bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell‐free synthesis of rat and human catechol O‐methyltransferase
Authors: ULMANEN Ismo , LUNDSTRÖM Kenneth
Published: March 2005

The protein‐coding capacities of rat and human catechol O‐methyltransferase (COMT) DNA clones were analysed by in vitro transcription and translation using bacteriophage RNA polymerase and rabbit reticulocyte lysate. Two types of clones corresponding to the structures of human placental cDNA clones were used. The shorter clones, containing the 663‐residue open reading frame for the soluble COMT (S‐COMT), produced 24‐kDa (rat) and 26‐kDa (human) polypeptides. Translation of the longer clones, containing 43 (rat) or 50 (human) amino acid amino‐terminal extensions to the S‐COMT polypeptides, yielded 28‐kDa (rat) and 30‐kDa (human) putative membrane‐bound COMT (MB‐COMT) polypeptides as the main products. These clones also yielded low amounts of the S‐COMT polypeptides. Labelling time or ionic conditions during translation did not eliminate the shorter products, suggesting translation initiation from the second S‐COMT AUG codon. In accordance with this postulation, the relative amount of S‐COMT could be affeted by changing the translation initiation contexts preceding the first AUG codon. The 28‐kDa and 30‐kDa products, but not the 24‐kDa and 26‐kDa products, associated with microsomal membranes cotranslationally, indicating that the amino‐terminal extensions were functional signal sequences. However, the presence of membranes did not affect the mobilities of the proteins in SDS/polyacrylamide gels. The MB‐COMT polypeptides could not be released from the microsomes by treatments with phospholipase C or alkali and were not protected by the microsomes against proteinase K digestion. These results indicate that MB‐COMT synthesized in vitro is an integral membrane protein having an aminoterminal signal‐anchor sequence.
Two different genes encode fibronectin binding proteins in Staphylococcus aureus
Authors: JÖNSSON Klas , SIGNÄS Christer , MÜLLER Hans‐Peter , LINDBERG Martin
Published: March 2005

A gene encoding a fibronectin binding protein (FnBP) has recently been isolated and sequenced from Staphylococcus aureus strain 8325–4. In the same bacterial strain, 682 bp downstream to the stop codon of this gene (fnbA), a second gene termed fnbB has now been discovered, encoding another FnBP (FnBPB). The two genes show in large parts striking sequence homologies. The complete amino acid sequence encoded by fnbB has been deduced and compared to that deduced from fnbA. In FnBPB a stretch of 66 amino acids downstream to the signal peptide has 75% identity with the corresponding region in FnBPA. At the C‐terminal site another 394 amino acid stretch is almost identical in both gene products. This stretch contains the 38 amino acid long D repeats, the wall spanning Wr repeats and the hydrophobic membrane spanning domain. In FnBPA each of the three D repeats has been identified as a fibronectin binding structure. These structures are highly conserved in FnBPB and most likely represent the major Fn‐binding domain of this protein. However, a subclone of gene fnbB lacking the coding region for the D repeats also clearly expresses fibronectin binding activity. This additional binding site is so far unique for FnBPB and interacts like the D domains with the N‐terminal 24–31‐kDa fragment of fibronectin. The purified recombinant FnBP fragment (not containing the D repeats) completely inhibits the binding of fibronectin to whole cells of S. aureus.
Oxidation‐reduction potentials of ferredoxin‐NADP+ reductase and flavodoxin from Anabaena PCC 7119 and their electrostatic and covalent complexes
Authors: PUEYO José J. , GOMEZ‐MORENO Carlos , MAYHEW Stephen G.
Published: March 2005

The oxidation‐reduction potentials of ferredoxin‐NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone couple (E2) and the flavodoxin semiquinone/hydroquinone couple (E1) were – 212 mV and –436 mV, respectively. E1 was independent of pH above about pH 7, but changed by approximately –60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox‐linked pKa at about 6.1, similar to those of certain other flavodoxins. E2 varied by – 50 mV/pH in the range pH 5–8. The redox potential for the two‐electron reduction of ferredoxin‐NADP+ reductase was –344 mV at pH 7 (ΔEm=– 30 mV/pH).

In the 1:1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by + 8 mV and E1 was shifted by – 25 mV; the shift in potential for the reductase was + 4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by – 21 mV and E1 shifted by + 20 mV; the reductase potential shifted by + 2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin‐NADP+ reductase from Anabena variabilis was complexed with flavodoxin from Azotobacter vinelandii.

The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.
Inhibitory activity and conformational transition of α1‐proteinase inhibitor variants
Authors: SCHULZE Andreas J. , HUBER Robert , DEGRYSE Eric , SPECK Denis , BISCHOFF Rainer
Published: March 2005

