BC) by HPLC peptide fingerprinting and structural analysis revealed peptide segments identical in amino acid sequence. Consequently, direct protein analyses and Northern blots show the presence of only one primary translation product. The data suggest that lagomorphic alcohol dehydrogenase, like the rodent enzyme, is not as isozyme rich as it may appear superficially, and that secondary modifications contribute substantially to mammalian alcohol dehydrogenase multiplicity. The active center of the rabbit enzyme suggests similarities to the horse S, human γ, and rat enzyme structures, compatible with a steroid dehydrogenase activity shown experimentally. Typical class‐I properties were established by direct analysis and confirmed by structural properties (Km for cyclohexanol 0.8–1.1 mM, for ethanol 1.6–1.9 mM). The isozyme versus species differences mark the variability of class‐I alcohol dehydrogenase versus class III and suggest a parallelism between rapid mutational differences and frequent duplicatory events.
Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK‐21 cells
Authors: NIMTZ Manfred , MARTIN Wolfgang , WRAY Victor , KLÖPPEL Klaus‐Dieter , AUGUSTIN Jan , CONRADT Harald S.
Published: March 2005

The native structures of the Asn‐linked oligosaccharides and the O‐glycans at Ser126 of human erythropoietin expressed from recombinant BHK cells have been elucidated. Enzymatically released N‐glycans were studied by methylation analyses, fast‐atom‐bombardment mass spectrometry as well as one‐ and two‐dimensional 1H‐NMR spectrometry at 600 MHz. Many (82.7%) were found to be tetraantennary N‐acetyllactosamine‐type (22.8% with one, 3.6% with two and 0.4% with three N‐acetyllactosamine repeats) being tetrasialylated (41%), trisialylated (29.6%) and disialylated (12.2%). A few (9.7%; 4.1% 2,4‐branched, 5.6% 2,6‐branched) of the chains were triantennary (5.4% trisialyl, 4.3% disialyl) and 4.6% were of the disialyl diantennary type. Almost all of the innermost GlcNAc residues were α1–6 fucosylated and NeuAc was exclusively α2–3 linked to Galβ1–4GlcNAc‐R; 60% of the protein was found to be O‐glycosylated at Ser126; structures were monosialylated (70%) or disialylated (30%) forms of the Galβ1–3GalNAc core type. Glycosylation patterns at individual Asn‐Xaa‐Thr/Ser sites were determined by analytical high‐pH anion‐exchange chromatography with pulsed amperometric detection. Only tetraantennary chains with 0–3 N‐acetyllactosamine repeats were detected at Asn38 and Asn83, while almost all of the di‐ and triantennary oligosaccharides were attached to Asn24. Batch analysis of different preparations of recombinant erythropoietin revealed the high reproducibility of the production procedure. Structures containing terminal GalNAc–GlcNAc were detected in small amounts in a few batches.
Phosphorylation and activation of the arachidonate‐mobilizing phospholipase A2 in macrophages in response to bacteria
Authors: SVENSSON Ulf , HOUWELING Martin , HOLST Elisabet , SUNDLER Roger
Published: March 2005

The role of potential target enzymes in the protein‐kinase‐C‐independent eicosanoid response triggered by certain bacteria in murine peritoneal macrophages [Svensson, U., Holst, E. & Sundler, R. (1991) Eur. J. Biochem. 202, 699–705] has been investigated. The eicosanoid response was found to be due to an increase in the mobilization of arachidonate rather than to inhibition of arachidonate esterification or activation of the cyclooxygenase pathway and to be accompanied by a persistent increase in the activity of the arachidonate‐mobilizing phospholipase A2 (PLA2−85). Also, down‐regulation of protein‐kinase C by prolonged treatment with 4β‐phorbol 12‐myristate 13‐acetate did not reduce the bacterial activation of PLA2‐85. The increase in activity of PLA2−85, like the increase in eicosanoid formation, showed a lag period of approximately 10 min.