Several variants of α1‐proteinase inhibitor (α1‐PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in α1‐PI containing an Arg residue in position 358 (yielding [Thr345 Arg, Met358 Arg]α1‐PI) results in complete loss of its inhibitory activity against human α‐thrombin; whereas an exchange of residue Met351 (P8) by Glu ([Met351 Glu, Met358 Arg]α1‐PI) does not alter activity. [Thr345 Arg, Met358 Arg]α1‐PI is rapidly cleaved by thrombin, while [Met358 Arg]α1‐PI and [Met351 Glu, Met358 Arg]α1‐PI form stable proteinase‐inhibitor complexes. The stability of [Thr345 Arg, Met358 Arg]α1‐PI against guanidinium chloride denaturation is significantly enhanced compared to wild‐type α1‐PI, and does not change after cleavage, resembling ovalbumin, a serpin with no inhibitory activity, from which the Thr345 Arg amino acid exchange had been derived. [Met351 Glu, Met358 Arg]α1‐PI and [Met358 Arg]α1‐PI resemble the wild‐type protein in this respect. The CD spectra of intact and cleaved α1‐PI variants do not compare well with the wild‐type protein, probably reflecting local structural differences. Insertion of a synthetic peptide, which corresponds to residues Thr345 Met358 of human α1‐PI, leads to the formation of binary complexes with all variants having the characteristic features of the binary complex between peptide and wild‐type protein.
Over‐production, purification and properties of the uridine diphosphate N‐acetylmuramoyl‐l‐alanine: d‐glutamate ligase from Escherichia coli
Authors: PRATVIEL‐SOSA Flore , MENGIN‐LECREULX Dominique , van HEIJENOORT Jean
Published: March 2005

The UDP‐N‐acetylmuramoyl‐l‐alanine: d‐glutamate ligase of Escherichia coli was over‐produced in strains that harbour recombinant plasmids bearing the mur D gene under the control of the lac or PR promoter. Purification to homogeneity was achieved by a two‐step procedure from a 181‐fold over‐producing strain. The N‐terminal sequence of the purified protein was determined and correlated with the nucleotide sequence of the murD gene. The purified activity was highly dependent on the concentration of potassium phosphate and Mg2+. The enzyme also catalysed the reverse reaction. The Km values for UDP‐N‐acetylmuramoyl‐l‐alanine; d‐glutamate and ATP/Mg2+ were estimated at 7.5, 55 and 138 μM, respectively. Under the most optimal in vitro conditions determined, a turnover number of 931 min‐1 was estimated. When considering the plasmid‐free parental strain, the copy number of the murD gene product was not more than 1000 · cell−1.
Properties to two high‐molecular‐mass forms of glyceraldehyde‐3‐phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity
Authors: CLASPER Steven , EASTERBY John S. , POWLS Roy
Published: March 2005

Two High‐Mr forms of chloroplast glyceraldehyde‐3‐phosphate dehydrogenase from spinach leaf can be separated by DEAE‐cellulose chromatography. One form, the high‐Mr glyceraldehyde‐3‐phosphate dehydrogenase, resembles an enzyme previously described [Yonuschot, G. R., Ortwerth, B. J. & Koeppe, O. J. (1970) J. Biol. Chem. 245 4193–4198]. The other, a glyceraldehyde‐3‐phosphate dehydrogenase/phosphoribulokinase complex, is characterised by possession of latent phosphoribulokinase activity, only expressed following incubation with dithiothreitol.

This complex is composed not only of subunits A (39.5 kDa) and B (41.5 kDa) characteristic of the high‐Mr glyceraldehyde‐3‐phosphate dehydrogenase, but also of a thirt subunit, R (40.5. kDa) comigrating with that from the active phosphoribulokinase of spinach.

Incubation of the complex with dithiothreitol markdely stimulated both its phosphoribulokinase and NADPH‐dependent dehydrogenase activities. This dithiothreitol‐induced activation was accompained by depolymerisation to give two predominantly NADPH‐linked tetrameric glyceraldehyde‐3‐phosphate dehydrogenases (the homotetramer, A4, and the heterotetramer, A2B2) as well as the active dimeric phosphoribulokinase. Incubation of the high‐Mr glyceraldehyde‐3‐phosphate dehydrogenase with dithiothreitol promoted complete depolymerisation yielding only the heterotetramer (A2B2).

Possible structures suggested for the glyceraldehyde‐3‐phosphate dehydrogenase/phosphoribulokinase complex are (A2B2)2A4R2 or (A2B2)(A4)2R2.
Substrate specificity of rat liver cytosolic α‐d‐mannosidase Novel degradative pathway for oligomannosidic type glycans
Authors: HAEUW Jean‐Françis , STRECKER Gerard , WIERUSZESKI Jean‐Michel , MONTREUIL Jean , MICHALSKI Jean‐Claude
Published: March 2005

The substrate specificity of rat liver cytosolic neutral α‐d‐mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(α‐2)Man(α‐3)[Man(α1–2)Man(α1–6)]Man(α1–2)Manα1–2)Man(α1–3)]Man(β‐4)GlcNAc; Man7GlcNac, Man(α1–3)[Man(α1–2)Man(α1–6)]Man(α1–6)[Man(α1–2)Man(α1–3]Man(β1–4)GlcNAc; Man5GlcNAc I, Man(α1–3)[Man(α1–6)]Man(α1–6)Man(α1–3)]Man(β1–4)GlcNAc; Man5GlcNAc II, Man(α1–2)Man(α1–2)Man(α1–3)[Man(α1–6)]Man(β1–4)GlcNAc; Man4GlcNAc, Man(α1–2)Man(α1–2)Man(α1–3)Man(β1–4)GlcNAc. The different oligosaccharide isomers resulting from α‐d‐mannosidase hydrolysis were analyzed by 1H‐NMR spectroscopy after HPLC separation. The cytosolic α‐d‐mannosidase activity is able to hydrolyse all types of α‐mannosidic linkages found in the glycans of the oligomannosidic type, i.e. α‐1,2, α‐1,3 and α‐1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble α‐d‐mannosidase acting on Man5GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(α1–6)[Man(α1–3)]Man(β1–4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(α1–2)Man(α1–2)Man(α1–3)[Man(α1–6)]Man(β1–4)GlcNAc, represents the ‘construction’ Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N‐glycosylprotein glycans. The cytosolic α‐d‐mannosidase is activated by Co2+, insensitive to 1‐deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal α‐d‐mannosidases [Michalski, J. C., Haeuw, J. F., Wieruszeski, J. M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369–379]. A possible complementarity between cytoslic and lysosomal α‐d‐mannosidase activities in the catabolism of N‐glycosylprotein is proposed.
Mechanisms of dramatic fluctuations of ornithine decarboxylase activity upon tonicity changes in primary cultured rat hepatocytes
Authors: TOHYAMA Youichi , KAMEJI Takaaki , HAYASHI Shin‐ichi
Published: March 2005

We investigated the mechanisms underlying the marked induction of ornithine decarboxylase (ODC) activity by hypotonic treatment and its rapid decay upon reversal to isotonicity in primary cultures of adult rat hepatocytes. Upon hypotonic treatment, ODC synthesis rate increased progressively whereas the amount of ODC mRNA increased only about twofold. In addition, ODC was stabilized severalfold. ODC activity rapidly decreased upon restoration of isotonicity, owing to immediate and nearly complete suppression of ODC synthesis and 3–6‐fold stimulation of ODC decay. The stimulation of ODC decay caused by restoration of isotonicity was mostly independent of time and protein synthesis. ODC decay was also stimulated by putrescine, even under hypotonic conditions, depending on time and new protein synthesis. Restoration of isotonicity and putrescine treatment together caused a synergistic stimulation of ODC decay, confirming that these act by different mechanisms.
Analysis of calcium binding to α‐lactalbumin using a fluorescent calcium indicator
Authors: EBERHARD Marc , ERNE Paul
Published: March 2005

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo‐3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo‐3 and bovine α‐lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of α‐lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the α‐lactalbumin–Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25°C, was found to be 123 ± 2 nM and 103 ± 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to α‐lactalbumin did not depend on pH in the range 6.6–8.4 and was differently affected by Na+ and K+. EDTA‐agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 ± 13 nM and the number of Ca2+ bound/molecule α‐lactalbumin was lowered to 0.069 ± 0.006. No interaction between fluo‐3 and α‐lactalbumin could be discerned from spectral analysis and fluorescence anisotrophy measurements.
Transcriptional regulation of interferon‐stimulated genes
Authors: WILLIAMS Bryan R. G.
Published: March 2005

Interferons (IFN) stimulate the expression of a number of genes following interaction with specific high‐affinity plasma membrane receptors. The products of these genes either singly or coordinately mediate the antiviral, growth inhibitory or immunoregulatory activities attributed to IFN. While the gene products in some cases have been well characterized, other IFN‐regulated genes encode proteins whose functions have yet to be elucidated. A feature common to all IFN‐stimulated genes characterized thus far is the presence of a DNA element which constitutes an IFN‐responsive enhancer, usually present in the 5′ upstream region of the genes. This element, termed interferon‐stimulated response element (ISRE) binds a nuclear factor(s) translocated from the cytoplasm to the nucleus following IFN‐receptor‐triggered signal transduction. The binding of these factors to the ISRE represents the initiating event in stimulating RNA‐polymerase‐II‐mediated transcription from IFN‐responsive genes. Depending on the nature of the cells responding to IFN and the genes involved, induced transcription may be prolonged or rapidly terminated. The rapid termination of transcription is dependent in some cases on IFN‐induced protein synthesis and also involves factor binding to the ISRE. Recent progress in detailing these events will be discussed including IFN‐receptor interactions, signal‐transduction pathways, comparing and contrasting IFN‐regulated genes and elucidation of IFN‐regulated factors.
Elongation factor 2 from Artemia salina embryos and its affinity for ribosomes
Authors: ZAMBONI Mariacristina , BRIGOTTI Maurizio , MONTANARO Lucio , SPERTI Simonetta
Published: March 2005

Crude extracts from Artemia salina undeveloped embryos do not contain detectable elongation‐factor‐2 (EF2) kinase and endogenous ADP‐ribosylating activities. Accordingly, EF2 purified from this source is an enzyme relatively free from phosphorylated and ADP‐ribosylated forms. Endogenous ADP‐ribosyltransferase activity appears only after purification of EF2. The affinities of EF2 and of ADP‐ribosyl‐EF2 for ribosomes from A. salina undeveloped embryos have been calculated by measuring the ability of the factors to inhibit the N‐glycosidase activity of ricin on ribosomes.
Differential scanning calorimetry of bovine rhodopsin in rod‐outer‐segment disk membranes
Authors: KHAN Saber M. A. , BOLEN Wayne , HARGRAVE Paul A. , SANTORO Marcelo M. , McDOWELL J. Hugh
Published: March 2005

Rhodopsin‐containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 ± 0.4°C and a thermal unfolding calorimetric enthalpy change (ΔHcal) of 700±17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9±0.3°C and a ΔHcal of 520±17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4–9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed ΔHcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. ΔHcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the ΔHcal values while the Tm values remain fairly constant. This shows that the ΔHcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.
Inhibition of glucose production during hepatic nerve stimulation in regenerating rat liver perfused in situ
Authors: IWAI Masaru , MIYASHITA Tetsukazu , SHIMAZU Takashi
Published: March 2005

To explore the possible role of gap junctions in neural regulation of hepatic glucose metabolism, the effects of hepatic nerve stimulation on metabolic and hemodynamic changes were examined in normal and regenerating rat liver which was perfused in situ at constant pressure via the portal vein with a medium containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate.