Furthermore, exposure of 32P‐labeled macrophages to either bacteria (Gardnerella vaginalis) or the protein‐phosphatase inhibitor okadaic acid caused an increase in the phosphorylation of PLA2−85. Okadaic acid (0.5 μM), which itself caused arachidonate mobilization and activation of PLA2−85 after a lag period of approximately 45 min, greatly promoted the response to bacteria even at earlier time points. This study provides strong evidence that the eicosanoid response to bacteria in macrophages occurs via a protein‐kinase‐C‐independent activation of PLA2−85 and that this activation is due to an increase in the phosphorylation of the enzyme.
Intron‐exon structure, alternative use of promoter and expression of the mouse collagen X gene,Col10a‐1
Authors: KONG Richard Y. C. , KWAN K. M. , LAU Elizabeth T. , THOMAS J. Terrig , BOOT‐HANDFORD Raymond P. , GRANT Michael E. , CHEAH Kathryn S. E.
Published: March 2005

The entire mouse collagen X gene (Col10a‐1) has been isolated. The gene is composed of three exons and two introns spanning 7.0 kb of the DNA sequence. Exons 2 and 3 together encode 15‐bp of 5′ untranslated sequence, a 2040‐bp open readin frame and an 895‐nucleotide 3′ non coding region. In the 5′ flanking region of the gene, two consensus TATA‐box sequences were found. Identification of the first exon by ribonuclease‐protection assays and the determination of the 5′ end ofCol10a‐1mRNA transcripts by primer‐extension analyses show that the more 3′ TATA box is probably predominantly used and that there are at least three transcription start sites in the exon 1 sequence 3′ to this, resulting in 5′ untranslated regions of 78, 77 and 55 nucleotides. By means of rapid amplification of cDNA ends by polymerase chain reaction, an additional mRNA species was detected which overlapped the otherCol10a‐1transcripts, including the 3′ TATA box sequence, giving a 5′ untranslated sequence of approximately 235 bases. This latter transcript starts approximately 20 bp 3′ to the more 5′ TATA box. The data suggest alternative use of promoters and transcription starts for theCol10a‐1gene. Comparison of the combined nucleotide and deduced amino acid sequences of exons 2 and 3 with chicken, bovine and human collagen X genes, showed a high degree of similarity indicating conservation of this gene throughout evolution. MouseCol10a‐1mRNA was shown to be approximately 3.0 kb and the pepsinized protein, as detected by SDS/PAGE, was ∼ 45 kDa. The mRNA and protein sizes correlate with that predicted by the open reading frame. Reverse‐transcription polymerase chain reaction assays indicate that the mouse collagen X gene is first expressed at 13.5 dayspost coitum, temporally preceding the onset of endochondral ossification. In agreement with the generally accepted association of type‐X collagen with endochondral ossification,in situhybridization analyses indicate thatCol10a‐1mRNA are restricted to the hypertrophic regions of growth cartilage.
8‐Dimethylamino‐1‐methyladenine, a novel potent antagonist of the 1‐methyladenine receptor in starfish oocytes
Authors: MONSEES Thomas , MEIJER Laurent , JASTORFF Bernd
Published: March 2005

Starfish oocytes are naturally arrested in the late G2 phase of the first meiotic division. The follicle‐cell‐derived hormone, 1‐methyladenine, is responsible for highly synchroneous induction of oocyte maturation. A series of systematically modified 1‐methyladenine analogues was used to map the essential molecular interactions between 1‐methyladenine and its stereospecific receptors, which are localized on the oocyte plasma membrane. The earlier hypothesis about structure/activity relationships has been confirmed. Quantum‐chemical calculations indicated additional dipole–dipole interactions and presumably a charge‐transfer interaction, with the nucleobase as π‐electron donor. Among the 49 compounds tested, a series of novel inhibitors of 1‐methyladenine‐induced maturation was found. With the synthesis of 8‐dimethylamino‐1‐methyladenine (concentration for 50% inhibition 1 μM), a very potent antagonist of the natural hormone was obtained which may become an important tool for investigating the mechanism of 1‐methyladenine‐induced maturation of the starfish oocytes, an important model for cell‐cycle‐control studies. The results are discussed in the context of preexisting biological data. In conclusion, we propose a modified model of the molecular interactions between 1‐methyladenine and its receptor.
A Candida albicans homolog of CDC25 is functional in Saccharomyces cerevisiae
Authors: GOLDBERG Doron , MARBACH Irit , GROSS Eitan , LEVITZKI Alexander , SIMCHEN Giora
Published: March 2005

We have cloned, by functional complementation of the cdc25‐2, mutation of Saccharomyces cerevisiae, a homolog of CDC25 from the pathogenic yeast Candida albicans. The new gene, named CSC25, codes for a 1333‐amino‐acid protein. The full length gene, as well as a truncated form coding for 795 amino acids, suppresses the thermosensitive phenotype of cdc25ts mutants. Biochemical analysis has shown that Csc25 activates the Ras/adenylyl cyclase pathway in S. cerevisiae at a rate two to three times faster than Cdc25, under the same conditions. The C‐terminal domain of Csc25 is highly similar to the C‐terminal domain of Cdc25, to almost the same extent as the C‐terminus of the endogenous Cdc25 homolog Sdc25. We show that polyclonal anti‐Cdc25 antibodies interact with Csc25 expressed in S. cerevisiae. In addition to the full length protein (∼ 150 kDa), we have found a ∼ 50‐kDa polypeptide which seems to include the C‐terminus of the CSC25 gene product.
Purification and characterization of cytochrome P450RR1 from Rhodococcus rhodochrous
Authors: ELTIS Lindsay D. , KARLSON Ulrich , TIMMIS Kenneth N.
Published: March 2005

A soluble cytochrome P450 whose synthesis is induced by and that binds 2‐ethoxyphenol was purified to apparent homogeneity from Rhodococcus rhodochrous strain 116. The enzyme had a subunit molecular mass of 44.5 kDa as determined by SDS/PAGE and a pI of 5.2. The electronic absorption spectrum indicates that the native cytochrome in the absence of substrate is predominantly in the low‐spin state (13% high‐spin state in 50 mM Mops, pH 7.0, 25 °C). 2‐Methoxyphenol binds to the cytochrome with a macroscopic dissociation constant of 0.53 +/– 0.03 μM (50 mM Mops, pH 7.0, 25 °C) and induces a 99.7% transition of the heme iron to the pentacoordinate high‐spin form. Using a reconstituted in‐vitro activity assay, it was demonstrated that P450RR1 catalyzed the O‐dealkylation of 2‐ethoxyphenol and 2‐methoxyphenol to produce catechol. The cytochrome binds other ortho‐substituted phenols, including 2‐ethoxyphenol, 2‐methylphenol (o‐cresol) and 2‐chlorophenol. The affinity of P450RR1 for these compounds is lower than that of 2‐methoxyphenol and they are less effective than 2‐methoxyphenol at inducing a transition in the heme iron to the high‐spin state. Para‐substituted and meta‐substituted ether phenols did not induce a spin transition.
Role of acidic lipids in the translocation and channel activity of colicins A and N in Escherichia coli cells
Authors: VAN DER GOOT F. Gisou , DIDAT Nathalie , PATTUS Franc , DOWHAN William , LETELLIER Lucienne
Published: March 2005

Colicins A and N are pore‐forming bacterial toxins that kill Escherichia coli cells. Their mode of action involves three steps; binding to specific receptors located in the outer membrane, translocation through this membrane and the periplasm, and channel formation in the inner membrane. In‐vitro studies have shown that negatively charged phospholipids are an absolute requirement for the channel formation of colicin A. Using HDL11 strain, in which the phosphatidylglycerol (PtdGro) content was altered by varying the synthesis of the PtdGro‐phosphate synthase, the effect of envelope PtdGro content on the activity of colicin A was studied in vivo. The formation by colicin A of a voltage‐gated channel in the cytoplasmic membrane results in an efflux of cytoplasmic potassium. This efflux is preceded by a lag time which is related to the time needed by the toxin to cross the cell envelope. This lag time is higher when the cells have a reduced PtdGro level, suggesting that the receptor/translocation machinery of colicin A (OmpF, BtuB and Tol QRAB proteins) is altered in the absence of PtdGro. The rate of potassium efflux is also greatly reduced when the PtdGro content is decreased, suggesting that a certain level of PtdGro is indeed required for proper insertion of the colicin‐A channel. In contrast, the activity of colicin N does not show any PtdGro dependence. The difference between the behavior of colicin A and that of colicin N is discussed.
Stimulatory and inhibitory guanine‐nucleotide‐binding regulatory protein involvement in stimulation of arachidonic‐acid release by N‐formyl‐methionyl‐leucyl‐phenylalanine and platelet‐activating factor from guinea‐pig alveolar macrophages
Authors: LEVISTRE Romuald , MASLIAH Joëlle , BEREZIAT Gilbert
Published: March 2005