1. The content of connexin 32, a major component of gap junctions in rat liver, decreased transiently to about 25% of the control level in regenerating liver 48–72 h after partial hepatectomy and recovered to normal by the 11th day after the operation.

2. In normal liver, electrical stimulation of the hepatic nerves (10 Hz, 20 V, 2 ms) and infusion of noradrenaline (1 μM) both increased glucose and lactate output and reduced perfusion flow.

3. In early stage of regenerating liver 48 h and 72 h after partial hepatectomy, the increase in glucose output in response to nerve stimulation was almost completely inhibited, whereas the change in lactate balance was partially suppressed and the reduction of flow rate was retained. The response of glucose output to nerve stimulation recovered by the 11th day after partial hepatectomy. In contrast, exogenous application of noradrenaline increased glucose output even in the early stage of regenerating liver.

4. The increase in noradrenaline overflow during hepatic nerve stimulation in the early stage of regenerating liver was approximately the same as in normal liver. Liver glycogen was sufficiently preserved in the early stage of regenerating liver. However, noradrenaline infusion could no more increase glucose output both in normal and in regenerating livers after 24 h of fasting that depleted liver glycogen.

These results suggest that the impaired effects of sympathotic nerve stimlation on glucose metabolism observed in regenerating liver are derived neither from reduced release of noradrenaline nor from depletion of liver glycogen, but rather from transient reduction of gap junctions which assist signal propagation of the nerve action through intercellular communication in rat liver.
Adrenocorticotropin‐dependent regulation of glutathione transferase subunit 4 in cultured rat adrenal cells
Authors: MANKOWITZ Louise , RYDSTRÖM Jan , DEPIERRE Joseph W.
Published: March 2005

After hypophysectomy, the level of glutathione transferase subunit 4 increases in the adrenal, as well as in the liver, as do those of several other forms of glutathione transferase. This increase in subunit 4 can subsequently be down‐regulated by administration of adrenocorticotropin. The present investigation demonstrates that also in primary cultures of female rat adrenal cells an increase in the level of glutathione transferase subunit 4 (as shown by immunoblotting) occurs in the absence of adrenocorticotropin. When adrenocorticotropin or dibutyryladenosine 3′, 5′‐phosphate was administered to these cells, a down‐regulation of this enzyme level was observed, in agreement with the in vivo situation. This down‐regulation was not affected by aminoglutethimide, an inhibitor of the cholesterol‐side‐chain‐cleavage enzyme (cytochrome P‐450scc) which is the rate‐limiting step in the biosynthesis of steroids. Hence adrenal steroid production is not involved in the down‐regulation of glutathione transferase subunit 4 by adrenocorticotropin.
Regulation of transcription of the gene coding for peroxisomal 3‐oxoacyl‐CoA thiolase of Saccharomcyes cerevisiae
Authors: EINERHAND Alexandra W. C. , VOORN‐BROUWER Tineke M. , ERDMANN Ralf , KUNAU Wolf‐H. , TABAK Henk F.
Published: March 2005

Transferring Saccharomyces cerevisiae cells from glucose‐ to oleate‐containing growth media results in a significant increase in the number and volume of peroxisomes. To investigate this proliferation process we studied the transcriptional regulation of the gene coding for peroxisomal 3‐oxoacyl‐CoA thiolase (EC 2.3.1.16) in response to the switch in carbon source. Expression was proved to be repressed during growth on glucose, derepressed during growth on glycerol, and induced during growth on oleate as the sole carbon source. By deletion and mutational analysis of sequences upstream of this gene, we have identified a region which is involved in the regulation of transcription. It is contained within a 52‐base‐pair sequence, UAST52 (upstream activation sequence thiolase 52), located between 203 and 151 nucleotides upstream of the translational initiation codon. This sequence proved to be required for repression, derepression and induction of transcription, and was able to activate transcription from the truncated version of the heterologous iso‐1‐cytochrome‐c (CYCI) promoter in a similar way as in the wild‐type promoter context.

Sequence comparison revealed that the UAST52 contained a sequence motif (‘β‐oxidation box’, that is very similar to sequences located in the 5′‐upstream regions of the genes coding for two other β‐oxidation enzymes of S. cerevisiae: the peroxisomal acyl‐CoA oxidase and the peroxisomal trifunctional β‐oxidation enzyme of S. cerevisiae. Mutational analysis of the ‘β‐oxidation box’ indicates that this sequence motif acts as a UAS in vivo.