The involvement of guanine‐nucleotide‐binding regulatory proteins (G proteins) in the regulation of arachidonic‐acid release induced by N‐formyl‐methionyl‐leucyl‐phenylalanine (fMet‐Leu‐Phe) or platelet‐activating factor (PAF) was examined in guinea‐pig alveolar macrophages. We report that maximal release of arachidonic acid in permeabilized cells requires the simultaneous addition of the agonist (fMet‐Leu‐Phe or PAF) and of GTP (or GTP[S]). Prior treatment of cells with increasing concentrations of pertussis toxin induces a parallel decrease of arachidonic‐acid release and of the labeling of a 40‐kDa protein in membranes incubated with [32P]NAD and pertussis toxin. fMet‐Leu‐Phe, but not PAF, allows the ADP‐ribosylation of a 40‐KDa protein by cholera toxin in the presence of Mg2+. This effect is prevented by guanyl nucleotides and by prior treatment with pertussis toxin. The 40‐kDa protein ADP‐ribosylated seems to be αi1 and/or αi2. Stimulation of GTPase activity by fMet‐Leu‐Phe and PAF has the same amplitude and is completely inhibited by pertussis toxin, but only in part by cholera toxin. Prior treatment of alveolar macrophages with cholera toxin, which ADP‐ribosylates Gs, inhibits PAF‐stimulated and fMet‐Leu‐Phe‐stimulated arachidonic‐acid release to the same extent, via a cAMP‐protein‐kinase‐A cascade. The decreased responsiveness of alveolar macrophages previously treated with cholera toxin to fMet‐Leu‐Phe and PAF is associated with a strong increase of in‐vitro [32P]NAD labeling of Gi proteins either by pertussis or by cholera toxin. This effect is mimicked by prior treatment of the cells with dibutyryl cAMP and okadaic acid, a protein‐phosphatase inhibitor, suggesting the involvement of protein‐kinase A in this process.

In conclusion, our results demonstrate that fMet‐Leu‐Phe and PAF receptors interact differently with Gi1/2 proteins in guinea‐pig alveolar macrophages. Gi1/2 proteins are a possible target of the cross‐regulation of arachidonic‐acid release by a Gs‐mediated pathway.
1H‐NMR spectroscopy of bovine lens β‐crystallin
Authors: COOPER Philip G. , CARVER John A. , TRUSCOTT Roger J. W.
Published: March 2005

1H‐NMR spectroscopic studies of bovine eye lens β‐crystallin aggregates (dimer, trimer and octomer) are presented. The NMR spectra for all three β‐crystallin aggregates are dominated by resonances from the βB2 subunit, particularly from the N‐ and C‐terminal extensions of this subunit. Resonances from other β subunits, which all have terminal extensions, are, in general, absent from spectra of the β‐crystallin aggregates. Therefore, the βB2 subunit and, in particular its terminal extensions, has enhanced flexibility compared to the other β‐crystallin subunits. Furthermore, resonances arising from the C‐terminal extension of βB2‐crystallin are not present in the spectrum of the octomer, which is consistent with the C‐terminal extension binding in this aggregate and hence being involved in large aggregate formation. A possible interaction between the C‐terminal extension of βB2 and the hydrophobic βB1 subunit, which is only found in the octomer, is discussed. At higher temperatures (45°C) in the octomer, partial exposure of the C‐terminal extension of βB2 occurs indicating that the octomer may be starting to break up into smaller aggregates.
Induction of protein phosphorylation, protein synthesis, immediate‐early‐gene expression and cellular proliferation by intracellular pH modulation
Authors: ISFORT Robert J. , CODY David B. , ASQUITH Thomas N. , RIDDER Gregg M. , STUARD Sharon B. , LEBOEUF Robert A.
Published: March 2005