Sequence comparison also revealed that just upstream of the ‘β‐oxidation box’, between positions – 213 and – 201, a potential binding site occurred for the yeast multifunctional autonomously replicating sequence binding factor ABF1. Gel‐retardation‐competition experiments indicate that ABF1 binds specifically to this sequence.
Purification and characterization of phosphatidylinositol 4‐kinase from human erythrocyte membranes
Authors: WETZKER Reinhard , KLINGER Reinhard , HSUAN Justin , FRY Michael J. , KAUFFMANN‐ZEH Andrea , MÜLLER Elke , FRUNDER Horst , WATERFIELD Michael
Published: March 2005

Two species of PtdIns 4‐kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X‐100, followed by Cibacron‐blue–Sephadex, phosphocellulose and Mono Q anion‐exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000‐fold purification of the enzyme from erythrocyte membranes.

Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D‐4 position of the inositol ring.

The Km values of both PtdIns 4‐kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. the enzymes are both activated by Mg2+. and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.
Inhibition of the Na+/I‐ symporter by harmaline and 3‐amino‐1‐methyl‐5H‐pyrido(4,3‐b)indole acetate in thyroid cells and membrane vesicles
Authors: KAMINSKY Stephen M. , LEVY Orlie , GARRY Mark T. , CARRASCO Nancy
Published: March 2005

Novel inhibitors of the Na+/I− symporter were identified using rat‐thyroid‐derived FRTL‐5 cells and sealed vesicles from calf thyroid as model systems. Na+‐dependent 125I− uptake was inhibited by the hallucinogenic drug harmaline and by a chemically related convulsive agent, 3‐amino‐1‐methyl‐5H‐pyrido(4,3‐b)indole acetate (TRP‐P‐2). TRP‐P‐2 (Ki= 0.25 mM) was tenfold more effective as an inhibitor than harmaline (Ki= 4.0 mM). Inhibition by TRP‐P‐2 was competitive with respect to Na+ and was fully reversible. Although TRP‐P‐2 is a Inhibitively low‐affinity inhibitor, its affinity for the Na+ site of the Na+/I− symporter is over 100 times higher than that of Na+ (Km= 50 mM). 45Ca2+‐efflux rates in calf thyroid membrane vesicles were not affected by TRP‐P‐2, indicating that membrane integrity is not disrupted by the drug. These findings show that TRP‐P‐2 may be a potentially useful tool for the identification and characterization of the Na+/I− symporter.
Identification of carrot cDNA clones encoding a second putative proliferating cell‐nuclear antigen, DNA polymerase δ auxiliary protein
Authors: HATA Shingo , KOUCHI Hiroshi , TANAKA Yoshiyuki , MINAMI Eiichi , MATSUMOTO Takashi , SUZUKA Iwao , HASHIMOTO Junji
Published: March 2005

The proliferating cell‐nuclear antigen (PCNA) plays a key role in the control of eukaryotic DNA replication. We have isolated two cross‐hybridizing groups of cDNA encoding carrot homologs of PCNA. Sequence analysis and Southern‐blot experiments showed that the cDNA were derived from two distinct genes. One corresponded to the typical PCNA, which is known to be highly conserved in eukaryotes from yeast to man; its mRNA is 1.2 kb in size and the calculated molecular mass of the protein is 29 kDa. The other encoded a larger PCNA homolog which has not previously been reported; the mRNA is 1.5 kb in size, the N‐terminal three quarters (calculated molecular mass, 29 kDa) of the protein product is 88% identical at the amino acid level to the typical PCNA, but the protein has an extra C‐terminal domain of 11 kDa. Both PCNA homologs were apparently coexpressed concomitant with somatic embryogenesis. The mRNA level of the novel homolog is 10–20% that of the typical PCNA in the embryos. The presence of the second putative PCNA may provide new insight into studies on the mechanism of DNA replication in eukaryotes.
An intestinal galactose‐specific lectin mediates the binding of murine IgE to mouse intestinal epithelial cells
Authors: BRASSART Dominique , KOLODZIEJCZYK Eric , GRANATO Dominique , WOLTZ Arlette , PAVILLARD Michel , PEROTTI Fabienne , FRIGERI Luciano G. , LIU Fu‐Tong , BOREL Yves , NEESER Jean‐Richard
Published: March 2005

A number of lactose‐binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C‐terminal domain of IgE‐binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose‐specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE‐binding protein (ɛBP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant ɛBP. This intestinal form of ɛBP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal ɛBP. The carbohydrate‐dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood‐group‐A‐active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE‐binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE‐mediated disorders, such as food allergy.
An oxidized analog of α‐human atrial natriuretic polypeptide is a selective agonist for the atrial‐natriuretic‐polypeptide clearance receptor which lacks a guanylate cyclase
Authors: KOYAMA Satoshi , TERAI Takao , INOUE Takeshi , INOMATA Kaoru , TAMURA Kouichi , KOBAYASHI Yuji , KYOGOKU Yoshimasa , KOBAYASHI Masakazu
Published: March 2005