In Syrian hamster embryo cells, intracellular acidification (but not alkalization) results in proliferation, immediate‐early‐gene expression and tyrosine phosphorylation. In addition, both intracellular acidification and alkalization result in serine/threonine phosphorylation and de novo protein synthesis of specific proteins. Calcium is not mobilized in response to either intracellular alkalization or acidification. Neither intracellular acidification nor alkalization altered the serum proliferative signal while intracellular alkalization (but not acidification) reduced the epidermal‐growth‐factor‐induced proliferative signal, tyrosine phosphorylation and immediate‐early‐gene expression. Finally, intracellular acidification (but not alkalization) could induce immediate‐early‐gene expression in cells growing in the presence of serum, indicating that the pH signalling pathway is not down modulated by the serum signalling pathway. These results, while indirect, indicate that hydrogen ions may play an important role in mitogen‐signal transduction in Syrian hamster embryo cells.
Molecular cloning of a cDNA showing alternative splicing of the 5′‐untranslated sequence of mRNA for human aromatase P‐450
Authors: TODA Katsumi , SHIZUTA Yutaka
Published: March 2005

A new type of full‐length cDNA clone encoding human aromatase P‐450 was isolated from a human placental cDNA library. The clone, designated as pES‐4, has a 3130‐bp insert. The nucleotide sequences of the translated region and the 3′‐untranslated region of the insert of pES‐4 are exactly identical with those of the cDNA clone characterized previously. However, the sequence of the 5′‐untranslated region of the insert has characteristic feature, i.e. an extra sequence of 109 bp is present at a junction between exon 1 and exon 2 on the processed human aromatase mRNA. Analysis of the genomic clones containing the region between exon 1 and exon 2 of the human aromatase P‐450 gene reveals that the 109‐bp genomic segment, encoding the same sequence as the extra sequence observed in pES‐4, is located approximately 10‐kbp downstream of exon 1 and that the nucleotide sequences of the 5′‐flanking and the 3′‐flanking regions of the segment conform to the GT‐AG rule for RNA splicing. By means of reverse transcription and polymerase chain reaction, relative amounts of the pES‐4‐type mRNA are estimated to be approximately 4.8% and 2.3% of the processed aromatase P‐450 mRNA in human placenta and human BeWo choriocarcinoma cells, respectively. These results indicate that the segment of 109 bp between exon 1 and exon 2 is a new exon hitherto unidentified and that heterogeneity observed in the 5′‐untranslated sequence of human aromatase P‐450 mRNA is, at least in part, caused by alternative splicing of this new exon.
Two‐dimensional NMR assignment of hyperfine‐shifted resonances of very fast relaxing metal binding sites of proteins by NOE spectroscopy
Authors: SETTE Marco , PACI Maurizio , DESIDERI Alessandro , ROTILIO Giuseppe
Published: March 2005

The isotropically shifted NMR resonances of bovine Cu, Co superoxide dismutase, arising from the imidazole protons of the metal binding site of the native Cu, Zn enzyme when Zn(II) is replaced by Co(II), were investigated by two‐dimensional NMR. NOE spectra have been measured at different mixing times in order to obtain a good set of interproton dipolar connectivities. A number of NOEs were obtained for very fast relaxing resonances close to the paramagnetic copper ion and new isotropically shifted resonances were detected in the proximity of the diamagnetic region. The recently obtained original X‐ray coordinates of the Cu, Co enzyme were used in order to give the appropriate values to the interproton distances of the imidazole rings of the copper‐coordinated histidines. The data allowed an unequivocal and definitive assignment of the isotropically shifted resonances to be done, with respect to previous results obtained using the coordinates of the Cu, Zn enzyme and monodimensional NMR techniques, which contain a potential source of artifacts in the selective excitation required prior to acquisition of spectra.
Structural determnation of the capsular polysaccharide produced by Klebsiella pneumoniae serotype K40
Authors: CESCUTTI Paola , TOFFANIN Renato , KVAM Bjarne J. , PAOLETTI Sergio , DUTTON Guy G. S.
Published: March 2005

The Klebsiella pneumoniae K40 capsular polysaccharide has been isolated and investigated by use of methylation analysis, specific degrations and NMR spectroscopy. The polysaccharide was depolymerised by a bacteriophage‐associated endogalactosidase, and the resulting oligosaccharide was characterised by one‐dimensional and two‐dimensional NMR spectroscopy and direct chemical ionisation MS. The repeating unit of the K40 capsular polysaccharide was shown to be a linear hexasaccharide with the composition

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(Rha, rhamnose).