The differences in biological functions between α‐human atrial natriuretic polypeptide (α‐hANP) and its oxidized analog, MetSO‐α‐hANP, have been investigated. Analysis of the ANP receptor subtypes by affinity labeling has shown that a bovine pulmonary aortic endothelial cell line (CPAE cells) primarily expresses ANP‐R1 (R, receptor) coupled to particulate guanylate cyclase, while Hela cells from human cervical carcinoma predominantly express ANP‐R2, which lacks a guanylate cyclase. α‐hANP could bind to both ANP receptor subtypes with high affinity, while MetSO‐α‐hANP showed more selective binding to ANP‐R2 than to ANP‐R1. The activity of MetSO‐α‐hANP for stimulation of guanylate cyclase coupled to ANP‐R1 was about 520‐fold less than that of α‐hANP (median effective dose = 2.5 nM for α‐hANP, 1.3 μM for MetSO‐α‐hANP), indicating that MetSO‐α‐hANP was a partial agonist for this receptor. While this oxidized analog could inhibit the cAMP production through ANP‐R2, with 0.15 times the activity of α‐hANP (median concentration = 0.31 nM for α‐hANP, 2.0 nM for MetSO‐α‐hANP). In in vivo studies, the diuretic activity of MetSO‐α‐hANP was 25–100‐fold less than that of α‐hANP. In addition, MetSO‐α‐hANP could potentiate the diuretic activity of α‐hANP that was also caused by C‐ANF4–23, a specific agonist for ANP‐R2.

These results demonstrate that MetSO‐α‐hANP can act as an agonist more selective for ANP‐R2 than for ANP‐R1, both in vivo and in vitro. The relationship between receptor selectivities and the conformation of α‐hANP or MetSO‐α‐hANP was also discussed.
Estrogen induction of alcohol dehydrogenase in the uropygial gland of mallard ducks
Authors: HIREMATH Leena S. , KESSLER Patricia M. , SASAKI Glenn C. , KOLATTUKUDY Pappachan E.
Published: March 2005

Treatment of mallard ducks with estradiol, or a combination of estradiol and thyroxine, has been shown to result in the proliferation of peroxisomes and production of diesters of 3‐hydroxy fatty acids, the female pheromones, in the uropygial gland of male and female mallard ducks. Such a treatment results in the induction of a unique set of proteins. A cDNA library enriched in hormone‐induced transcripts was subjected to differential screening. The nucleotide sequence of one of the two unique cDNA clones, DGH1, had high similarity to the Human class I alcohol dehydrogenase (ADH) gamma subunit and represented the carboxy‐terminus of the protein from amino acid 190–374. SDS/PAGE and Western blot analysis of the proteins indicated that the level of a 38‐kDa protein that cross‐reacted with antibodies prepared against the chicken ADH was increased 5–7‐fold by hormone treatment. Assays for ADH activity in the uropygial gland extracts of male mallards showed a 5–7‐fold induction of the enzyme by hormone treatment. The 1.9‐kb ADH mRNA levels were increased 12–14‐fold under these conditions. Of all the tissues tested, the uropygial gland had the highest levels of ADH mRNA. Induction of ADH by estradiol treatment occurred only in this tissue. Elevated levels of ADH were also observed in the glands of male mallards in eclipse, the post‐nuptial condition when the hormonal balance is shifted to higher estrogen levels, suggesting that this enzyme is regulated by estrogens in this period. Estradiol treatment caused an 80% decrease in the NAD+/NADH ratio in the uropygial gland and a twofold increase in the fatty alcohol oxidation rate catalyzed by the gland extract. These observations could help explain how increased levels of ADH could contribute to the production of the diesters.
Localization of a vitamin‐D‐binding protein interaction site in the COOH‐terminal sequence of actin
Authors: HOUMEIDA Ahmed , HANIN Veronique , CONSTANS Jacques , BENYAMIN Yves , ROUSTAN Claude
Published: March 2005

The serum vitamin D binding protein is the carrier of vitamin D and its derivatives in the plasma. One of the known roles of this protein is to sequester monomeric actin in the blood, therefore implicating this protein in actin elimination. However, its binding site at the surface of actin is poorly delimited. We report here the results of a study which locates, using several actin fragments together with immunological probes, a vitamin D binding protein site near the COOH‐terminal extremity. Thus, the interface is delimited by the sequence 360–372 in subdomain I of actin.
Drosophila acetylcholinesterase
Authors: FOURNIER Didier , MUTERO Annick , RUNGGER Duri
Published: March 2005

In insects, acetylcholinesterase is mainly found in the central nervous system. It is expressed in the synapse where it hydrolyzes the neurotransmitter acetylcholine. Maturation of this protein involves several post‐translational modifications. The precursor polypeptide is cut at three sites; the N‐terminal signal peptide is removed, the C‐terminal hydrophobic polypeptide is clipped off and replaced by a glycolipid anchor and the resulting peptide is cut into two polypeptides, corresponding to active subunits. Two of these active subunits are associated to form the final active glycosylated protein. We have expressed the protein via microinjection of an expression vector into Xenopus oocyte nuclei. When the complete cDNA is injected, the acetylcholinesterase formed is biochemically similar to the Drosophila‐head acetylcholinesterase. However, the hydrophobic C‐terminal peptide is not replaced by a glycolipid anchor. As a consequence, the enzyme is no longer externalized, the proteolytic cutting of the main peptide does not occur and a new polymerization form occurs. Although incompletely processed, this protein is enzymatically active. When a cDNA lacking the coding region of the C‐terminal hydrophobic peptide is injected, the resulting acetylcholinesterase is hydrophilic, cleaved into two subunits and secreted into the incubation medium free of contaminants.
Preparation and kinetic properties of 5‐ethylphenazine–lactate‐dehydrogenase–NAD+ conjugate, a semisynthetic lactate oxidase showing a hide‐and‐seek effect
Authors: YOMO Tetsuya , URABE Itaru , OKADA Hirosuke
Published: March 2005

5‐Ethylphenazine–lactate‐dehydrogenase–NAD+ conjugate (EP+‐LDH‐NAD+) was prepared by linking poly(ethylene glycol)‐bound 5‐ethylphenazine and poly(ethylene glycol)‐bound NAD+ to lactate dehydrogenase. The average number of the ethylphenazine moieties bound per molecule of enzyme subunit was 0.46, and that of the NAD+ moieties was 0.32. This conjugate is a semisynthetic enzyme having lactate oxidase activity using oxygen or 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) as an electron acceptor; to make such conjugates seems to be a general method for artificially converting a dehydrogenase into an oxidase. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis‐Menten equation with the following kinetic constants: for the reaction system with oxygen, the turnover number per subunit is 2.3 min−1 and Km for oxygen is 1.91 mM; and for the system with MTT, the turnover number is 0.25 min−1 and Km for MTT is 0.076 mM. At the initial steady state of the oxidase reaction, only 2.1% of the NAD+ moieties of the conjugate are in the free state (i.e. not bound in the coenzyme‐binding site of the lactate dehydrogenase moiety) and the rest are hidden in the coenzyme site; almost all the NAD+ moieties are in the reduced state. The apparent intramolecular rate constant for the reaction between a free NADH moiety and an oxidized ethylphenazine moiety is 2.3 s−1 and 2.1 s−1 for the systems with oxygen and with MTT, respectively. The apparent effective concentration of the free NADH moiety for the ethylphenazine moiety is 5.5 μM and is much smaller than that (0.34 mM) of the ethylphenazine moiety for the free NADH moiety; this difference is due to the effect of hiding the NADH moiety in the binding site, as the hidden NADH moiety cannot react with the ethylphenazine moiety.
The molecular and genetic analysis of mouse development
Authors: GOSSLER Achim , BALLING Rudi
Published: March 2005

This review describes some recent advances in the molecular‐genetic analysis of mouse development. Reversed genetics and gene assignment have been used to isolate genes affected in developmental mutations. The establishment of a high‐density molecular‐genetic map promises to facilitate cloning of additional genes with developmental functions. Based on molecular, biochemical or other biological criteria many mouse genes that code for transcriptional regulators, growth‐factor‐like molecules and their receptors have been isolated. The role of these genes during development can be analysed in vivo after producing targeted mutations. Mutations can be generated by homologous recombination in the genome of embryonic stem cells and can then be introduced into the mouse germ line by means of germ‐line chimaeras. Additional approaches employing stem cells to identify and mutate putative developmental genes are coming into use.
Cross‐species comparison of the sequence of the leukaemia inhibitory factor gene and its protein
Authors: WILLSON Tracy A. , METCALF Donald , GOUGH Nicholas M.
Published: March 2005

Leukaemia inhibitory factor (LIF) is a pleiotropic growth factor active in diverse cell systems in both the adult and the embryo. The LIF gene from a number of mammalian species is highly conserved. The ovine and porcine LIF genes were cloned, sequenced and compared to the previously published murine and human LIF gene sequences. While the coding regions of the LIF gene are highly conserved, the non‐coding regions are largely non‐conserved. In a region of ∼ 340 bp, the 5′ end of the translational initiation codon is highly conserved (84%). This region includes four conserved TATA boxes, two transcriptional start‐sites identified in the murine gene and the minimal region required to function as the LIF promoter. A sequence in the murine gene adjacent to this highly conserved region which appears to contain a negative control element is, however, poorly conserved between the four species compared, except for a sequence of 16 conserved nucleotides. Within the largely non‐conserved first intron, there is a block of ∼ 150 nucleotides which is highly conserved between all four species (∼ 72%). However, a sequence in intron 1 of the murine LIF gene which corresponds to an alternative exon of a putative variant LIF transcript is very poorly conserved between species, with only relics of this exon evident in the other three species. A comparison of the five LIF protein sequences available (murine, rat, human, ovine and porcine) revealed that the protein displays a high degree of similarity, ranging from 74% between mouse and sheep to 92% between rat and mouse. Several large blocks of absolutely conserved amino acid sequence were identified. The ovine LIF gene was modified to allow production of recombinant ovine LIF in yeast cells, which was shown to be biologically active on murine cells.
6‐Deoxyerythronolide‐B synthase 2 from Saccharopolyspora erythraea
Authors: BEVITT Debra J. , CORTES Jesus , HAYDOCK Stephen F. , LEADLAY Peter F.
Published: March 2005

Sequencing of the eryA region of the erythromycin biosynthetic gene cluster from Saccharopolyspora erythraea has revealed another structural gene (ORF B), in addition to the previously characterised ORF A, which appears to encode a component of 6‐deoxyerythronolide‐B synthase, the enzyme that catalyses the first stage in the biosynthesis of the polyketide antibiotic erythromycin A. The nucleotide sequence of ORF B, which lies immediately adjacent to ORF A, has been determined. The predicted gene product of ORF B is a polypeptide of 374417 Da (3568 amino acids), which is highly similar to the product of ORF A and which likewise contains a number of separate domains, each with substantial amino acid sequence similarity to components of known fatty‐acid synthases and polyketide synthases. The order of the predicted active sites along the chain from the N‐terminus is 3‐oxoacyl‐synthase–acyltransferase–acyl‐carrier‐protein–3‐oxoacyl‐synthase–acyltransferase–dehydratase–enoylreductase–oxoreductase–acyl‐carrier‐protein. The position of the dehydratase active site has been pinpointed for the first time for any polyketide synthase or vertebrate fatty‐acid synthase. The predicted domain structure of 6‐deoxyerythronolide‐B synthase is strikingly similar to that previously established for vertebrate fatty‐acid synthases. This analysis of the sequence supports the view that the erythromycin‐producing polyketide synthase contains three multienzyme polypeptides, each of which accomplishes two successive cycles of polyketide chain extension. In this scheme, the role of the O R F B gene product is to accomplish extension cycles 3 and 4.
Analysis of genes involved in the biosynthesis of lantibiotic epidermin
Authors: SCHNELL N. , ENGELKE G. , AUGUSTIN J. , ROSENSTEIN R. , UNGERMANN V. , GÖTZ F. , ENTIAN K.‐D.
Published: March 2005

The structural gene of the lanthionine‐containing peptide antibiotic epidermin is located on a 54‐kb plasmid of Staphylococcus epidermidis [Schnell et al. (1988) Nature 333, 276–278]. A 13.5‐kb DNA region neighbouring the epidermin structural gene (epiA) was subcloned and its sequencing revealed five additional open reading frames. Three of these reading frames, epiB, epiC and epiD shared no homology with previously described proteins stored in data bases. They were located 3′ adjacent to epiA. Using epiB as a probe, a 5‐kb mRNA was identified indicating that three or all four reading frames are transcribed as an operon. Additionally, a 0.3‐kb mRNA specific for epiA was identified. Two open reading frames (epiP and epiQ) were located 3′ to epiA, epiB, epiC and epiD, but in the reverse orientation. The epiQ gene product shows similarity to the positive regulatory factor PhoB. This might indicate a regulatory function of epiQ in epidermin biosynthesis. The epiP gene product shows striking similarity to several serine proteases which makes epiP a likely candidate for processing the epidermin prepeptide. Heterologous epidermin synthesis in the non‐producing organism Staphylococcus carnosus finally proved that these reading frames are necessary for epidermin biosynthesis.
Subunit composition of the untransformed glucocorticoid receptor in the cytosol and in the cell
Authors: ALEXIS Michael N. , MAVRIDOU Irene , MITSIOU Dimitra J.
Published: March 2005

We have used bifunctional reagents to examine the subunit composition of the non‐DNA‐binding form of the rat and human glucocorticoid receptor. Treatment of intact cells and cell extracts with a reversible cross‐linker, followed by electrophoretic analysis of immunoadsorbed receptor revealed that three proteins of apparent approximate molecular masses, 90, 53 and 14 kDa are associated with the receptor. The first of these was identified immunochemically as a 90‐kDa heat‐shock protein (hsp90). The complex isolated from HeLa cells contained 2.2 mol hsp90/mol steroid‐binding subunit. Cross‐linking of the receptor complex in the cytosol completely prevented salt‐induced dissociation of the subunits. The cross‐linked receptor was electrophoretically resolved into two oligomeric complexes of apparent molecular mass 288 kDa and 347 kDa, reflecting the association of the 53‐kDa protein with a fraction of the receptor. Since no higher oligomeric complexes could be generated by cross‐linking cell extracts under different conditions, we conclude that most of the untransformed cytosolic receptor is devoid of additional components.
The piscine plasma retinol‐binding protein
Authors: BERNI Rodolfo , STOPPINI Monica , ZAPPONI Maria Carla
Published: March 2005

1. Retinol‐binding protein (RBP) has been isolated from the pooled plasma or rainbow trouts (Oncorhinchus mykiss) by gel filtration, hydrophobic interaction chromatography and ion‐exchange chromatography. By this procedure two forms of the protein, both with a molecular mass (approximately 20 kDa) similar to that of mammalian RBP, were purified to homogeneity. Five amino acid substitutions have been found in the partial (about 60%) sequences of the two forms of trout RBP, which are presumably acetylated at their N terminus. The apparent participation of six conserved cysteines in the formation of disulphide bridges, as in human RBP, and the similarity (about 60%) of the amino acid sequence of trout and mammalian RBPs, indicate the existence of a similar overall structure organization in evolutionary distant RBPs.

2. Although the two forms of trout RBP are not physiologically involved in the formation of any protein‐protein complex in plasma, they are capable of interacting with mammalian transthyretin, albeit with a binding affinity (K′d= 15–40 μM) considerably lower than that of mammalian RBP. Our data indicate that the two forms of trout RBP also possess the region that in mammalian RBP has the functional role of binding transthyretin. It is suggested that transthyretin (or a homologous protein) was modified, during phylogenetic development of the non mammalian vertebrates, to acquire a binding site for such a region of the RBP molecule.